Enhancing the Efficacy of Anti-CD20 Monoclonal Antibody Therapy Through Manipulation of the Tumour Microenvironment

Enhancing the efficacy of anti-CD20 monoclonal antibody therapy through manipulation of the tumour microenvironment

A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health

2018

Ester Fagnano

Division of Cancer Sciences

School of Medical Sciences

Contents

List of contents 2

List of figures 7

List of abbreviations 12

Abstract 16

Declaration 17

Acknowledgements 19

1. Introduction 22

1.1. B-cell malignancies 23

1.2. Anti-CD20 monoclonal antibodies 28

1.3. Mechanisms of action of anti-CD20 mAbs 32

1.3.1. Complement-dependent cytotoxicity (CDC) 34

1.3.2. Antibody-dependent cellular cytotoxicity (ADCC) and

phagocytosis (ADCP) 36

1.3.3. Programmed cell death (PCD) 38

1.4. Rituximab and tumour resistance to treatment 39

1.5. Obinutuzumab (GA101) 41

1.6. The tumour micro-environment in B-cell lymphomas 45

1.6.1. Contact-dependent interactions of malignant B cells with stromal-like cells 48

1.6.2. Soluble factor-mediated initiation of pro-survival signalling pathways 49

1.6.3. Reciprocal interactions between TME and tumour B cells 49

1.6.4. CXCR-4/CXCL-12/CXCR-7 signalling axis 51

1.6.5. VLA-4/VCAM-1 signalling axis 52

2 1.7. Anti-CD20 mAbs and the tumour microenvironment 54

1.8. Induction of long-term anti-tumour immune responses 56

1.9. Project outline 57

2. Materials and methods 59

2.1. Cell lines 59

2.2. In vivo experiments 60

2.3. Isolation of PBMCs from whole blood and buffy coats 60

2.4. Antibodies and reagents 62

2.5. Flow cytometry 62

2.6. Co-culture experiments 63

2.6.1. Programmed cell death assay 64

2.6.2. Complement-dependent cytotoxicity assay 64

2.6.3. Antibody-dependent cellular phagocytosis assay 64

2.6.4. NK cell activation (IFN-γ release assay) 65

2.7. Migration assay 66

2.8. Receptor expression levels on tumour cell surface 66

2.9. Detection of protein expression through western blotting 67

2.10. Immunocytochemistry 68

2.11. Microscopy and time-lapse experiments 68

2.12. Stable isotope labelling by amino acids in cell culture (SILAC) 69

2.13. Mass spectrometry 69

2.14. Enrichment analysis of differentially expressed proteins 70

2.15. Statistical analysis 70

3. Stroma-mediated protection of tumour cells from anti-CD20 mAb-induced programmed cell death (PCD) 72

3.1. Effect of co-culture with stromal cells on mAb-induced PCD 74

3 3.2. Role of the CXCR-4/CXCL-12 pathway in stromal-tumour cell interactions 77

3.2.1. CXCR-4-dependent tumour cell migration 79

3.2.2. Effects of CXCR-4/CXCL-12 blockade on GA101-induced cell death 86

3.3. Role of stromal cell contact versus stroma-released soluble factors 90

3.3.1. GA101-induced PCD in the presence of medium conditioned by stromal cells 90

3.3.2. Non-contact co-culture of stromal cells and tumour cells 93

3.4. Role of the VLA-4/VCAM-1 pathway in stromal-tumour cell interactions 95

3.4.1. Effects of VLA-4 blockade on GA101-induced cell death 97

3.5. Effect of mAb pre-treatment on stroma-mediated protection 99

3.6. Effect of co-culture on tumour cell death after removal of contact 107

3.7. Effect of co-culture on actin remodelling after removal of contact 110

3.8. Comparison between culture on plastic, vs stroma, vs fibronectin 112

3.9. Discussion 120

4. Stroma-mediated protection of tumour cells from anti-CD20 mAb-induced antibody-dependent cellular phagocytosis (ADCP) 123

4.1. Stroma-mediated protection from monocyte-mediated ADCP 125

4.1.1. Influence of length of tumour/stroma co-culture times on protection from monocyte-mediated ADCP 131

4.1.2. Role of contact with stroma vs soluble factors 134

4.1.3. Effect of attachment on the ECM component fibronectin on ADCP 137

4.1.4. Role of CD47/CD172a (SIRP-α) signalling pathway in stroma-mediated protection from monocyte-mediated ADC 139

4.1.5. Effect of stroma on ADCP mediated by different subsets of monocytes 143

4.2. Stroma-mediated protection from macrophage-mediated ADCP 149

4.3. Stroma-mediated protection from neutrophil-mediated ADCP 152

4 4.4. Discussion 154

5. Stroma-mediated protection of tumour cells from other mechanisms of action of anti-CD20 mAbs 157

5.1. Stroma-mediated protection of tumour cells from anti-CD20 mAb- induced antibody-dependent cellular cytotoxicity (ADCC) 157

5.1.1. GA101-induced ADCC measured as NK cell activation 157

5.1.1.1. Effect of S-CM on NK cell activation 162

5.1.1.2. Effect of different tumour-stroma contact times on NK cell activity 164

5.1.1.3. Effect of attachment on the ECM component fibronectin on NK cell activity 167

5.2. Stroma-mediated protection of tumour cells from anti-CD20 mAb- induced complement-dependent cytotoxicity (CDC) 169

5.2.1. Role of CXCR-4/CXCL-12 axis in stroma-mediated protection from rituximab-induced CDC 172

5.3. Discussion 174

6. Functional changes in tumour cells’ proteome after co-culture with stroma 177

6.1. Mass spectrometry-based proteomics 177

6.1.1. SILAC experiment 178

6.2. In silico analysis of protein expression changes 179

6.2.1. DAVID (Database for Annotation, Visualization and Integrated Discovery) 179

6.2.2. IPA (Ingenuity Pathway Analysis) 183

6.2.2.1. Canonical pathway analysis in IPA 183

6.3. In vitro validation of enrichment analysis 191

6.3.1. Role of the B-cell receptor (BCR) signalling pathway 191

6.3.2. Effect of blockade of the BCR pathway on CD20 expression 196

5 6.3.3. Effect of co-culture with stroma on expression level of surface CD20 molecule 199

6.3.4. Role of cadherin-mediated cell-cell adhesion 202

6.4. Discussion 214

7. In vivo effect of immune cell mobilisation on mAb efficacy 218

7.1. Effect of CXCR-4 blockade in combination with GA101 in vivo 218

7.2. Effect of TGF-β blockade in combination with GA101 in vivo 229

7.3. Migration of tumour cells to the bone marrow in vivo 232

7.4. Discussion 238

8. Conclusions and future directions 240

8.1. Summary of main findings 241

8.2. Discussion of methodology and future work 244

8.3. Impact of research in the field 253

9. Bibliography 256

Word count: 58533

6 List of figures

Figure 1.1 Stages of B cell development and malignancies arisen from each of them 26

Figure 1.2 General structure of antibody molecules 30

Figure 1.3 Mechanisms of action exerted by anti-CD20 mAbs 33

Figure 1.4 Hypothesised CD20 binding model of type-I (A) and type-II (B) mAbs 35

Figure 1.5 The CD20 molecule and mAbs binding epitopes 43

Figure 1.6 B lymphocytes interact with the TME which provides them with pro-survival and 47 anti-apoptotic signals

Figure 2.1 Schematic diagram showing the separation of components in blood upon density 61 gradient centrifugation

Figure 3.1 Representative image of the gating strategy used in FlowJo 73

Figure 3.2 Stromal cells protect Raji (A) and Daudi (B) cells from anti-CD20 mAb-induced 75 direct cell death

Figure 3.3 Human stromal cells HS-5 protect Raji (A) and Daudi (B) cells from GA101- 76 induced direct cell death

Figure 3.4 CXCR-4 and CXCR-7 are highly expressed on Raji and Daudi cell surface 78

Figure 3.5 Daudi cells migrate through a transwell membrane in the presence of SDF-1α 80

Figure 3.6 Daudi cells migration in response to SDF-1α is inhibited by the CXCR-4 82 antagonist plerixafor

Figure 3.7 Tumour cell migration toward a layer of stromal cells is blocked upon CXCR-4 84 inhibition

Figure 3.8 Tumour cell migration toward a layer of stromal cells is blocked upon CXCL-12 85 inhibition

Figure 3.9 Blockade of CXCR-4 on cell surface does not abrogate stromal-mediated 87 protection from GA101-induced PCD

Figure 3.10 Blockade of CXCL-12/CXCR-4 and CXCL-12/CXCR-7 interactions does not 89 abrogate stromal-mediated protection from GA101-induced PCD

Figure 3.11 Stromal cell-CM does not protect Raji and Daudi cells from GA101-induced PCD 91

Figure 3.12 Tumour/stromal cell-CM does not protect Raji and Daudi cells from GA101- 92 induced PCD

Figure 3.13 Stromal cells protection of tumour cells is lost in non-contact conditions 94

7 Figure 3.14 VLA-4 is expressed on Raji and Daudi cell surface 96

Figure 3.15 Blockade of VLA-4/VCAM-1 interactions does not impair stromal-mediated 98 protection from GA101-induced PCD

Figure 3.16 Representative images of Raji cells pre-treated with GA101 and then poured onto 100 stroma

Figure 3.17 Representative images of Daudi cells pre-treated with GA101 and then poured 101 onto stroma

Figure 3.18 Representative images of Raji-GFP-actin cells pre-treated with GA101 and then 103 poured onto stroma

Figure 3.19 Representative images of Raji cells pre-treated with GA101 and then poured onto 104 stroma

Figure 3.20 Pre-incubation of tumour cells with GA101 followed by pouring cells onto the 106 stromal layer does not reduce stroma-mediated protection from PCD

Figure 3.21 Representative dot plots showing absence of contaminating stromal cells 108

Figure 3.22 Stroma-mediated protective effect lasts after removal of contact 109

Figure 3.23 Representative images of actin remodelling in Raji cells after treatment 111

Figure 3.24 Attachment to the ECM component fibronectin does not mediate relevant 113 protection from GA101-induced PCD

Figure 3.25 Representative images of tumour cells pre-treated with GA101 and cultured on 115 plastic, stroma or fibronectin

Figure 3.26 A higher concentration of the ECM component fibronectin does not increase 117 protection of tumour cells from GA101-induced PCD

Figure 3.27 Representative images of tumour cells pre-treated with GA101 and cultured on 118 plastic, stroma or fibronectin (10 µg/cm2)

Figure 3.28 Representative images of tumour cells treated with GA101 on fibronectin (10 119 µg/cm2)

Figure 4.1 Representative image of the gating strategy used in FlowJo 124

Figure 4.2 Stromal cells decrease the ability of monocytes to phagocyte tumour cells 126

Figure 4.3 Monocyte-mediated ADCP of tumour cells cultured on plastic increases over time 128

Figure 4.4 Stromal cells’ ability to decrease monocyte-mediated phagocytosis of Raji cells 129 lasts over time

Figure 4.5 Stromal cells’ ability to decrease monocyte-mediated phagocytosis of Daudi cells 130 lasts over time

Figure 4.6 Protection from monocyte-mediated ADCP in Raji cells is only observed after a 132 pre-contact time of at least 1 hour

8 Figure 4.7 Protection from monocyte-mediated ADCP in Daudi cells is only observed after a 133 pre-contact time of at least 1 hour

Figure 4.8 Culture of tumour cells in S-CM does not decrease monocyte-mediated ADCP 135

Figure 4.9 Culture of tumour cells in T/S-CM does not decrease monocyte-mediated ADCP 136

Figure 4.10 Culture of tumour cells on fibronectin does not lead to decreased monocyte- 138 mediated ADCP

Figure 4.11 Anti-mouse CD172a antibody efficiently blocks its binding epitope on the 140 CD172a molecule

Figure 4.12 Blockade of CD172a fails to abrogate protection from monocyte-mediated ADCP 142

Figure 4.13 Representative plot showing three different subsets of monocytes based on 144 CD14/CD16 expression

Figure 4.14 Stromal cells strongly decrease ADCP mediated by the classical subset of 145 monocytes

Figure 4.15 Stromal cells do not induce a strong protective effect from non-classical 147 monocyte-mediated ADCP

Figure 4.16 Representative dot plots showing the percentage of CD14+ and CD16+ cells 148 within the CellVue+-isolated cells

Figure 4.17 Stromal cells mediate protection of tumour cells from human macrophage- 150 mediated ADCP

Figure 4.18 Stromal cells mediate protection of tumour cells from murine macrophage- 151 mediated ADCP

Figure 4.19 Stromal cells mediate protection of tumour cells from human neutrophil-mediated 153 ADCP

Figure 5.1 Representative image of the gating strategy used in FlowJo 159

Figure 5.2 Stromal cells decrease NK cells’ activity in the presence of GA101 161

Figure 5.3 Culture and treatment in S-CM does not impair the ability of GA101 to activate 163 NK cells

Figure 5.4 Stroma-mediated decrease of NK cell activation is independent of contact times 165 between stromal and Raji cells

Figure 5.5 Stroma-mediated decrease of NK cell activation is independent of contact times 166 between stromal and Daudi cells

Figure 5.6 Adhesion of tumour cells to fibronectin does not reduce NK cell activation upon 168 GA101 treatment

Figure 5.7 Representative graph showing M2-10B4 sensitivity to human serum 170

Figure 5.8 Stromal cells protect tumour cells from rituximab-induced CDC 171

9 Figure 5.9 Inhibition of CXCR-4 does not sensitise Raji cells to death by CDC and does not 173 reduce survival mediated by stromal cells

Figure 6.1 The five most enriched functional clusters identified by DAVID 181

Figure 6.2 Functional clusters with enrichment score higher than 6.5 identified by DAVID 182

Figure 6.3 Canonical pathway analysis in IPA showing the most significantly enriched 185 pathways (1 to 20)

Figure 6.4 Canonical pathway analysis in IPA showing the most significantly enriched 186 pathways (21 to 40)

Figure 6.5 P-values and Z-scores for each canonical pathway analysed by IPA 187

Figure 6.6 Overlapping pathway analysis performed by IPA showing the 20 most 189 significantly enriched pathways, interconnected if shared proteins are present

Figure 6.7 Overlapping pathway analysis performed by IPA showing the 20 most 190 significantly enriched pathways that share at least 15 proteins

Figure 6.8 Blockade of Syk or PI3Kδ does not impair stromal-mediated protection from 193 GA101-induced PCD

Figure 6.9 Combination of GA101 with the BTK inhibitor ibrutinib does not abrogate 195 stroma-mediated protection of tumour cells

Figure 6.10 Pre-treatment of Raji cells with ibrutinib leads to a reduction in the surface level 197 of CD20

Figure 6.11 Pre-treatment of Daudi cells with ibrutinib leads to a reduction in the surface level 198 of CD20

Figure 6.12 Co-culture of Raji cells with stromal cells significantly decreases surface levels of 200 the CD20 molecule

Figure 6.13 Co-culture of Daudi cells with stromal cells significantly decreases surface levels 201 of the CD20 molecule

Figure 6.14 Culture of tumour cells on stromal cells increases the expression of total cadherin 204

Figure 6.15 Culture of tumour cells on the ECM component fibronectin does not lead to an 206 increase in the amount of total cadherin content

Figure 6.16 Culture of tumour cells on stromal cells, but not on the ECM component 207 fibronectin, leads to an increase in the amount of total cadherin content

Figure 6.17 E-cadherin expression on the surface of Raji, but not Daudi, tumour cells 209 decreases after co-culture with stromal cells

Figure 6.18 N-cadherin expression on the surface of Raji, but not Daudi, tumour cells 210 decreases after co-culture with stromal cells

Figure 6.19 VE-cadherin is not expressed on the surface of Raji and Daudi cells 212

Figure 6.20 P-cadherin is not expressed on the surface of Raji and Daudi cells 213

10 Figure 7.1 Treatment schedule used to analyse in vivo efficacy of combination between PXF 220 and GA101

Figure 7.2 Sysmex count of immune cell subsets at day 1 demonstrates that plerixafor is able 222 to mobilise CD34+ cells

Figure 7.3 Sysmex count of immune cell subsets at day 7 demonstrates that plerixafor is able 223 to mobilise CD34+ cells

Figure 7.4 Sysmex count of immune cell subsets at day 14 demonstrates that plerixafor is 224 able to mobilise CD34+ cells

Figure 7.5 Combination of GA101 and PXF did not lead to significant survival advantages in 226 a syngeneic model of T-cell lymphoma

Figure 7.6 Treatment with GA101 and combination of GA101 and PXF did not lead to 227 significant survival advantages in a syngeneic model of B-cell lymphoma

Figure 7.7 Earlier treatment with GA101 and PXF did not lead to significant survival 228 advantages in a syngeneic model of CD20-expressing EL4-huCD20

Figure 7.8 Treatment schedule used to analyse in vivo efficacy of combination between 230 1D11 and tositumumab

Figure 7.9 Combination of Tositumumab and 1D11 did not lead to significant survival 231 advantages in a syngeneic model of B-cell lymphoma

Figure 7.10 Schematic treatment schedule and survival of NSG mice after tumour inoculation 234

Figure 7.11 Images showing localisation of tumour burden in NSG mice at day 1 235

Figure 7.12 Images showing localisation of tumour burden in NSG mice at day 7 236

Figure 7.13 Images showing localisation of tumour burden in NSG mice at day 14 237

11 List of abbreviations

7-AAD 7-Aminoactinomycin D ADCC antibody-dependent cellular cytotoxicity

ADCP antibody-dependent cellular phagocytosis

Akt protein kinase B

ALL acute lymphoblastic leukaemia

ANOVA analysis of variance

APC allophycocyanin Ara-C arabinosylcytosine (cytarabine) ATCC American Type Culture Collection BAFF B-cell activating factor Bcl-2 B-cell lymphoma 2

BCR B-cell receptor bFGF basic fibroblast growth factor

BL Burkitt’s lymphoma

BM bone marrow

BMSC bone marrow stromal cell

BTK Bruton’s tyrosine kinase

CAF cancer-associated fibroblast

CD cluster of differentiation

CDC complement-dependent cytotoxicity

CHOP cyclophosphamide, doxorubicin, vincristine, prednisone

CLL chronic lymphocytic leukaemia

CM conditioned media CML chronic myeloid leukaemia CXCL-12 C-X-C motif chemokine ligand 12

CXCR-4 C-X-C motif chemokine receptor 4

DCs dendritic cells

12 DLBCL diffuse large B-cell lymphoma

ECM extracellular matrix ERK extracellular-signal-regulated kinase

ES enrichment score

FACS fluorescence-activated cell sorter FAP fibroblast activation protein FC fludarabine, cyclophosphamide

FcR Fc receptor

FcγR Fcγ receptor

FL follicular lymphoma

GSK-3β glycogen synthase kinase-3β

HCL hairy cell leukaemia

HL Hodgkin’s lymphoma

HRI heme-regulated eIF2α

HSC haematopoietic stem cell

IAP inhibitor of apoptosis protein

IFN interferon

Ig immunoglobulin

IgG immunoglobulin G

IL interleukin

ILK integrin-linked kinase

LAM lymphoma-associated macrophage

LC liquid chromatography

LL lymphocytic lymphoma

LMP lysosomal membrane permeabilization mAb monoclonal antibody

MAPK mitogen-activated protein kinase

MCL mantle cell lymphoma

13 Mcl-1 myeloid cell leukemia-1

MFI median fluorescence intensity MM multiple myeloma MSC mesenchymal stromal cell MS/MS tandem mass spectrometry

MZL marginal zone lymphoma

NADPH nicotinamide adenine dinucleotide phosphate

NFκB nuclear factor κ-light-chain-enhancer of activated B cells

NHL non-Hodgkin’s lymphoma

NK natural killer

NLC nurse-like cell

NSG NOD-SCID-γ chain-deficient

O/N overnight ORR overall response rate

PB peripheral blood

PBTC peripheral blood T-cell lymphoma

PCD programmed cell death

PCDH protocadherin

PE R-phycoerythrin PDGF platelet-derived growth factor PI3K phosphatidylinositol-4,5-bisphosphate 3-kinase

PMN polymorphonuclear neutrophil

ROS reactive oxygen species

RT room temperature

SDF-1 stromal-derived factor 1

SEM standard error of the mean SLL small lymphocytic lymphoma

SMA smooth muscle actin

SNP single nucleotide polymorphism

14 STAT signal transducer and activator of transcription

TGF-β transforming growth factor-β

TME tumour microenvironment

TNF-α tumour-necrosis factor-α

TSP-1 thrombospondin-1

VCAM-1 vascular cell adhesion molecule-1 VEGF vascular endothelial growth factor

VLA-4 very late antigen-4 XIAP X-linked inhibitor of apoptosis

15 Abstract

Anti-CD20 monoclonal antibodies have significantly improved outcomes in a wide range of B-cell malignancies over the last two decades. However, many patients still relapse, and develop progressive and refractory disease, ultimately dying of their disease. Such resistance to therapy is mainly caused by the ability of malignant B cells to migrate towards the bone marrow and home in the stromal layer. In the bone marrow, the tumour micro-environment components, namely stromal cells, extracellular matrices and soluble growth factors, promote the onset of tumour-stromal-tumour interactions, which ultimately mediate tumour cell survival and protection from therapies. The abrogation of such stroma- mediated increased survival could lead to higher responses to therapy. Therefore, the project aims to characterise the stroma-mediated tumour cell protection from the novel type-II antibody GA101 (obinutuzumab) and to develop strategies to block tumour-stromal interactions, in order to improve therapeutic efficacy.

Firstly, a stromal-tumour co-culture system has been established in vitro which was able to protect tumour cells from GA101-mediated programmed cell death (PCD), antibody- dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC). The basis of this protection was investigated, to understand whether such increased survival was due to direct contact between tumour cells and stromal layer, or to soluble components of the microenvironment. Some of the main pathways known to mediate interaction and migration of B cells towards the micro-environment were analysed, to investigate whether their inhibition could have led to the abrogation of stroma- mediated tumour cell protection. A mass spectrometry-based proteomic analysis was then performed to gain insights into pathways in tumour cells which were altered after co- culture with stromal cells.

It was shown that the presence of stromal cells was able to protect tumour cells from type- II antibody-induced PCD, ADCP and ADCC in vitro, and such protection seemed to require the contact between stroma and tumour cells. Stromal cells appeared to interfere with the GA101-mediated B cell homotypic adhesion that leads to PCD. The proteomic analysis of tumour cells after contact with stroma led to the identification of a number of altered pathways, some of which have been further explored in vitro to validate their role in mediating protection of tumour cells from type-II antibody GA101.

Characterisation of the stroma-mediated protection of malignant B cells and a deeper understanding of tumour-stroma interactions could prove a useful tool to define better strategies to target the micro-environment, ultimately improving therapeutic outcomes in B-cell lymphoma patients.

16 Declaration

I declare that no portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning

17 Copyright statement

The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes.

Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made.

The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions.

Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses

18 Acknowledgements

I am sat here writing the very last page of my PhD thesis, and I still hardly believe I have actually managed to reach this stage. The hardest and most important 3.5 years of my career have finally come to an end.

I would have never been able to climb this mountain that my PhD has been without your support.

My uttermost gratitude goes to my main supervisor, Prof Tim Illidge, for his constant help, guidance and advice. Tim gave me the opportunity to undertake this PhD and had confidence in my capacities even before I started doing so myself. His guidance made me grow both scientifically and personally.

None of this work would have been completed – and in fact, I would still be stuck in front of a 4-colour Calibur analyser, trying to figure out how to change the compensation settings – without the help of Dr Eleanor Cheadle. I came here with my very short 6-month previous experience in a lab, and she found the time and patience to teach me every single laboratory technique I now know. But most importantly, she has been an example of rigour, hard work and dedication, and her scientific acuity has been inspirational for me.

I would also like to thank my advisor Dr Claus Jørgensen, for his helpfulness and his advice with both the science and the future career.

I would like to express my deep gratitude to everyone in the Targeted Therapy group for the help, support, advices and the great time. I had three and a half amazing years, and both bad and good times helped me become who I am today.

I feel a special mention goes to these guys:

Dr Jamie Honeychurch, who has patiently answered my typical hundred thousand questions per day about actin remodelling and GA101 although being always super busy with his own students, science and career.

19 Dr Rick Walshaw, who from just a colleague became the best lab mate I could ever wish for. This PhD – and especially this last year – would have been much harder without his friendship and the endless banter, the chocolate club, the gym sessions and the countless tea breaks.

Federica Monaco, for being a constant support throughout my entire PhD, a team-mate both in and out the lab and a friend I know I could always count on.

Dr Swati Pendharkar, for the amount of chocolate bars, sandwiches, pastries & co that I would always find on my desk at the end of a very long, hard day in the lab.

Dr Sapna Lunj, for being a source of positivity every single day and for her precious help during the stressful times of thesis writing and lab moves.

I would like to thank all the students and clinical fellows who have been part of the lab during these 3.5 years, for their support, their input and their company (and for being patient while sharing the office with the professional loud chatter I am!).

My gratitude also goes to the Medical Oncology group, who have been a second home for me since the devastating fire of 26th April 2017 and until exactly a year later. Thank you for hosting our group for almost a year and me for even longer, for making me feel welcomed and supported during the difficult last months.

Many people have united and worked together to make sure the lab work kicked off again as promptly as possible after the fire. I would like to express my gratitude to all these people that made the completion of this PhD possible, starting from the CRUK MI, to the University of Manchester, to all the members of the public who donated to fund us. Thank you.

This PhD was not just hard work and mind-blowing science: I was fortunate enough to meet amazing people, many of which are now my friends, with whom I shared laughs, experiences and cheeky pints after work. So thank you Sakis, Denys, Amy, Luke, Joe, Sam for your friendship.

I would not have reached the finish line and obtained any of my success without the help of my lifelong friends, who have been supporting me from afar. Thank you Eric, Flavia, Andreana, Roberta, Mauro, Sara, Marta and Mario for having been and still being, despite the distance, a constant certainty in my life.

20 Last but not least, I would like to thank the two people I never thank enough: my parents. I could never find the right words to express all my love and gratitude. I would have been nowhere near who I am without them, without their help, their patience, their scowls and their love. All the goals I reach, all the success I obtain, I owe it to them. Thank you Mami and Papi.

Now for the fun part…!

21 1. Introduction

Haematological malignancies include those cancers that arise from cells in the blood, the immune system and lymphatic organs and as group have relatively high incidence in the UK. Leukaemias and lymphomas account for the majority of cancer cases in children under 14 years old, representing one of the principal causes of death, together with brain tumours [1]. Among the different classes of haematological cancers, non-Hodgkin’s lymphoma (NHL) is the most common type, with more than 10000 new cases expected in the UK each year and a 5-year relative survival of 66.1% [2].

Arguably the important advance in therapeutic strategies over the last two decades has been the introduction of anti-CD20 monoclonal antibodies (mAbs) for the treatment of B- cell malignancies, which has led to substantially improved outcomes and overall survival rates [3]. The introduction into clinics of the mAb rituximab, in 1997, has seen an annual decline in mortality rates in the United States by almost 3% for the two most common subtypes of NHL, follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) [3]. The activity of these monoclonal antibodies is based on their specificity for the surface antigen CD20, which is expressed in both malignant and normal B cells, but not in haematopoietic stem cells and differentiated plasma cells [4,5], allowing for the replenishment of depleted B cells and only mild adverse effects with no clinically significant increase in infections. Moreover, the addition of rituximab to standard chemotherapeutic agents was shown to be well tolerated, leading to improved overall survival rates in patients with DLBCL, follicular lymphoma (FL) and mantle cell lymphoma (MCL) [6,7], and more recently chronic lymphocytic leukaemia (CLL) [8,9].

Despite the progress made in the treatment of B cell malignancies since the approval of rituximab, most patients relapse and/or develop resistance. Numerous studies have been carried out to determine the mechanisms underlying rituximab-induced cell death resistance and several hypotheses have been proposed [10]; however, the pathways involved in this phenomenon remain unclear.

The low response rate of rituximab as a single agent and relapsed and refractory disease underline the need for new strategies to increase efficacy and overcome resistance. While, on the one hand, novel combination therapies have been designed to achieve a more complete clearance of tumour cells, on the other hand, the advances made in the field of

22 immunotherapy led to the development of a new monoclonal antibody targeting the CD20 antigen, namely GA101 (now called obinutuzumab). A phase III clinical trial analysed the effect of GA101 plus chlorambucil in patients with CLL, showing considerably increased survival rates, leading to the approval of GA101 by FDA in 2013 [11]. GA101 induced extensively higher rates of tumour cell death in both in vitro and in vivo studies [12], ensuring a more complete clearance of malignant B-cells, and recent clinical trials showed a strong activity of GA101 in refractory NHL patients [13]. Nevertheless, disease relapse is likely to continue to represent a critical problem. Malignant B cells circulating in the bloodstream are easily accessible for mAbs and likely to be targeted and killed. However, it is possible that some residual tumour cells are able to circumvent the treatment, thus leading to the possibility of tumour recurrence. One escape from therapy is caused by a cytokine-mediated, gradient-dependent migration of B cells from the bloodstream to the bone marrow (BM) [14,15], where tumour cells are able to infiltrate beneath the stromal layer, grow and proliferate by virtue of interactions with bone marrow stromal cells (BMSCs) [16]. BMSC, through the release of soluble factors in the microenvironment and the activation of a number of signalling cascades upon adhesion to tumour cells, mediate neoplastic B cell survival and resistance to drugs [16-18].

Stromal cells are documented to protect B-cell tumours from cell death by rituximab through different mechanisms, such as the CXCR-4/CXCL-12 and the VLA-4/VCAM-1 signalling pathways [19-21], but nothing is known about the impact of the stroma on GA101-mediated killing. Considering the higher effectiveness at inducing tumour cell death shown for GA101 in the treatment of B-cell malignancies, the idea of further increasing survival rates in patients through the combined use of GA101 and inhibitors of microenvironment/tumour interactions is a potentially attractive possibility. This strategy could ensure not only the clearance of circulating tumour cells, but also the killing of those cells which would otherwise be spared because of protection by the stromal environment.

1.1. B-cell malignancies

B-cell malignancies are neoplastic transformation of B cells of the lymphocyte lineage (also called B lymphocytes). B lymphocytes develop in the bone marrow, which along with the thymus forms the central lymphoid tissue, from lymphoid precursor cells arising from haematopoietic stem cells (HSCs). Maturation of B cells begins with the differentiation of lymphoid precursor cells into progenitor B cells (pro-B cells).

23 Interactions of pro-B cells with the bone marrow microenvironment, and specifically direct contact with stromal cell membrane receptors first and binding to cytokines such as IL-7 afterwards, determine the subsequent differentiation of pro-B cells into precursor B cells (pre-B cells) [22,23]. During the steps that lead to their differentiation, these premature B cells undergo several rounds of divisions which bring about functional changes in their cytology and gene expression [24]. Particularly relevant are the gene rearrangements that occur in the immunoglobulin (Ig) gene, as these determine the fate and features of the developing B cells. The first Ig-gene rearrangements occur at the pro-B cell stage, where the gene loci encoding the heavy chain (described in 1.2) undergoes a D-to-J and V-to-DJ rearrangement [25]. Upon completion of the VDJ recombination in the heavy chain, a pro- B cell develops into a pre-B cell. Pre-B cells then undergo V-to-J rearrangements in the gene loci encoding the light chain (described in 1.2), leading to the formation of a fully competent B-cell receptor (BCR) on the surface of the now called immature B cells [25]. Immature B cells are then released from the bone marrow and migrate into peripheral lymphoid tissues, such as spleen and lymph nodes, where they circulate as naïve B cells until the eventual encounter with an antigen. This event, in turn, leads to the migration of B cells into the germinal centre and to their proliferation and further differentiation into mature B cells, characterised by the expression of the fully functional molecules IgM and IgD on their surfaces [26]. The encounter with an antigen is a fundamental step of the developmental process, as this determines which B cell clone will undergo clonal expansion (proliferation of the B cell clone that reacts with the antigen), affinity maturation (where repeated mutational events in the Ig variable region lead to the selection of clones with increased affinity for the antigen) and class switching (which determines the expression of specific classes of heavy and light chains, described in 1.2) [27]. These events are followed by the differentiation of mature B cells into either memory B cells or antibody-producing plasma cells.

B-cell lymphomas and leukaemias are caused by erroneous mutations in either proto- oncogenes, tumour suppressor genes, or both, that arise during one of the stages of B-cell development. Based on the genetic features mirrored by neoplastic B cells, and specifically on mutations (or lack of) in the Ig variable region, each different B-cell malignancy can be linked to a certain stage of the developmental pathway and consequently classified [28]. In B-CLL, for instance, malignant mature B cells can present either mutated or non-mutated Ig variable regions, revealing that the uncontrolled expansion of clones can occur either before an encounter with the antigen (pre-germinal centre stage) or afterwards (post- germinal centre stage) [29,30]. B-NHL, on the other hand, often present mutated Ig

24 variable regions, indicating that encounter with an antigen has occurred before the clonal expansion of mature B cells. In fact, FL, Burkitt’s lymphoma (BL) and, in some cases, DLBCL all arise at different stages of the germinal centre B cell (extensively reviewed in [31]). Finally, B-ALL arises from non-mature B cells (blast B cells), and specifically from premature B-cells (pre-B-cells) and progenitor B-cells (pro-B-cells) (figure 1.1, [32]). Although originally arisen from different cellular stages, all B-cell malignancies, just as every other cancer type, are characterised by presenting altered patterns of gene expression, up-regulated pro-survival signalling pathways, higher resistance to the apoptotic signals and ability to evade the immune system [28]. These features are currently being studied with the objective of identifying putative novel targets for B-cell malignancies.

Figure 1.1. Stages of B cell development and malignancies arisen from each of them. Haematopoietic stem cells (HSC) that commit to the lymphoid lineage undergo several passages that lead to the development of a mature B cell. Based on the stage of B cell development at clonal expansion, each B-cell malignancy is shown in the upper panel. ALL = acute lymphoblastic leukaemia; NHL = non-Hodgkin lymphoma; CLL = chronic lymphocytic leukaemia; FCL = follicular centre lymphoma; BL = Burkitt’s lymphoma; DLBCL = diffuse large B-cell lymphoma. Figure adapted from [32].

26 A milestone in of the treatment of B-cell malignancies, and in particular B-NHL and B- CLL, was the identification of the CD20 antigen as a therapeutic target on the cell surface of B lymphocytes [4] – expressed from the pre-BI (large) stage until mature B cells (see figure 1.1). CD20 is a transmembrane phosphoprotein which contains three highly hydrophobic regions that span the cell membrane four times [33]. Both the amino and carboxyl termini are located within the cytoplasm, while the extracellular domain forms two loops, one bigger (between the transmembrane segments 3 and 4) and one much smaller (between segments 1 and 2) [34,35]. Through the activation of diverse intracellular signalling pathways upon binding of a ligand, CD20 appears to mediate a number of functions in B cells. For instance, inhibiting CD20 was shown to block RNA synthesis and B cell progression into the cell cycle [36]. Other studies investigated CD20 activity as an ion channel and showed that CD20 regulates Ca2+ conductance through the plasma membrane of B cells, therefore suggesting another role the protein could play [37]. However, despite the growing importance that has been delineated for the CD20 antigen in B lymphocytes, in vivo studies aimed at blocking its activity by generating mice carrying a CD20 gene disruption have not shown developmental problems in B lymphocytes or impaired B-cell functions [38]. Characterisation of the MS4A family of genes, which shares homologies with CD20 in terms of structure and conserved residues, has led to the speculation that either there is some redundancy, and CD20 functions might be covered by different proteins [39], or CD20 may be non-critical for B cell development. More recently, a case study reported by Kuijpers et al. highlighted a new role for CD20. In fact, a patient presenting CD20 deficiency, despite displaying normal B cell development, had impaired ability to produce T-cell independent antibody responses and a lowered amount of IgG molecules after encounter with T-cell independent antigens [40]. The group was able to reproduce the same results in CD20-deficient mice, thus establishing a new role for CD20.

The CD20 antigen has been shown to be expressed in both normal and malignant B cells, but its presence has not been observed in other cell types including plasma cells or haematopoietic stem cells [4,5]. The absence of CD20 in the latter constitutes a crucial finding, because it allows targeting of CD20-positive cells despite the subsequent depletion of normal B lymphocytes, as they would be eventually replaced by new cells arisen from primary stem cells. CD20 is highly expressed on the cell surface and not present as a circulating protein in the serum, thus reducing the amount of antibody required [35]. These observations have deeply influenced, in terms of therapeutic strategies, the treatment of B-

27 cell lymphomas, and prepared the ground for the advent of monoclonal antibodies targeting the CD20 antigen, currently in use in patients with B-cell malignancies.

1.2. Anti-CD20 monoclonal antibodies

Therapeutic monoclonal antibodies (mAbs) are artificially-made antibodies that target specific antigens of interest. Just like B cell-produced antibodies, mAbs consist of two long heavy chains and two short light chains that are bound to each other through disulphide bonds. The amino-terminal regions of each chain are highly variable regions (VH, VL) and are responsible for the binding to specific epitopes on the antigen, while the remaining domains, called constant regions (CH, CL), tend to be similar in each different antibody

[41]. Based on the different heavy chain constant regions (CH) expressed, both naturally- and artificially-made antibodies can be classified in 5 main subtypes, called isotypes: IgG (expressing the γ heavy chain); IgM (µ heavy chain); IgD (δ heavy chain); IgA (α heavy chain); IgE (ε heavy chain). Each isotype then pairs with a light chain, which can be of either κ or λ type, to form the final antibody molecule [42]. The different isotypes, together with exhibiting structural diversities, also display different biological features: while antibodies of the IgG class are able to activate the classical pathway of the complement system and initiate phagocytosis by binding to receptors on phagocytic cells, for instance, IgM and IgD are the only isotypes which are present on the surface of mature B cells as membrane-bound antibody molecules. IgM antibodies can also be secreted as soluble molecules, and are found in the serum as pentamers which can strongly activate the complement system. IgA molecules play their major roles in the mucous membranes, representing a first-line defence from invading pathogens, while antibodies of the IgE class are mainly involved in the onset of hypersensitivity reactions [42].

When digested by the protease papain, antibodies are fragmented into three portions, of which two identical portions are called Fab (fragment antigen-binding) regions and one is called Fc (fragment crystallisable) region (figure 1.2). The Fab regions include the highly variable domains VH and VL which bind the antigen; on the other hand, the Fc region is responsible for binding to specific receptors on immune cells, called Fc receptors (FcRs). Such receptors are membrane glycoproteins which, upon interacting with their Fc regions, mediate some of the main functions of antibody molecules: for instance, the binding of the Fc region to FcRs is responsible for transporting antibodies across membranes, as well as triggering the antibody’s effector functions through the recruitment of immune effector

28 cells of both innate and adaptive immunity [43]. The Fc receptors exist in several forms, which have different structures and different affinities for each antibody class. For example, antibodies of the IgG class bind to specific Fc receptors called Fcγ receptors (FcγRs), and each of them can have either activating or inhibiting roles (recently reviewed in [44]), thus mediating diverse biological functions. Therefore, through this mechanism antibodies are able to regulate immune responses.

Figure 1.2. General structure of antibody molecules. An antibody molecule of the immunoglobulin G (IgG) family is constituted of two heavy chains (blue) and two light chains (orange). Disulphide bonds (-SS-) bind each heavy chain to a light chain and each heavy-light pair to the other pair. The region where the two pairs connect to each other is known as a hinge region. Each chain consists of variable (VH, VL) and constant

(CH, CL) regions. Digestion by papain causes the mAb molecule to fragment into two identical Fab regions and one Fc region, called in the figure Fab1, Fab2 and Fc.

30 Rituximab was the first mAb targeting the CD20 antigen to be approved by the US FDA for the treatment of cancer in 1997. Since that time, important progress has been made in the development of numerous different anti-CD20 mAbs. Based on preclinical investigations into the mechanisms of action they employ, anti-CD20 mAbs can be subdivided into two categories, namely type I and type II anti-CD20 mAbs.

Type I anti-CD20 mAbs are characterised by their ability to induce complement-dependent cytotoxicity (CDC) through the classical pathway of the complement system. On the other hand, type II mAbs induce very low degrees of CDC; however, high levels of B cell death are still achieved by virtue of their greater capacity to induce programmed cell death (PCD) through the activation of intracellular signalling pathways upon mAb binding [12]. Both type I and type II, in addition, are able to induce antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by recruiting effector cells of the immune system through their Fc regions [45].

Structural differences between the several anti-CD20 mAbs developed also contribute to their cytotoxicity. First-generation anti-CD20 mAbs are characterised by their chimeric nature: rituximab, for instance, presents murine light- and heavy-chain variable regions and human light- and heavy-chain constant regions; second-generation mAbs, which include ofatumumab, veltuzumab and ocrelizumab, are humanised or fully-human Abs and have reduced immunogenicity, but induce prolonged B-cell depletion in vivo compared to first generation mAbs [46-48]. The latest years have seen the development of a third generation of anti-CD20 mAbs, the most representative of which is GA101 (now called obinutuzumab). These latest antibodies are also humanised, but also present an engineered Fc region developed in order to increase the binding to Fc receptors on immune effector cells, therefore enhancing ADCC and ADCP [49-51]. GA101 has also been shown to potently induce PCD, leading to a greater overall efficacy in B-NHL cell lines and in in vivo models of B-cell lymphoma [52]. Intriguingly, in vivo studies have also shown that anti-CD20 mAbs are able to trigger the induction of long-term anti-tumour responses in treated mice, leading to extended survival and response to tumour re-challenge [53,54]. The immune responses that are at the basis of such phenomenon are currently being investigated; however, the possibility of generating a vaccine-type effect after mAb therapy [55] constitutes a promising perspective which could lead to important improvements in B-NHL treatment.

31 1.3. Mechanisms of action of anti-CD20 mAbs

The last two decades have seen the introduction into the clinic of several anti-CD20 mAbs, which are now employed as frontline therapies in patients with B-NHL. Recent insights into their mechanisms of action have identified four major modalities by which mAbs induce cell lysis: complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody dependent cellular phagocytosis and programmed cell death (figure 1.3). Each mAb can strongly induce one -or more- of these pathways, depending on the mAb type, on B-cell surface markers, but also on the genetic characteristics of the patient [56]. The way all these factors influence mAb-mediated mechanisms of action is described in the next sections.

Figure 1.3. Mechanisms of action exerted by anti-CD20 mAbs. Anti-CD20 mAbs induce killing of target B cells via four main mechanisms upon mAb binding. CD20 molecules can cluster into lipid rafts, which lead to recruitment of C1q complement and triggering of CDC; mAb ligation through Fc regions activates immune effector cells that present FcγRs (i.e. neutrophils, NK cells), ultimately leading to ADCC; Fc regions can activate macrophages and trigger phagocytosis (ADCP); finally, mAb binding can cause PCD via homotypic adhesion (HA) and actin reorganisation. This step is followed by lysosomal membrane permeabilisation (LMP) and subsequent release of cathepsins. These events lead to the generation of ROS, ultimately mediating tumour cell death. CDC: complement-dependent cytotoxicity; ADCC: antibody- dependent cellular cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; PCD: programmed cell death.

33 1.3.1. Complement-dependent cytotoxicity (CDC)

The potential role of CDC is controversial, with some considering this mechanism one of the principal modes of action in type-I mAb-mediated B-cell death [57]. Indeed the type-I mAb ofatumumab was specifically designed to increase CDC in an effort to improve anti- CD20 efficacy further [46]. Upon mAb binding, CD20 molecules are reorganised into detergent-insoluble lipid micro-domains, and this specific configuration is able to augment recruitment of C1q complement, leading to the activation of the classical pathway of the complement system and the subsequent killing of target cells [58]. Such an arrangement is typically observed in type-I mAbs, but it is not induced after the binding of type-II mAbs. Indeed, the lack of CD20 clustering into lipid rafts is thought to account for the inferior induction of CDC by type-II mAbs [58,59]. One reason that might explain the strong activation of CDC after treatment with mAbs is the proximity of the antibody to the CD20 molecule. The limited extension of the large and the small extracellular loops of CD20, which contain the binding epitopes recognised by the mAbs, constricts the latter close to the B-cell surface. The proximity to the surface might play a role in inducing a strong activation of the complement cascade [60].

The specific conformations acquired by CD20 molecules after mAb binding are also due to diversities in the CD20:mAb binding ratio in type-I and type-II mAbs with a 2:1 binding stoichiometry for type I mAbs [61]. It has been hypothesised that such different binding capacity is due to the possibility that type-I mAbs bind between tetramers of CD20 molecules (inter-tetramer) such that each mAb can therefore bind to two CD20 tetramers. Conversely, type-II mAb ligation might occur within a CD20 tetramer (intra-tetramer), resulting in only one antibody bound per tetramer [62,63] (figure 1.4). This peculiar geometry could influence CD20 conformations upon antibody ligation, with the bivalent binding capacity of rituximab leading to engagement of numerous CD20 molecules and formation of the lipid micro-domains [62]. Type-II mAbs, on the contrary, would not exhibit such characteristics, thus displaying a decreased ability to induce CDC.

Figure 1.4. Hypothesised CD20 binding model of type-I (A) and type-II (B) mAbs. Type-I mAb binding (A) is inter-tetrameric with a binding ratio of 2:1 (2 mAb per 1 CD20 tetramer). Type-II binding (B) is intra- tetrameric and this leads to a halved binding capacity (binding ratio 1:1) compared to type-I mAbs. Figure adapted from [64].

35 The relative importance of CDC to type I anti-CD20 mAb activity remains undetermined and controversial. CDC induces heterogeneous degrees of B-cell lysis based on the B-cell line considered, ranging from a high susceptibility to complement after addition of low concentrations of serum, to a complete resistance despite the presence of high serum concentrations [65]. These differences were attributable to the expression of specific molecules on the cell surface of B-cell tumour cell lines, and specifically the complement defence molecules CD55 and, to a lesser extent, CD59; their blockade through the addition of antibodies, in fact, triggered higher levels of cell death in those cell lines which were less sensitive to the mAb [65]. Although many questions remain unanswered, a greater understanding could ultimately be exploited to develop novel therapeutic strategies aimed at increasing CDC potency and potentially treatment efficacy, as this was not associated with increased toxicity.

1.3.2. Antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP)

ADCC is dependent on the ability of mAbs to interact, through their Fc regions, with the Fc receptors (FcγRs) located on the cell surface of immune effector cells. Effector cells, such as macrophages, natural killer (NK) cells, neutrophils, dendritic cells (DCs) and mast cells, express a wide range of FcγRs that can either activate or suppress their immune activity. The balance between activating (FcγRI, FcγRIIA, FcγRIIIA) and inhibitory (FcγRIIB) receptors determines the initiation of a signalling cascade of events [45,66]. These lead to the release of cytokines, such as IFN-γ, which recruit other immune cells and thus initiate an inflammatory immune response, and cytotoxic granules, such as granzymes and perforins, which directly cause disruption of the cell’s membrane, ultimately inducing death of target cells [67,68]. Based on the different IgG subclasses, each mAb might possess a higher or lower binding affinity for each receptor, and this could influence the final outcome of mAb-CD20 interaction [69]. Both type-I and type-II anti-CD20 mAbs have been shown to induce high levels of ADCC [12,57]; however, the cytolytic effect could be either boosted or dampened depending on the FcγR expression level on immune cells. For instance, deletion or blockade of the inhibitory receptor FcγRIIB has been shown to increase killing of target cells in in vitro models [70,71]. Enhancement of ADCC efficacy in anti-CD20 mAb therapy can also be achieved through manipulation of the mAb Fc portion, by adding or deleting specific chemical groups: for instance, the removal of a fucose residue from the mAb Fc region increased the binding affinity of the therapeutic mAb obinutuzumab to FcγRIIIA, therefore making it able to induce higher Fc-mediated

36 effector functions such as ADCC [72]. Other studies showed that sialic acid content is inversely correlated with the ability of the mAbs to interact with activating FcγRs and generate an immune response, revealing an anti-inflammatory role for Fc sialylation [73].

Another important mechanism of action which is dependent on the interactions between anti-CD20 mAbs and the Fc receptors on immune effector cells is antibody-dependent cellular phagocytosis (ADCP). ADCP is initiated upon the binding of Fc regions on mAbs to their cognate receptors on phagocytic cells, such as macrophages or neutrophils. This binding, in turn, leads to the engulfment and phagocytosis of the target cell by phagocytic cells. Such a mechanism has been shown to be highly effective, with high percentages of B-cell killing induced in the presence of extremely low concentrations of effector cells. In fact, in an in vitro experiment, at a 1:1 ratio of tumour to immune cell (ex vivo activated macrophages) the death of 80% of B-CLL cells was observed [74]. More recent studies have suggested that ADCP might be one of the most effective mechanisms of action in depleting B cells in in vivo models [75]. Because ADCP, just as ADCC, is dependent on the binding of the antibody Fc region to the immune cell Fc receptors, the increased affinity between those could lead to an enhanced therapeutic efficacy [51,76].

Although the importance of ADCC in anti-CD20 mAb therapy is well established, it is still unclear as to the importance of the contributions of specific immune effector cell populations to such mechanisms. As mentioned above, a number of studies have highlighted the fundamental role played by macrophages in the in vivo depletion of B cells [75,77]. On the other hand, in vitro analyses carried out by several groups revealed the involvement of NK cell-mediated ADCC, because dysfunctions in NK cells caused decreased levels of cell death [78,79]. Lately, a study investigated the role of different immune cell subsets in mAb-induced survival in lymphoma-bearing mice. This highlighted the importance of NK cells and NK cell-mediated release of interferon (IFN)-γ in modulating the balance of type-1 T helper (Th1)/regulatory T (Treg) cells and thus ultimately in enhancing anti-CD20 mAb therapy [80].

These studies suggest that different modes of action are likely to contribute to in vivo and in vitro Fc-dependent effector mechanisms; additional insights could be useful to understand this pathway and increase its efficacy in mAb therapy.

37 1.3.3. Programmed cell death (PCD)

PCD takes place upon direct contact of the mAb with target cells through the induction of intracellular signalling pathways, ultimately causing cell cycle arrest and death. Whether this mechanism induces cell death via activation of the apoptotic pathway has been largely discussed, leading to controversial results and discordant opinions [81]. On the one hand some groups reported the involvement of caspase cleavage in mAb-induced B cell death, supporting the hypothesis of the execution of an apoptotic mechanism [82,83]. However, on the other hand, subsequent studies showed that initiation of mAb-induced PCD does not involve the activation and cleavage of caspases, is not dependent on mitochondria and does not lead to DNA fragmentation, therefore underlining the lack of the typical apoptotic features [61]. These controversial results, however, could be explained when considering that distinct research groups have analysed different mAbs in different conditions (for instance, free mAb vs cross-linked mAb) and these might act through diverse mechanisms and pathways. In support of this hypothesis, several studies have focused their attention on type-II mAbs and have shown their ability to evoke a non-apoptotic, non-autophagic PCD that, differently from type-I mAb- and rituximab-induced PCD, involved cell-cell homotypic adhesion and swelling of lysosomes [84]. The important role of such a non- apoptotic PCD has also been highlighted in in vivo conditions, where type-II mAbs were able to induce a strong Fc-independent death of tumour cells, while type-I mAbs’ efficacy in vivo was mainly due to the induction of Fc-dependent mechanisms of action [59].

Recently, new important insights on type-II-mediated PCD, have shed light on the signalling events that lead to B-cell death. Consistent with the finding that type-I mAbs do not evoke great degrees of PCD, type-I-induced rearrangement of CD20 molecules into lipid rafts does not appear to be relevant in PCD [61]. Instead, the induction of high levels of direct cell death with type II mAbs was correlated with large amounts of homotypic cellular adhesion [61]. Such cellular aggregation followed alteration of the cytoskeletal structure and reorganisation of actin, which redistributed to the sites of cell-cell contact [84]. Redistribution of actin filaments was followed by enlargement of lysosomes and the subsequent release of lysosomal content into the cytoplasm (lysosomal membrane permeabilisation, LMP) [84], an early event of the lysosome-triggered non-apoptotic pathway [85]. More recent studies, performed by using the novel type-II mAb GA101 (obinutuzumab), have clarified the mechanisms by which PCD is exerted and underlined the important role that such a mechanism plays in type-II-mediated cell death: mAb ligation causes homotypic cellular adhesion, which is followed by reorganization of actin

38 filaments; this triggers LMP and subsequent release of lysosomal content, such as cathepsins, that ultimately mediate the generation of reactive oxygen species (ROS) through the NAPDH oxidase, leading to non-apoptotic direct cell death [86,87]. The deeper understanding achieved on PCD might lead to the identification of modalities to increase the mAb potency through the augmentation of direct cell death, thus obtaining a higher therapeutic efficacy.

1.4. Rituximab and tumour resistance to treatment

Rituximab has been the first anti-CD20 monoclonal antibody approved by the FDA for the treatment of relapsed, refractory and follicular B-cell lymphoma, and the last decades have seen its use increasing substantially in B-NHL cases, in combination with chemotherapy and as maintenance treatment after initial induction chemotherapy.

The mAb rituximab was developed in 1994 from the murine monoclonal antibody 2B8, which specifically binds to CD20. The variable regions of murine 2B8 were cloned and transferred into a cDNA expression vector containing four separate human genes, encoding the IgG1 heavy chain and the κ-light chain constant regions [88]. Early experiments in macaque cynomolgus monkeys showed a depletion of nearly 50% of peripheral blood (PB) B cells at the lowest concentrations with a complete recovery of B-cells achieved within six months of the end of treatment, while symptoms of toxicity or adverse effects were not observed [88].

While the efficacy of rituximab has been observed in several lymphoma subtypes, in approximately 50% of cases of certain B-cell malignancies (i.e. relapsed follicular lymphoma, small lymphocytic leukaemia) there is no detectable response to the therapy [89]; in addition to this primary unresponsiveness, numerous patients develop relapses after treatment, and only 40% of those respond to a further treatment with rituximab [90]. The observation of such a decrease in response rates after therapy suggested the existence of mechanisms of resistance that may hamper mAb efficacy, with much research focused on the CD20 molecule. CD20 is expressed in all B-cell malignancies; however, expression levels are not constant between malignancy subtypes, so while FL cells express a high percentage of CD20 on their surfaces, CLL or small lymphocytic lymphoma (SLL) only display a low expression, and this difference seems to correlate with diverse therapeutic outcomes [91]. Nevertheless, such correlation has not been observed in DLBCL or MCL, which present high CD20 expression but do not exhibit a better response to treatment

39 [92,93], therefore pointing out the existence of additional and distinct mechanisms. Cases of CD20-negative relapses, developed after rituximab therapy, have also been observed [94,95] and loss of CD20 might be one of the ways tumour cells could escape the treatment. This phenomenon has been reported by several studies, and some groups have explained it by postulating the existence of a “shaving reaction”: monocytes, in fact, seemed to be able to remove the rituximab-CD20 complexes from the B-cell surface upon mAb binding [96], thus generating CD20-negative B lymphocytes which would therefore not be targeted by anti-CD20 therapeutic antibodies. The loss of CD20 could also be explained by a different phenomenon, namely trogocytosis. Trogocytosis is a FcγR- mediated event whereby immune cells, upon binding to the antibody/antigen complex, take the latter up by incorporating fragments of the target cell’s membrane, therefore generating CD20-lacking tumour cells [97]. Such a mechanism has been described to be mediated by both activatory and inhibitory receptors [98].

Modulation of the CD20 antigen might be another mechanism by which B-NHL cell lines acquire resistance to rituximab treatment. A study recently performed observed that after type-I mAb treatment, but not type-II, CD20 levels on the cell surface lower significantly. This phenomenon was observed in mice lacking activatory FcγRs; therefore, Fc-FcγR interactions and mobilisation of effector cells were not involved in CD20 modulation. The observed decrease in CD20 levels, thus, is not dependent on a macrophage-mediated shaving reaction or trogocytosis, but is likely due to a rituximab-mediated internalisation of the antigen, which traffics to early endosomes and is degraded in lysosomes [99]. Such down-modulation was remarkably strong in CLL, followed by MCL and FL, while DLBCL exhibited only little internalisation [99]. These diversities, dependent on tumour subtypes, could explain the conflicting results obtained by different groups, further supporting the existence of this phenomenon.

Whilst these mechanisms could interfere with rituximab binding to CD20 antigen on B cells, there might be other ways for malignant cells to achieve resistance to therapy, which involve the events following mAb binding. Rituximab induces B-cell killing mainly through CDC and ADCC. The potency of ADCC could be dependent on individual genetic characteristics. FcγRs on immune effector cells, in fact, are not genetically identical between different patients, because of the presence of single nucleotide polymorphisms (SNPs) in the genes encoding those receptors. These SNPs have been found to have an impact on FcγR functions, and specifically on mAb binding through Fc regions: for instance, a nucleotide substitution in the FcγRIIIA-encoding gene leads to the existence of

40 two different forms of this receptor, displaying either a valine or a phenylalanine at residue 176 [100]. Such a SNP could interfere with mAb therapeutic efficacy, with patients exhibiting a valine shown to achieve better treatment outcomes; the replacement of valine with a phenylalanine, on the other hand, determined a worse prognosis, due to the weaker binding of mAb Fc regions to FcγRIIIA expressed on monocytes and NK cells and, thus, weaker induction of ADCC [56]. A following study, carried out in patients with FL, together with confirming this finding also associated polymorphisms in the FcγRIIA, and specifically the histidine/arginine SNP at position 131, with better therapeutic outcomes upon rituximab treatment [101]. Controversial results, however, were observed in different studies: in patients with B-CLL, polymorphisms in neither FcγRIIIA nor FcγRIIA appeared to correlate with treatment outcome, with rituximab plus chemotherapy improving outcomes over chemotherapy alone in all the analysed arms (i.e. homozygous vs heterozygous vs intermediate genotype) [102]. The same observations were made in following analyses, performed in patients with FL [103,104]. These genetic dissimilarities, nonetheless, at least in some patients could be relevant in terms of different responsiveness to mAbs and could partly account for the cases of resistance observed during treatment.

1.5. Obinutuzumab (GA101)

Obinutuzumab (initially labelled GA101 in development) is a humanised, third-generation, type-II IgG1 mAb with impaired CDC but increased PCD compared to other type-I mAbs, and increased ADCC compared to both type-I and type-II mAbs. Such increased cytotoxic activity is due to two main characteristics: the glycoengineered Fc regions (specifically the removal of a fucose residue) which displays an enhanced binding affinity to FcγRIIIA, thus leading to increased ADCC and ADCP, and the modification of an elbow-hinge sequence within the variable regions, which result in a stronger induction of PCD [49] compared to rituximab. Following greater anti-tumour activity in xenograft models [12,105,106], obinutuzumab was tested in a number of clinical trials and was ultimately approved by the FDA in 2013 for use in B-CLL [11].

The reason for such a stronger and intrinsically different execution of B-cell death lies in the structural characteristics of obinutuzumab. Its molecular structure and epitope binding was investigated by site-directed mutagenesis and crystallization, demonstrating that, although obinutuzumab binds a similar epitope as rituximab on the CD20 molecule, different residues are involved in the binding (172-178 for obinutuzumab vs. 168-175 for

41 rituximab), regulating the distinct affinity of each mAb (figure 1.5) [63]. The recognition of slightly different amino-acids is probably due to structural properties, since obinutuzumab has a 30° wider elbow-hinge angle than rituximab [63]: such characteristic causes a slightly different orientation of the two mAbs, therefore determining the involvement of different residues in the binding event. This specific epitope recognition and the subsequent mAb orientation, in turn, facilitate the formation of distinct 3D rearrangements of the bound CD20 molecules – and this has indeed been observed, as rituximab ligation provoked the engagement of CD20 into lipid rafts, while obinutuzumab did not: on the contrary, it mainly induced homotypic cellular adhesion.

Figure 1.5. The CD20 molecule and mAbs binding epitopes. CD20 is a transmembrane protein that spans the cell membrane four times. Both the N- and C-termini are located within the cytoplasm, whereas two loops, one smaller (residues 74 to 80) and one much bigger (145 to 185), are exposed to the extracellular space. The overlapping, but distinct, binding epitopes for rituximab (168-175) and obinutuzumab (172-178) are shown in red and yellow, respectively. Figure adapted from [107].

43 The greater efficacy of obinutuzumab in inducing ADCC, ADCP and PCD compared to rituximab has been shown in numerous studies in vitro. For instance, obinutuzumab was found to induce stronger direct cell death than rituximab in CLL cell lines, as well as higher ADCC in whole blood samples from CLL patients [105]; obinutuzumab-mediated cell death also appeared to be less dependent on the presence of complement [105]. In a more recent study, the ability of obinutuzumab to induce ADCC has been compared to both ofatumumab (a humanised type-I mAb, [108]) and rituximab in CLL primary cell lines isolated from patients [109]. Obinutuzumab, by virtue of its afucosylated Fc region, was able to bind with increased affinity to the FcγRIIIA receptor, which is highly expressed on NK cells. Such enhanced binding led to a better recruitment and activation of NK cells and to the subsequent higher ability of obinutuzumab to induce NK cell-mediated ADCC [109].

In addition to this, other studies have compared the ability of both wild-type and glycoengineered rituximab and obinutuzumab to activate immune effector cells in CLL whole blood samples, showing that the glycoengineered versions of both the two mAbs are able to bind to the receptor FcγRIIIB with much greater affinity than the wild-type mAbs [110]. Because FcγRIIIB is highly expressed on the surface of polymorphonuclear neutrophils (PMNs), such higher affinity of glycoengineered mAbs ultimately leads to mobilisation of PMNs. PMNs, in turn, undergo degranulation, followed by release of inflammatory molecules, such as tumour-necrosis factor α (TNF-α) and interleukins, leading to strong induction of ADCP [110]. Thus, the glycoengineering of obinutuzumab plays a fundamental role in enhancing its phagocytic abilities ultimately leading to higher therapeutic efficacy when compared with rituximab and other non-glycoengineered mAbs.

Recently, the effects of complement activation on rituximab- and obinutuzumab-mediated ADCC was evaluated in Burkitt’s lymphoma cell lines, as the presence of the C3b complement had been previously shown to interfere with mobilisation of immune effector cells and specifically NK cells [111]. Whilst the addition of complement blocked the interaction of rituximab with NK cells, impairing ADCC-mediated cell death, there was no effect on obinutuzumab-mediated activation of NK cells and ADCC [112]. This revealed that the inability of obinutuzumab to fix complement and induce CDC makes the antibody more efficient at inducing ADCC through interaction with NK cells in the presence of complement, and such characteristics might contribute to the overall higher efficacy of obinutuzumab observed in numerous studies. Moreover, recently one study has analysed the activity of obinutuzumab, rituximab and ofatumumab in killing B-lymphoma cells in

44 vitro [113], taking into account the possible internalisation of the CD20 antigen after mAb binding. Interesting, obinutuzumab induced inferior internalisation, while both rituximab and ofatumumab ligation led to reduced levels of CD20 on B-cell surface [113]. Such findings suggest that the use of obinutuzumab in clinical settings could probably decrease the development of resistance to treatments, due to the longer persistence of the antigen on cell surface.

Finally, a recent study has highlighted the important role that execution of PCD by GA101 plays in in vivo settings: in fact, by introducing a specific mutation in their Fc regions, Herter et al. generated Fc region-inactive variants of both GA101 and rituximab, which were called GA101-P329GLALA and rituximab-P329GLALA respectively. Because of their mutated Fc regions, both these forms of mAbs were unable to mediate Fc-dependent recruitment of immune cells and of components of complement system, but could only provoke cell death by induction of PCD [114]. The killing efficacy of GA101- P329GLALA was compared to that of glycoengineered GA101, wild-type GA101, wild- type rituximab and rituximab-P329GLALA in whole blood B cell depletion assays and in xenograft models of DLBCL. Interestingly, while rituximab-P329GLALA was not able to induce measurable degrees of cell death, GA101-P329GLALA had an efficacy which was comparable to that of wild-type rituximab. This study further supports the important role played by GA101-induced PCD in mediating B cell clearance, and also highlights the dependency of rituximab on Fc region-mediated functions [114].

Overall, these studies highlight the greater efficacy of obinutuzumab in depleting B cells compared to other anti-CD20 mAbs.

1.6. The tumour microenvironment in B-cell lymphomas

Despite the improved response rates and outcome with the addition of rituximab for B-cell malignancies, many patients still relapse or become refractory to anti-CD20 therapy. Whilst the mechanisms of resistance remain still poorly understood, a potentially important factor in malignant B lymphocyte survival is the tumour microenvironment (TME). TME is now considered as an integral part of neoplastic malignancies, and its components actively take part in the establishment and maintenance of all the hallmarks of cancers [115]. All the cellular components of the TME, such as mesenchymal stromal cells, lymphoma-associated macrophages (LAM), monocyte-derived nurse-like cells (NLC),

45 endothelial stromal cells, NK cells and T cells, are able to interact with malignant B cells, promoting their growth and suppressing the immune response [116].

The important role played by the stromal microenvironment has been described in CLL, where cells that normally would undergo spontaneous apoptosis in vitro showed prolonged survival in the presence of BMSCs [117,118]. Such a protective role exerted by the TME was further established with the observation that BMSCs are also able to promote B- NHL/B-CLL survival after treatment of B lymphocytes with cytotoxic drugs [18,119]. The adhesion of tumour cells to the stromal cells and extracellular matrix (ECM) components, such as fibronectin, initiates a cascade of signalling pathways through the integrin transmembrane receptors that leads to up-regulation of anti-apoptotic proteins and ultimately determines B-cell survival to drugs (cell adhesion-mediated drug resistance, CAM-DR) [16,120] (figure 1.6).

Another essential characteristic of B-cell malignancies is their ability to exit the circulation and migrate to distant organs and the TME plays a fundamental role in enhancing this metastatic potency. Fibroblast-like stromal components play an important role in stimulating B-cell migration to the bone marrow: once activated by malignant cells, stromal cells are able to release a number of soluble factors which induce further tumour growth. Of those soluble factors, chemotactic cytokines (chemokines) have an essential role in tumour migration and metastasis, which they exert through their ability to generate chemotactic gradients that attract their cognate receptors of target cells [121]. Malignant B lymphocytes normally circulate in the bloodstream and are therefore easily targetable with therapeutic agents. However, once in the bone marrow, B cells make contact with and migrate underneath the layer of stromal cells in a process called pseudoemperipolesis [15], thus accessing a growth-supportive environment where they are protected from drugs. Such a protection can be due to the physical barriers created by the presence of stromal cells, but also to specific signalling pathways which are activated within the microenvironment [119,122].

The various signalling cascades mediated by interaction with the TME components, which ultimately protect malignant B cells from therapeutic agents, are detailed in the following sections.

Figure 1.6. B lymphocytes interact with the TME which provides them with pro-survival and anti- apoptotic signals. The TME, composed of stromal cells, supportive matrices and soluble factors (right), is able to establish interactions with malignant B cells, mediated by cell surface receptors and adhesion molecules. This in turn leads to the activation of downstream signalling pathways, transduction of signals to the nucleus and subsequent alteration of gene expression and up-regulation of pro-survival and anti-apoptotic intracellular responses.

47 1.6.1. Contact-dependent interactions of malignant B cells with stromal-like cells

In different models, different mechanisms of stroma-mediated protection were observed. In order to develop a reliable system for the study of tumour-stromal interactions, Kurtova et al. assessed the ability of several human stromal cell lines, murine stromal cell lines and human primary mesenchymal stromal cells to protect B-CLL cells from spontaneous and drug-induced apoptosis [123]. In this study, all the stromal lines tested were able to protect B-CLL cells at any tumour to stroma ratios used, despite a greater ability of murine cells to increase survival compared to human ones. Interestingly, such protection seemed to be entirely dependent on direct contact, as the introduction in the wells of micropore inserts that separated tumour and stromal cells, allowing the passage of media and soluble factors, completely abrogated survival advantage [123].

In order to unravel the mechanisms of contact-dependent protection mediated by stromal cells, Tabe et al. co-cultured leukemic cell lines with BMSCs and showed suppression of drug-induced apoptosis through the ILK-mediated activation of the PI3K/Akt pathway. This in turn led to the inhibitory phosphorylation of GSK-3β, phosphorylation of ERK1/2 and translocation to the nucleus of β-catenin and STAT3, leading to regulation of gene expression and enhanced survival [124]. Disruption of this pathway through inhibition of either ILK or PI3K signalling was found to sensitise cells to chemotherapy, abrogating stromal-mediated protection of tumour cells [124].

Another interesting insight into the mechanisms of stroma-mediated protection was provided by Lwin et al, who showed that adhesion between DLBCL cell lines and the human bone marrow stromal line HS-5 led to the activation of the NFκB signalling axis [16]. Stroma-induced proteolytic cleavage of the p100-NFκB2 molecule, in fact, provoked p52 transcription factor accumulation and translocation to the nucleus, ultimately leading to the up-regulation of the inhibitor of apoptosis (IAP) family of proteins, and therefore to drug resistance and increased survival. Such protection was shown to be inhibited after blockade of the NFκB pathway in the presence of stromal cells [16]. Interestingly, activation of NFκB signalling axis could also be observed, even if only at a lower degree, in the presence of micropore inserts that separated stromal cells from tumour cells, suggesting that such mechanism of protection could be exerted in both direct contact and (even though partly) non-contact conditions [16].

48 1.6.2. Soluble factor-mediated initiation of pro-survival signalling pathways

Soluble factors released by stromal cells are active components of the TME. As previously mentioned, chemokines such as CXCL-12 are constitutively released by stromal (or stromal-like) cells and mediate not only migration of malignant B cells to the bone marrow, but also initiation of pro-survival signalling pathways: the inhibition of CXCL-12 through the use of a specific inhibitor, in fact, could abrogate the stroma-mediated protective effect and sensitise CLL cells to cytotoxic drugs [125]. Stromal cells are able to produce and release in the microenvironment a huge amount of factors other than chemokines, one of which is vascular endothelial growth factor (VEGF). Stroma-derived VEGF has been shown to mediate protection from spontaneous apoptosis of B-CLL cells, and blocking of secreted VEGF through the use of a monoclonal antibody abrogated protection [126]. In a recent study, Kay et al. cultured CLL cells with marrow stromal elements (MSE) and showed that both direct contact and soluble factors were responsible for the augmented survival of CLL after treatment [18]. Such protective effect was due to increased expression of the anti-apoptotic proteins XIAP, Mcl-1, Bcl-2 and survivin, but also to the up-regulation of the pro-angiogenic protein basic fibroblast growth factor (bFGF) and concomitant down-regulation of thrombospondin (TSP)-1 – thus mediating an “angiogenic switch” towards angiogenesis that could further boost tumour growth [18].

1.6.3. Reciprocal interactions between TME and tumour B cells

Recently, several reports have described how not only stroma influence on tumour cells, but also tumour influence on stromal cells can affect TME-mediated protection from therapeutic agents [127]. In fact, normal fibroblasts can be activated by tumour cells, leading to their transformation into cancer-associated fibroblasts (CAFs); such phenotypically altered stroma, in turn, is able to interact with tumour cells and modify the latter’s behaviour, increasing tumour cell proliferation, drug resistance and survival [128].

In a study performed by Ding et al, B cells from patients with B-CLL were co-cultured with mesenchymal stromal cells (MSCs), and the co-culture led to the up-regulation of Mcl-1 and XIAP anti-apoptotic molecules, which in turn mediated protection of CLL cells from both spontaneous and drug-induced apoptosis [129]. Interestingly, the group also looked at the changes in Akt and ERK phosphorylation status in stromal cells upon interaction with CLL cells, showing that culture of stromal and tumour cells either in direct contact or in transwell plates (i.e. using a micropore insert that separates the two cell types) strongly activated Akt and ERK. Whether the activation of these molecules could be

49 responsible for the ability of stromal cells to signal back to tumour cells and mediate the protective effect observed, however, remained an unanswered question. In a following study, the same group analysed the ability of CLL-conditioned media (CM) to activate MSCs and found that CLL-CM contained elevated levels of both VEGF and platelet- derived growth factor (PDGF) [130]. These factors were responsible for the activation of PDGF receptors in MSCs, which in turn mediated Akt phosphorylation and MSC proliferation [130]. Therefore, bidirectional interactions between stromal and tumour cells might cause resistance to cytotoxic drugs in patients with B-CLL.

A similar behaviour was observed in primary MCL cells: in fact, Medina et al. showed that viability and survival of primary MCL cells could be increased by culture with both BMSCs and the murine stromal line MS-5, and this was brought about by increased production of B-cell activating factor (BAFF) by stromal cells which in turn mediated activation of the anti-apoptotic NF-κB pathway in MCL cells [119]. Interestingly, co- culture of stromal cells with MCL cells resulted in a much stronger increase in BAFF production, compared to stromal cells cultured alone [119]. This suggested that, in order to mediate a strong protective effect, interactions between stromal and tumour cells were needed.

More recently, an additional report has further supported the importance of reciprocal interactions between tumour and stromal cells: in fact, Lutzny et al. showed that interactions between CLL B cells and primary human or murine stromal cells mediated the contact-dependent up-regulation of protein kinase C (PKC)-βII in the latter [131]. PKC- βII, in turn, led to activation of NF-κB signalling axis in stromal cells. Activation of NF- κB was then responsible for stromal production and secretion of various cytokines, such as IL-1 and IL-15, which ultimately induced soluble factor-dependent protection of CLL cells from cytotoxic drugs [131]. This study, thus, highlighted that tumour-mediated activation of stromal cells is required for TME-mediated drug resistance to occur. Moreover, both direct contact (for activation of stromal cells) and soluble factors (for the subsequent protection of tumour cells) were needed in order to achieve tumour cell survival.

Therefore, when investigating the crosstalk between TME and malignant B cells, it is important to take into account the bi-directionality of such interactions and the influence that each singular component could have on the more complex TME/tumour system.

50 1.6.4. CXCR-4/CXCL-12/CXCR-7 signalling axis

Stromal cells constitutively release the chemokine CXCL-12 (also called stromal derived factor [SDF]-1α) in the microenvironment [132]. Malignant B cells, on the other hand, generally express high levels of the chemokine’s cognate receptor CXCR-4 on their surface [133]. CXCR-4 or CXCL-12 gene knock-out experiments in mice have shown the importance of this signalling axis in embryonic development, since those mice exhibited lethal defects due to impaired haematopoiesis [134]. Studies on CXCR-4 expression have revealed it to be up-regulated in the majority of tumour subtypes, highlighting a strong correlation between over-expression and tumour invasiveness with subsequent metastasis ultimately provoking the onset of secondary tumours [135]. In the presence of CXCL-12, malignant B lymphocytes bind to the chemokine through CXCR-4. In turn, ligation to CXCL-12 down-regulates the expression of CXCR-4, via receptor endocytosis, on the surface of those cells that have migrated and have made contact with stromal cells [15,136]. This ultimately generates a chemotactic gradient-dependent migration of neoplastic B cells toward CXCL-12-releasing stromal cells [14], and this mechanism forms the basis of the tumour cell’s ability to metastasise.

CXCR-4, as well as other chemokine receptors, is a G-protein-coupled receptor (GPCR), therefore it is normally associated with a heterotrimeric protein complex composed by Gα, Gβ and Gγ subunits. The heterotrimer is linked, in its basal state, to a GDP molecule. Upon CXCL-12 binding, GDP is exchanged with GTP, and this leads to the dissociation of the trimer into Gα, bound to GTP, and the complex β+γ, which is released. Such events, in turn, activate a cascade of downstream pathways that eventually trigger the chemotactic migration of target cells to distant organs [137]. The activation of those signalling pathways potentially interferes with therapeutic outcomes and may lead to treatment resistance, resulting in tumour relapse and progression. For these reasons, efforts are now oriented towards the blockade of such pathways, with the aim of hampering TME- mediated drug and mAb resistance.

Disruption of the CXCR-4/CXCL-12 signalling axis, which provokes migration of malignant B cells to the bone marrow and the following initiation of intracellular signals, has been considered a promising approach to sensitise neoplastic cells to cytotoxic agents and mAbs, and has therefore been evaluated in numerous studies. In B-CLL, CXCR-4 inhibition through the use of small peptide inhibitors, such as T140, was found to block CXCL-12-induced chemotaxis and migration of malignant cells beneath the layer of stromal cells in vitro, and to prevent the activation of downstream signalling molecules

51 MAPK and STAT3 [138]. Moreover, treatment with CXCR-4 inhibitors was able to sensitise CLL cells to chemotherapy, increasing fludarabine-mediated apoptosis [138]. In B-cell ALL, CXCR-4/CXCL-12 axis disruption was achieved through both pharmacological (plerixafor, BKT140) and genetic (CRISPR-Cas9 deletion) inhibition of CXCR-4: both of these methods led to the inhibition of B-ALL migratory ability towards a BMSC layer and to the abrogation of stroma-mediated drug resistance [139]. Furthermore, in a xenograft mouse model inoculation of B-ALL cells where CXCR-4 had been genetically deleted conferred increased survival advantages compared to inoculation of wild-type B-ALL [139].

The chemokine CXCL-12 has been recently shown to be able to interact with another transmembrane chemokine receptor, namely CXCR-7 [140]. Several studies reported the over-expression of CXCR-7 in many cancers, highlighting the role that the receptor might play in driving tumour progression (reviewed in [141]). Similarly, a key role for CXCR-7 in tumour progression was also observed in B-cell malignancies: for instance, the expression of CXCR-7 in DLBCL was linked to tumour spread in lymph nodes, bone marrow and brain. CXCR-7 knock-out strongly reduced the ability of tumour cells to migrate into other organs in DLBCL-bearing mice [142]. Like CXCR-4, CXCR-7 drives chemotaxis and adhesion of tumour cells to the chemokine-releasing stromal environment through the interaction with CXCL-12. Such an activity was observed in several different cancers, including multiple myeloma (MM) [143], ALL [144] and AML [145]. Moreover, some evidence suggested that the blockade of CXCR-7/CXCL-12 interactions might lead to increased apoptosis of cancer cells [146-148], highlighting the important role of this axis in mediating drug resistance.

1.6.5. VLA-4/VCAM-1 signalling axis

Very late antigen-4 (VLA-4, CD49d) is a heterodimeric integrin (α4 + β1) that mediates both cell-cell and cell-ECM adhesion and is highly expressed on normal B cells and B-cell lymphoma cell surfaces [149,150]. VLA-4 has been shown to be fundamental in B cell growth, providing pro-survival and anti-apoptotic signals at the very early stages of B cell development [151]. Vascular cell adhesion molecule-1 (VCAM-1, CD106) is the cognate ligand of VLA-4, and it is constitutively released by the bone marrow microenvironment, mediating the adhesive interaction of the latter with haematopoietic cells [152].

Several studies have highlighted correlations between VLA-4 overexpression and poor prognosis, suggesting its targeting as an attractive strategy to avoid drug resistance and

52 achieve higher therapeutic efficacy [153,154]. It was demonstrated that the VLA-4 receptor ligand VCAM-1 was able to dramatically reduce the sensitivity of Burkitt’s lymphoma cells to the drug etoposide, and this effect was accompanied by conformational changes in the pro-apoptotic protein Bax [155]. In addition to this, interaction between VLA-4 receptor and the ECM component fibronectin was shown to be responsible for protection of B-CLL cells from fludarabine-induced cell death. In this study, resistance to fludarabine was due to the increased expression of the anti-apoptotic molecules Mcl-1 and Bcl-xL and the decreased expression of the pro-apoptotic molecule Bax [156]. A second study showed that VLA-4 was also able to induce fludarabine resistance through a decrease in the levels of p53, which in turn led to reduced cell death [157]. More recently, VLA-4 was shown to be involved in a novel pro-survival pathway: in fact, the inhibition of VLA-4 caused the down-regulation of the PI3K/Akt signalling axis and in turn a decreased release of the IL-10 cytokine in both T- and B-cell lymphoma cell lines. This in turn abrogated an autocrine loop whereby IL-10 augmented STAT3 activity and survivin expression, thus mediating drug resistance [158]. These findings made clear the fundamental role of the VLA-4/VCAM-1 pathway in mediating B cell resistance to treatments when in the presence of components of the TME.

Interestingly, several studies underlined that the chemokine CXCL-12 might be able to modulate VLA-4 expression, as VLA-4 adhesive and migratory functions were enhanced in the presence of CXCL-12 [159,160]. It was also demonstrated, by performing co- immunoprecipitation assays, that the two receptors CXCR-4 and VLA-4 directly interact when stimulated by CXCL-12 [161]. Moreover, the NF-κB signalling pathway was recently shown to be activated following VLA-4/VCAM-1 interaction in both AML and ALL cell lines, suggesting that the CXCR-4/CXCL-12 and VLA-4/VCAM-1 axes also share common downstream effectors [162]. More recently, it has been demonstrated that in MCL cells the simultaneous inhibition of CXCR-4 and VLA-4 through combination of anti-VLA-4 and anti-CXCR-4 antibodies led to a diminished migratory ability [163] and to an increased tumour cell death after Ara-C treatment compared to the single inhibition of either receptor [164]. Both antibodies were able to block the activation of the downstream signalling pathways ERK1/2, Akt and NF-κB, and such inhibitory activity was even stronger in the presence of BMSCs [164]. Furthermore, a decreased adhesion to stromal cells following the combinatorial inhibition of CXCR-4 and VLA-4 was also observed in HCL [165].

53 1.7. Anti-CD20 mAbs and the tumour microenvironment

The observation of TME-mediated mechanisms of drug resistance has led to the idea that stromal cells could exert the same effect on B cells after mAb therapy. Indeed, recent analyses have underlined the reduced efficacy of rituximab-mediated direct cell death [21], CDC [166], and ADCC [167] in the presence of BMSCs in vitro, therefore unravelling the ability of stromal TME to protect B cells and promote their escape from treatments. Such TME-induced resistance to rituximab has been shown to be partly due to the down- regulation of the CD20 molecules on B-cell surfaces, observed in vitro after co-culture of tumour cells and mesenchymal stromal cells in CLL [167]. Therefore, the TME might play a crucial role in determining the susceptibility of malignant B cells to chemo- and mAb therapy.

The blockade of CXCR-4 through the antagonist TN14003 was shown to abrogate stroma- mediated protection of B cells from both type-I antibodies alemtuzumab and rituximab by enhancing the efficacy of passive, but not active, immunotherapy, leading to B-cell depletion through the strong induction of CDC [166]. This work also underlined the importance of Mcl-1 and Akt pathways in the acquisition of resistance to apoptosis upon contact with BMSCs [166]. In more recent work, the CXCR-4/CXCL-12 axis was inhibited through the use of cell-penetrating pepducins in systemic lymphoma mouse models, and the higher efficacy of rituximab therapy after combination was highlighted [168]. A recent study combined the CXCR-4 antagonist BKT140 with rituximab and looked at the induction of direct cell death, observing a significant reduction (93%) in the number of lymphoma cells in primary samples from bone marrow of patients with B-NHL [19]. Similar results were obtained in vitro and in vivo, by using the Raji cell line and B104 lymphoma model: the addition of the CXCR-4 antagonists plerixafor and GENZ-644494 in combination with rituximab or alemtuzumab was able to reverse the protective effect of stromal microenvironment and increase B-cell depletion in vivo and in vitro through direct cell death [20]. The treatment also led to an increase in the number of circulating tumour cells due to disruption of the CXCR-4/CXCL-12 axis and subsequent blockade of B-cell migration and homing to the bone marrow. Importantly, CXCR-4 blockade also led to the mobilisation of immune effector cells, and especially neutrophils, into the circulation, and this increase in the number of available immune cells was shown to be a determinant in the greater therapeutic efficacy observed in mice: depletion of neutrophils markedly impaired the efficacy of the combination therapy [20]. The egress of a greater number of neutrophils from the bone marrow to the circulation, thus, enhances mAb potency, probably through a

54 stronger induction of ADCP/ADCC. Such effects suggest that CXCR-4 blockade could act not only through the inhibition of protective signals from the microenvironment, but also by actively improving mAb therapy through the enhancement of mAb effector mechanisms.

The binding of VLA-4-expressing cells to VCAM-1 has been found to promote stromal- mediated B-cell protection not only from cytotoxic drugs, but also from anti-CD20 mAbs. A recent paper showed that B-cell lymphoma cells are protected from cytotoxic drugs by the stromal microenvironment in vitro and that stromal-cell interactions are dependent on CXCR-4 and VLA-4 receptors [150]. More recently, it was demonstrated that the TME protects B cells from the type-I anti-CD20 mAb rituximab and that this protection is mediated by the VLA-1/VCAM-1 signalling axis [21]. Natalizumab, a monoclonal antibody against the α4 integrin, was shown to be able to abrogate the stromal cell- mediated protective effect and to significantly increase B cell death when in combination with rituximab [21].

The published literature suggests that VLA-4 might be an attractive target, and its blockade could enhance the therapeutic efficacy of anti-CD20 mAbs. Combination between VLA-4 and CXCR-4 inhibition might be even more effective, and it would be interesting to determine whether such a combination approach could improve mAb efficacy further.

55 1.8. Induction of long-term anti-tumour immune responses

The potency of anti-CD20 mAb therapy might not be limited to the induction of cytotoxicity against B cells during treatment: in fact, there is some evidence for the existence of a long-term anti-tumour immunity which takes place after mAb therapy and is long-lasting, suggesting a mAb-induced “vaccinal” effect. One study first showed that tumour cells undergoing apoptosis after treatments are phagocytosed by DCs, leading to DC maturation and presentation of lymphoma-associated antigens on DC surface (phenomenon called cross-presentation) [169]. Then, the consequences of this event on immunity were investigated, and it was demonstrated that co-culture of primed DCs with T cells led to the generation of cytotoxic T cell responses against malignant B cells [170]. To investigate whether the immune system influences the therapeutic outcome of mAb treatment, a recent work characterised the T-cell subsets from both blood and tumour microenvironment in 250 patients with FL. Both tumour and blood CD3+, CD4+ and CD8+ T cells were positively associated with a good prognosis after rituximab treatment, and the CD8+ subset was shown to determine a prolonged therapeutic effect, increasing the time to next treatment [171]. Since T cells are activated by DCs, the study also suggested that such T cell-dependent higher efficacy after treatment could be boosted in the presence of antigen-specific DCs. Recently, a paper analysed the response of T cells to stimulation with DCs in blood from FL patients before and after rituximab monotherapy: DCs were pulsed with patient-specific idiotype proteins and then used to stimulate T lymphocytes [55]. Only after rituximab treatment, the presence of Id-pulsed DCs led to an increase in IFNγ-producing T cells, suggesting that treatment with mAbs induces generation of an idiotype-specific T cell-mediated immune response [55]. Furthermore, immune-competent mice which rejected a syngeneic lymphoma following anti-CD20 mAb treatment survived a subsequent tumour re-challenge, showing that mAb therapy induced long-lasting survival even after re-inoculation of tumour cells [53]. Such long-term survival was T-cell mediated, and was dependent on the presence of the CD4+ subset at the beginning of the therapy, while both CD4+ and CD8+ T cells were required after tumour re-challenge [53]. Additional studies have further detailed the characteristics of such mAb-induced long-term adaptive immunity, identifying the Th1 subset of CD4+ T cells as a fundamental effector in establishing a pro-inflammatory microenvironment [80]. Furthermore, a recently published study investigated the ability of the type-II anti-CD20 antibody obinutuzumab to eradicate B-cell lymphomas when in combination with immuno-modulatory agents, and showed that obinutuzumab combined to a toll-like receptor (TLR) 7 agonist was able to induce long-

56 term anti-tumour immune responses in immuno-competent mice [54]. Whilst primary immune responses against T-cell lymphoma cells were found to be mediated by NK cells and CD4+ T cells, immune responses against a second tumour re-challenge were dependent on both CD4+ and CD8+ T cells [54].

Altogether, those findings suggest the possibility to generate a “vaccinal effect” through anti-CD20 mAb treatments, ensuring long-term protection through the induction of an adaptive anti-tumour T cell-mediated immune response.

Further insights into these mechanisms of action are potentially important in developing novel therapeutic strategies in B-cell lymphomas that target not only the tumour cells but the microenvironment as well.

1.9. Project outline

Anti-CD20 mAb therapy has been highly successful in improving outcome for patients with B-cell malignancies, in combination with chemotherapy, targeted radiotherapy (radio- immunotherapy) or with other immunotherapy approaches. Despite the significant improvements and progresses made in the last years in this field, patients still relapse or develop progressive disease. New strategies that specifically target those mechanisms which are at the basis of tumour escape from treatment are urgently needed.

Several studies have highlighted the importance that stromal components of the tumour microenvironment might play in mediating B-cell malignancy resistance to therapies, and have identified several signalling axes as main mediators of B-cell contact with and migration underneath the bone marrow stromal cells and induction of pro-survival pathways. Thus, those pathways might be determinants of stroma-induced protection. Blockade of these axes could lead to the inhibition of such protective effects, to B-cell mobilisation to blood vessels and to an overall better efficacy of type-II mAb killing of B cells.

The central hypothesis of the thesis is that the inhibition of interactions between tumour and stromal cells will enhance the therapeutic efficacy of type-II anti-CD20 mAbs.

57 The aims of this project are:

1- To investigate how the presence of the tumour stromal microenvironment impacts on the induction of cell death and on the mechanisms of action of the type-II mAb obinutuzumab in B-cell lymphoma cell lines; 2- To determine which mechanisms are at the basis of the tumour stromal microenvironment-mediated protection of B-cell lymphoma cell lines from obinutuzumab; 3- To investigate whether inhibition of stroma-tumour signalling axes can enhance anti-CD20 mAb effector mechanisms (PCD, CDC, ADCC) in B-cell lymphoma cell lines in the presence of stromal cells.

In patients with B-cell malignancies, where tumour relapses and resistance to treatments are among the principal causes of death, specific targeting of the microenvironment in order to improve therapeutic outcomes and potentially avoid development of secondary tumours appears a promising approach. Further insights into these mechanisms of action are potentially important in developing novel therapeutic strategies in B-cell malignancies that target not only the tumour cells but the microenvironment as well. Therefore an enhanced understanding and developing therapeutics that target this interplay between the tumour and the TME may inform clinical translation and ultimately lead to improvements in treatment approaches.

58 2. Materials and methods

2.1. Cell lines

The B-cell lymphoma cell lines Raji and Daudi (Burkitt’s lymphoma) were purchased from the American Type Culture Collection (ATCC) Company. The murine fibroblast-like stromal cell line M2-10B4 was kindly provided by Claire Hart (University of Manchester, UK). The human fibroblast-like cell line HS-5 and the murine macrophage RAW cell line were purchased from ATCC. EL4-huCD20 and Eµ-myc-huCD20 (expressing a human CD20 surface receptor, described in [54]) were kindly provided by Dr Eleanor Cheadle (University of Manchester, UK). The Eµ-myc cell line, generated by Dr Jamie Honeychurch (University of Manchester, UK), was derived from B-cell tumours developing in the lymph nodes and spleens of Eµ-myc mice by maintaining single cell suspensions in complete RPMI supplemented with 20% FCS at a density of 2 x 106/ml, until stable cell lines were successfully generated. The GFP-actin-expressing Raji cell line (called Raji-GFP-actin in this report), which was transfected to express a GFP-actin fusion protein [84], was kindly provided by Dr Andrejs Ivanov (University of Manchester, UK). The luciferase-iRes-GFP-expressing Raji (Raji-luc-iGFP) cell line, made as described in [172], was kindly provided by Dr Eleanor Cheadle. The luciferase-iRes-GFP-expressing EL4-huCD20 (EL4-huCD20-luc-iGFP) cell line was kindly provided by Dr Grazyna Lipowska-Bhalla (University of Manchester, UK). The human prostate cancer cell line DU145 was kindly provided by Dr Debayan Mukherjee (University of Manchester, UK).

o The cell lines were cultured under standard conditions (5% CO2, 37 C) either in RPMI- 1640 media (Gibco, Life Technologies, Thermo Fisher Scientific, Paisley, UK) supplemented with 10% heat-inactivated Foetal Bovine Serum (Invitrogen, Life Technologies, Thermo Fisher Scientific, Paisley, UK) and 2 mM L-glutamine (Invitrogen), or in RPMI-1640 media (Gibco, Life Technologies) supplemented with 10% heat- inactivated Foetal Bovine Serum (Invitrogen), 2 mM L-glutamine (Invitrogen), 25 mM Hepes (Sigma-Aldrich, Poole, UK) and 50 nM 2-mercaptoethanol (Invitrogen), or in DMEM media (Gibco, Life Technologies) supplemented with 10% heat-inactivated Foetal Bovine Serum and 2 mM L-glutamine. Cells were routinely screened to confirm negativity to mycoplasma infection.

59 2.2. Animals used in in vivo experiments

C57Bl/6 mice were purchased from Envigo (Loughborough, UK). NSG mice were obtained from Jackson Laboratories (Bar Harbor Road, USA) and bred in-house in the Cancer Research UK Manchester Institute. All animals used were female and weighed at least 18-20 g on arrival. Mice were housed in specific pathogen-free facilities. For each experiment, each treatment group included at least 7 mice. Each experiment was performed at least once. Mice were culled when moribund signs were shown, 20% weight loss, or at the development of hind leg paralysis.

All the experiments involving animals were carried out under the regulations of the 1986 ASPA act and EU directive 2010/63, UKCCCR guidelines. All in vivo studies were performed under a UK Home Office license and were approved by an ethical committee.

2.3. Isolation of PBMCs from whole blood and buffy coats

Whole blood was kindly donated by healthy donors within the Cancer Research UK, Manchester Institute. Buffy coats of blood from healthy donors were purchased from the Manchester Plymouth Grove Blood Donor Centre, Manchester, UK. Blood was obtained with ethical consent from the South Manchester Ethics committee in accordance with the declaration of Helsinki.

In order to isolate peripheral blood mononuclear cells (PBMCs), blood and buffy coats were diluted 1:4 with phosphate buffered saline (“PBS”, Thermo Fisher Scientific, Altrincham, UK) and then carefully layered on Lymphoprep density gradient media (Stemcell Technologies, Grenoble, France) – 25 ml of diluted blood layered onto 20 ml of Lymphoprep media in 50 ml tubes (Corning, Amsterdam, The Netherlands). The tubes were then centrifuged at 580g at room temperature for 30 minutes without brake. This density gradient centrifugation leads to the separation of blood into four different layers: from bottom to top, erythrocytes and neutrophils, Lymphoprep media, a cloudy layer that contains PBMCs and a final layer of plasma (figure 2.1). Plasma was discarded and the cloudy layer of PBMCs was collected through the use of 3.5 ml transfer pipettes (Sarstedt, Leicester, UK); PBMCs were then washed in PBS, centrifuged at 800g at room temperature for 10 minutes with brake, before being re-suspended in FBS containing 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich) and stored at -80°C until use.

Figure 2.1. Schematic diagram showing the separation of components in blood upon density gradient centrifugation. Whole blood or buffy coats of blood were diluted 1:4 with PBS, then carefully layered onto 20 ml Lymphoprep media. After centrifugation, blood components separate into four different layers: from bottom to top, erythrocytes and neutrophils, density gradient media, a cloudy layer containing the PBMCs and a final layer of plasma.

61 2.4. Antibodies and reagents

Rituximab and Herceptin were purchased from the Christie Hospital NHS Trust (Manchester, UK). GA101 (obinutuzumab), GA101-m2a (containing a murine IgG2a Fc region) and anti-SDF-1α antibody (clone 2A5) were kindly provided by Roche Innovation Center, Zurich (Switzerland). Tositumumab (Bexxar) and ocaratuzumab (AME-133v) were kindly provided by GlaxoSmithKline (GSK, Uxbridge, UK) and Mentrix Biotech respectively. The mouse anti-human CD49d (VLA-4, clone 9F10) and rat anti-mouse CD172a (SIRP-α) monoclonal antibody (clone P84), functional grade, was purchased from eBioscience, Thermo Fisher Scientific. Mouse SDF-1α (CXCL-12) cytokine and human fibronectin were obtained from Miltenyi Biotec Ltd (Bisley, UK) and Scientific Laboratory Supplies (Orchard House, UK) respectively. CountBright™ Absolute Counting Beads were purchased from Life Technologies. In-solution CXCR-4 antagonist plerixafor (AMD- 3100) was purchased from Merck Chemicals (Hull, UK). CellVue Lavender cell labelling kit was purchased from eBioscience. The Bruton’s tyrosine kinase inhibitor PCI-32765 (Ibrutinib) and the PI3Kδ inhibitor CAL-101 (Idelalisib, GS-1101) were purchased from ApexBio Technology (Houston, USA). The Syk inhibitor R406 was obtained from InvivoGen SAS (Toulouse, France).

2.5. Flow cytometry

The BD FACS (fluorescence-activated cell sorter) Calibur cell analyser (Becton Dickinson), the BD FACS Canto II cell analyser (Becton Dickinson) and the ACEA NovoCyte flow cytometer (ACEA Biosciences, San Diego, USA) were used to analyse cell viability and to confirm receptor expression on cell surface. The BD Influx cell sorter (Becton Dickinson) and BD FACS Aria II cell sorter (Becton Dickinson) were used to separate different cell populations previously co-cultured and then analysed after being exposed to different conditions. BD CellQuest Pro™ software (Becton Dickinson), BD FACS Diva software (Becton Dickinson) and NovoExpress software (ACEA Biosciences) were used to acquire data from the cytometers.

M2-10B4 stromal cells were distinguished from tumour B cells Raji and Daudi by staining them with the green fluorescence linker PKH67 (emission peak: 502 nm) or with the green fluorescence dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (emission peak: 521 nm, Thermo Fisher Scientific). Cell viability was analysed by staining cells with

62 AnnexinV-Cy5.5/7-AAD (emission peaks: 670 nm and 617 nm respectively). Expression levels of surface receptors on Raji and Daudi cell lines were calculated as either median fluorescence intensity or geometric mean fluorescence intensity of either APC+ cells (excitation peak: 650 nm, emission peak: 660 nm), PE+ cells (excitation peak: 496 nm, emission peak: 580 nm), PE-Cyanine 7 (PE-Cy7)+ cells (excitation peak: 496 nm, emission peak: 785 nm), Alexa Fluor 488+ cells (excitation peak: 495 nm, emission peak: 519 nm) or FITC+ cells (excitation peak: 494 nm, emission peak: 520 nm), based on the antibody used, after gating in only the live cell population on the basis of its forward scatter/side scatter characteristics. Cytometric analyses were performed by using FlowJo data analysis software (version 10, TreeStar, Miltenyi Biotec Ltd).

2.6. Co-culture experiments

For co-culture experiments with stromal cells, M2-10B4 cells (2.5 x 104 cells/1 ml/well) or HS-5 cells (either 5 or 8 x 104 cells/1 ml/well) were stained with the green fluorescence linker PKH67 (Sigma-Aldrich) for general cell membrane labelling and then seeded on 24- well plates (Falcon, Copenhagen, Denmark and Starlab, Blakelands, UK) for 72 hours, to ensure the formation of a confluent stromal layer on the plastic. After 72 hours, the medium was carefully removed and tumour cells were added in fresh medium onto the stromal layer (1.25 x 105 cells/ml/well) at a ratio Raji/Daudi:M2-10B4 of 5:1. For stromal- conditioned medium experiments, M2-10B4 were cultured for 72 hours (2.5 x 104 cells/ml/well), medium collected, filtered through a 0.45 μm filter (purchased from Appleton Woods Ltd, Catesby Park, UK) and used to culture Raji or Daudi cells (1.25 x 105 cells/ml/well) for 24 additional hours. For tumour/stromal-conditioned medium experiments, M2-10B4 were cultured for 72 hours (2.5 x 104 cells/ml/well), then Raji or Daudi cells (1.25 x 105 cells/ml/well) were added in fresh medium for 24 additional hours. After 24 hours of co-culture, medium conditioned by the co-culture conditions was collected and used to culture new Raji or Daudi cells (1.25 x 105 cells/ml/well). For non- contact co-culture experiments, 24-well polycarbonate transwell plates (Appleton Woods) with a diameter of 6.5mm and pore size of 0.4 μm were used. Stromal cells were plated onto the bottom compartments at a concentration of 2.5 x 104 cells/well in 1 ml RPMI for 72 hours, then Raji and Daudi cells were added on the top compartments at a ratio tumour:stromal cells of 5:1 in 100 μl RPMI for 24 additional hours. For culture of tumour cells on fibronectin, 1 mg human fibronectin was purchased from Thermo Fisher Scientific

63 and re-suspended in distilled H2O. Fibronectin was diluted in PBS to a final concentration of 5 µg/cm2 (unless differently stated) and incubated on wells for 1 hr at room temperature.

The wells were then rinsed with distilled H2O and used to plate cells for 24 hours (unless differently stated).

2.6.1. Programmed cell death assay

To measure the degree of programmed (direct) cell death induced by the treatments, cells were treated after 1 hour of tumour/stromal cell co-culture with anti-CD20 monoclonal antibodies at a concentration of 10 μg/ml. After 23 additional hours, tumour and stromal cells were aspirated from the wells, washed with PBS and stained for cell viability by re- suspending samples in Annexin V-Cy5.5 (used at 1 µg/ml, diluted in 1X Annexin V buffer, both from Becton Dickinson, Oxford, UK) and 7-Aminoactinomycin D (7-AAD eBioscience, used at 0.5 µg/ml) for 15 minutes in the dark, at room temperature. Samples were then analysed by flow cytometry. Tumour cells were gated as PKH67- cells and survival was calculated as % of tumour cells negative for Annexin V and 7-AAD.

2.6.2. Complement-dependent cytotoxicity assay

To assess the degree of CDC after rituximab treatment, tumour cells were co-cultured with M2-10B4 or HS-5 in 24-well plates and human serum collected from healthy donors was added to the wells (at concentration of either 5, 10 or 20%). Serum was separated from whole blood by allowing the blood to clot and centrifugation at 900g at 4oC for 20 minutes. The upper phase was then collected and centrifuged again to ensure removal of any residual red blood cells (13000g, 1 minute). After 72 hours of single culture of M2-10B4, tumour cells and human serum were added to the wells. Then, cells were treated with rituximab (10 μg/ml). After 24 additional hours, cells were removed from the wells and stained for cell viability with Annexin V-Cy5.5 and 7-AAD, as previously described (see 2.6.1).

2.6.3. Antibody-dependent cellular phagocytosis assay

To measure the ability of anti-CD20 monoclonal antibodies to induce tumour cell death through phagocytosis by monocytes, neutrophils or macrophages, tumour cells were labelled with the red fluorescence linker PKH26 (Sigma-Aldrich) for general cell membrane labelling and co-culture with PKH67-labelled stromal cells for either 24 hours or different time-points. PBMCs were isolated either from whole blood or buffy coats as described in 2.3, while immune effector cells were isolated by using specific isolation kits

64 (pan monocyte isolation kit, neutrophil isolation kit, CD16+ monocyte isolation kit, all from Miltenyi Biotec Ltd, as per the manufacturer’s protocol). Human macrophages were derived from pan monocytes which were cultured in T75 flasks for 6 days in complete RPMI media supplemented with 50 µg/ml recombinant human M-CSF (macrophage colony-stimulating factor, Bio-Rad Laboratories Ltd, Maxted Road, UK) on day 0 and day 4. The murine RAW macrophage cell line was also used as an effector immune population. Effector cells were then added to the co-culture at specified effector:target ratios. Three- way cultures were treated with antibodies (at either 0.1 μg/ml or 10 μg/ml) and, at the stated time-points, were collected, centrifuged at 400g for 3 minutes, washed in FACS buffer (made with PBS + 1% Fetal Bovine Serum) and re-suspended in the appropriate antibody diluted in FACS buffer at 4°C for 30 minutes. Samples were then centrifuged at 400g for 3 minutes, washed with FACS buffer, re-suspended in 1% paraformaldehyde (PFA, Sigma-Aldrich) and analysed by flow cytometry. The following antibodies were used to label specific sets of effector cells: anti-human CD11b-APC antibody, clone ICRF44; anti-mouse CD11b-APC antibody, clone M1/70; anti-human CD14-APC antibody, clone 61D3; anti-human CD16-APC antibody, clone eBioCB16; anti-human CD16-PE-Cyanine7 antibody, clone eBioCB16; all from eBioscience. Concentration of each antibody is specified in figure legends.

2.6.4. NK cell activation (IFN-γ release assay)

To assess the degree of NK cell activation induced by treatments, tumour cells were co- cultured with PKH67-labelled stromal cells for 24 hours. Immune effector cells (NK cells) were isolated from PBMCs through the use of a CD56+ NK cell isolation kit (Miltenyi Biotec Ltd, as per the manufacturer’s protocol), treated with an inhibitor of intracellular protein transport (Brefeldin A solution 1000X, eBioscience) and added to the co-culture at specified effector:target ratios. Cells were treated with antibodies at either 0.1 μg/ml or 10 μg/ml for 4 hours. Then, cells were harvested, centrifuged at 400g for 3 minutes, washed in PBS and re-suspended in an anti-human CD56-APC antibody (clone TULY56, eBioscience) at 4°C for 30 minutes, in order to label NK cells. Samples were then centrifuged at 400g for 3 minutes, washed with FACS buffer and permeabilised and fixed by using a Foxp3/transcription factor staining buffer set (eBioscience, as per manufacturer’s protocol). The production of IFN-γ was measured through flow cytometry, after intracellularly staining cells with an anti-human IFN-γ-PE antibody (clone 4S.B3, eBioscience) and re-suspending them in 1% PFA.

65 2.7. Migration assay

Tumour cell migration towards the chemotactic gradient was measured through the use of 24-well polycarbonate transwell plates (Appleton Woods) with a diameter of 6.5 mm and pore size of 5 μm. Either mouse SDF-1α (CXCL-12) recombinant protein (at 10, 30 or 50 ng/ml) or M2-10B4 (2.5 x 104 cells/well) were seeded in the bottom compartment of the transwell plates. After coating the inserts with human fibronectin (10 μg/cm2), Raji or Daudi cells (either 3 or 1.25 x 105 cells/well) were pre-treated with plerixafor for 30 minutes and then seeded into the upper compartment of the transwell plates in 100 μl RPMI. After 4 hours, migration was assessed by collecting the cells in the bottom chamber and adding CountBright counting beads (4000/sample). The number of cells migrated towards the bottom compartment was measured by flow cytometry using the following equation:

(Number of migrated cells detected / number of beads detected) * total number of beads.

2.8. Receptor expression levels on cell surface

To evaluate CXCR-4/CXCR-7 expression levels on Raji and Daudi cell surfaces, tumour cells (1 x 106 cells) were harvested and centrifuged at 400g for 3 minutes, washed in FACS buffer and re-suspended with anti-human CXCR-4 (CD184)-APC (clone 12G5, Miltenyi Biotec Ltd) or mouse anti-human CXCR-7-APC (clone 10D1-J16, BioLegend, Greenwood Place, UK) antibodies, diluted in FACS buffer to a final concentration of 50 μg/ml. Cells were kept at 4°C for 30 minutes. Samples were then centrifuged at 400g for 3 minutes, washed with FACS buffer, re-suspended in 1% paraformaldehyde (PFA, Sigma-Aldrich) and analysed by flow cytometry. Receptor expression (measured as median fluorescence intensity, MFI, unless differently stated) was detected by flow cytometry (see 2.5).

The same protocol was used to determine the following cell surface receptors’ expression levels: CD172a (SIRP-α) on M2-10B4 cell surface, using the anti-mouse CD172a-APC (clone P84) or rat IgG1-κ-APC isotype control (clone eBRG1) at 2 μg/ml (eBioscience); CD20 on Raji and Daudi cell surface, using the anti-human CD20-APC (clone 2H7, eBioscience) and mouse IgG2b-APC isotype control (clone eBMG2b, eBioscience), both used at 0.24 μg/ml; integrin VLA-4 on Raji and Daudi cell surface, using the mouse anti- human VLA-4 (CD49d)-PE (clone 9F10, eBioscience) antibody and mouse IgG1-κ-PE isotype control (clone P3.6.2.8.1) at 0.25 μg/ml; E-cadherin (CD324) and N-cadherin

66 (CD325) on Raji and Daudi cell surface, using the mouse anti-human CD324-PE antibody (clone 67A4, Becton Dickinson) and the mouse anti-human CD325-PE antibody (clone 8C11, eBioscience), and mouse IgG1-κ-PE isotype control (clone MOPC-21 (RUO), Becton Dickinson and clone P3.6.2.8.1, eBioscience); P-cadherin and VE-cadherin (CD144) on Raji and Daudi cell surface, using the mouse anti-human P-cadherin-APC antibody (clone CSTEM29) and mouse IgG2a-κ-APC isotype control (clone eBM2a) for P-cadherin, mouse anti-human VE-cadherin-PE-Cy7 (clone 16B1) and mouse IgG1-κ-PE- Cy7 isotype control (clone P3.6.2.8.1) for VE-cadherin, both from eBioscience.

2.9. Detection of protein expression through western blotting

To detect the expression of specific proteins in tumour cells, lysates were prepared by using Cell Lysis buffer (10X, Cell Signalling Technology, Danvers, USA) and Protease Inhibitor Cocktail (100X, Cell Signalling Technology), diluted to a 1X working solution by adding distilled H2O. Samples were washed twice in ice-cold PBS, centrifuged at 400g for 3 minutes and re-suspended in the 1X working solution. Cells were kept on ice for 10 minutes, then centrifuged at 13000g for additional 10 minutes. Supernatant from each sample was then collected and the protein content was measured by using a BCA protein assay kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. Briefly, protein samples and standard samples containing different known amounts of bovine albumin serum (BSA) are mixed with an alkaline reagent containing bicinchoninic acid (BCA) and copper sulfate. In an alkaline environment, proteins will reduce Cu2+ to Cu1+. BCA then binds to Cu1+, forming a complex that can absorb light at wavelength 562 nm. Absorbance is then measured through a FLUOstar Omega microplate reader (BMG Labtech, Aylesbury, UK). Because the amount of reduced Cu1+ is proportional to the amount of proteins in samples, by knowing the amount of protein contained in the BSA standards, their absorbance and the absorbance of the samples, it is possible to calculate the amount of protein contained in each sample. Samples were then stored at -20°C.

For each western blot experiment, approximately 15-20 μg of proteins were then denatured by adding reducing Laemmli SDS sample buffer (6X, Alfa Aesar, Thermo Fisher Scientific) for 3 minutes at 96°C, loaded onto and separated by 4-20% SDS-PAGE gels (Novex WedgeWell 4-20% Tris-Glycine mini gels, Life Technologies) by using 10X Tris-

Glycine running buffer (Alfa Aesar, Thermo Fisher Scientific) diluted in dH2O. Proteins were transferred to Immun-Blot Polyvinylidene Difluoride (PVDF) membranes (Bio-Rad

67 Laboratories Ltd, activated in 100% methanol) through electro blot, by using 10X electro blot transfer buffer (Alfa Aesar, Thermo Fisher Scientific) and 5X methanol diluted in dH2O. Membranes were then blocked in 5% powdered skimmed milk (Marvel, UK) for 1 hour at room temperature and incubated overnight at 4°C with the appropriate primary antibodies (GAPDH rabbit monoclonal antibody, clone 14C10; pan-cadherin rabbit polyclonal antibody; both from Cell Signalling Technology) diluted 1:1000 in 0.1% milk. The following day, membranes were washed and visualised by electrochemiluminescence (using the Western Lightning plus-ECL kit, PerkinElmer) after adding an appropriate IgG (H+L) HRP-conjugated secondary antibody (Life Technologies) diluted 1:2500 in 0.5% milk.

2.10. Immunocytochemistry

To visualise the actin cytoskeleton in GA101-treated cells after FACS separation from stromal cells, Raji-GFP-actin cells were treated for 4 hours with 10 µg/ml GA101 and then poured onto Poly-L-Lysine slides (Thermo Fisher Scientific). Slides were incubated at 37°C for 30 min to allow adhesion. Cells were then fixed by addition of 4% paraformaldehyde and washed three times in PBS. Slides were then mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and visualised under a Leica gSTED confocal nanoscope (63X oil objective). DAPI nuclear staining was acquired by using the DAPI filter, actin was visualised under the GFP filter.

2.11. Microscopy and time-lapse experiments

To determine the ability of tumour cells to reorganise the actin cytoskeleton when in co- culture with stromal cells or cultured on fibronectin, cells were pre-treated with GA101 at 10 µg/ml for two hours, then poured onto 24-well plates and imaged either immediately after pouring or after 24 hours using a ZEISS lowlight microscope (either 10X or 20X air objectives). For time-lapse experiments, M2-10B4 (2.5 x 104 cells/ml) were seeded into 24-well plates for 72 hours. Raji and Daudi cells were pre-treated with GA101 and then poured onto the stromal layer (1.25 x 105 cells/ml). Cells were kept in an environmental chamber at 37°C, 5% CO2 and imaged every 30 minutes for 24 hours with a ZEISS lowlight microscope (either 10X or 20X air objectives). Movies were developed using the

68 MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices, USA).

2.12. Stable isotope labelling by amino acids in cell culture (SILAC)

To detect changes in protein expression in tumour cells after contact with stroma, the stable isotope labelling by amino acids in cell culture (SILAC) technique has been used. Tumour cells were cultured in SILAC RPMI-1640 medium (Gibco, Life Technologies), which is modified to lack L-lysine, L-arginine and L-glutamine. SILAC RPMI medium was supplemented with 10% heat-inactivated dialysed Fetal Bovine Serum, 2 mM L- 13 glutamine (Invitrogen), and isotopically labelled by adding either 0.3 mM L-Lysine- C6 13 13 15 and 0.3 mM L-Arginine- C6 (medium isotope), or 0.3 mM L-Lysine- C6, N2 and 0.3 13 15 mM L-Arginine- C6, N4 (heavy isotope). 200 µg/ml L-Proline was added to both medium and heavy media to prevent conversion of Arginine to Proline. Isotopically labelled amino- acids, L-Proline (both Sigma-Aldrich) and dialysed FBS (Invitrogen) were kindly provided by Dr Claus Jørgensen (CRUK Manchester Institute). Cells were then grown in either medium or heavy RPMI media for 5 passages prior to a labelling efficiency test performed to ensure that adequate labelling had been achieved.

Medium- and heavy-labelled cells were then lysed as described in 2.9. The content of proteins in the lysates was measured by performing a BCA assay (described in 2.9). 10 µg were taken from both medium-Raji and heavy-Raji and mixed 1:1. The resulting 20 µg of proteins were run on a 4-20% SDS-PAGE gel, the lane produced in the gel was stained with SimplyBlue SafeStain (Thermo Fisher Scientific) for 1 hour, sliced into six bands and proteins contained in each band were analysed by mass spectrometry (see 2.13).

2.13. Mass spectrometry

Mass spectrometry analysis of protein content in medium- and heavy-labelled cells was performed by the Biological Mass Spectrometry facility at the Cancer Research UK Manchester Institute.

Briefly, proteins acquired from each of the gel bands were cleaved by trypsin digestion. Trypsin cleaves proteins C-terminal to Arginine and Lysine residues except when followed by Proline, thus ensuring the presence of either one or both of the labelled amino acids in

69 each resulting peptide. The peptides were then subject to nano-liquid-chromatography (LC), which involves the use of a gradient of organic solvent to ensure the elution of peptides from a reversed phase to a stationary phase in tiny volumes, thereby maximising peptide concentration that elutes into the mass spectrometer. The mass spectrometer used is a quadrupole/Orbitrap/Ion trap mass spectrometer (MS). The instrument detects each peptide fragment’s mass and, through electrospray ionization, further fragments each peptide into peptide’s ions. The mass/charge ratio is therefore calculated for each peptide and each derived peptide’s ion. The corresponding intensity is proportional to the amount of peptide detected.

Spectra generated by these cycles of LC-MS/MS are then matched to a database containing theoretically determined peptide fragments’ mass/charge ratios by using Peaks Protein Identification Software (Bioinformatic Solutions Inc., Waterloo, Canada). Peaks calculates a p-value, expressed as significance score (equal to -10Log(P-value)) for each matched protein and, based on the intensity of each analyte, also calculates a heavy- to medium- labelled protein ratio. Such a ratio will therefore be a measure of the relative abundance of each protein in tumour cells cultured on stroma compared to tumour cells cultured on plastic.

2.14. Enrichment analysis of differentially expressed proteins

The enrichment analysis of the dataset of proteins obtained from mass spectrometry was performed by using the Database for Annotation, Visualisation and Integrated Discovery (DAVID, available at https://david.ncifcrf.gov/) and Ingenuity Pathway Analysis (IPA, QIAGEN Bioinformatics, license purchased by The University of Manchester, UK).

In DAVID, for each group of proteins, an enrichment score (ES) is shown. This is the geometric mean of each EASE score calculated for each single protein ID (see 2.15).

2.15. Statistical analysis

Statistical analyses were performed using GraphPad Prism software (version 7.00). p- values (statistical significance: p<0.05) were calculated using the two-way Analysis of Variance (Anova) test (unless differently stated). Results are the mean ± standard error

70 (SEM), geometric mean ± SEM or medians ± SEM of three independent experiments (unless differently stated in the figure legends).

In general, the two-way Anova test was used to analyse statistical difference in data that was normally distributed, where the analysis required multiple comparisons, i.e. both within and between different groups. The one-way Anova test was used to analyse normally distributed data where more than two groups were present, that only required comparison between different groups (i.e. not within each group). Non-parametric tests, such as the Mann-Whitney U test, were used when the data was not considered normally distributed, and therefore statistical difference was calculated for medians, instead of means. Finally, the Mantel-Cox test was used to determine statistical differences between different survival curves in in vivo experiments.

Statistical analyses in mass spectrometry-based proteomics were performed by using Peaks (where the p-value is defined as the probability that a false protein identification achieves the same or better matching score than a real protein identification, [173]) and DAVID or IPA (where the p-value is the probability that the overlap between experimentally observed proteins and a given pathway is due to random chance). In IPA, p-values are calculated using a right-tailed Fisher’s Exact Test. In DAVID, p-values are calculated using a modified Fisher’s Exact Test, namely EASE score ([174], [175] and [176]). Statistical significance was p<0.05, or ES>1.3.

71 3. Stroma-mediated protection of tumour cells from anti- CD20 mAb-induced programmed cell death (PCD)

To assess the ability of stroma to protect tumour cells from anti-CD20 monoclonal antibody-induced mechanisms of action, a co-culture system was established. Murine fibroblast-like bone marrow stromal cells M2-10B4 were seeded onto 24-well plates for 72 hours, to ensure the formation of a confluent stromal layer in each well. The cell line M2- 10B4 was chosen based on previous studies which reported their ability to replicate the protective microenvironment [150,177]. Human Burkitt’s lymphoma cells Raji or Daudi were then added to the plates at concentration Raji/Daudi:M2-10B4 5:1. After 1 hour, cells were treated with the monoclonal antibodies AME-133v (type-I mAb), rituximab (type-I mAb), tositumumab (type-II mAb) or GA101 (type-II mAb) at 10 μg/ml to test the antibody-induced PCD. Herceptin (anti-CD340/Her2 antibody) was used as a negative control. After 23 additional hours (total co-culture time of 24 hours) cells were removed from the wells and analysed by flow cytometry using the viability dyes 7-AAD and Annexin V to assess cell death induced by each antibody in the presence and in the absence of stromal cells. The gating strategy used to distinguish tumour and stromal cells in the co- culture experiments is shown below (figure 3.1).

A B

Figure 3.1. Representative image of the gating strategy used in FlowJo. A) shows tumour cells positivity for 7-AAD (X-axis) and Annexin V (Y-axis), with the respective percentages, in a non-treated control sample. B) shows tumour cells positivity for 7-AAD (X-axis) and Annexin V (Y-axis), with the respective percentages, in a GA101-treated sample in the absence of stroma. Survival is calculated as percentage of cells negative to 7-AAD and Annexin V. C) shows the gating strategy used to separate tumour cells (inside the gate) from stromal cells based on the positivity of the latter for PKH67 prior to analysis of cell viability.

73 3.1. Effect of co-culture with stromal cells on mAb-induced PCD

To investigate whether stromal cells were able to increase tumour cell survival from anti- CD20 mAb-induced PCD, the co-culture system established has been used. Tumour cells were treated with the mAb herceptin (negative control), AME-133v, rituximab, tositumumab or GA101 for 23 hours and cell viability was measured by flow cytometry. As expected, GA101 had the greatest ability to cause cell death in both Raji (figure 3.2 A) and Daudi (figure 3.2 B) cell lines (% of survival: 27.55 for Raji; 28.28 for Daudi). When B cells were co-cultured with M2-10B4, however, the levels of PCD were considerably lower. In the presence of M2-10B4, the percentage of live cells was 2-fold higher when cells were treated with GA101 (% of survival: 27.55 vs 52.85 in the presence of stromal cells, p=0.0002 for Raji; 28.28 vs 56.35 in the presence of stromal cells, p<0.0001 for Daudi), and significantly higher for tositumumab (p=0.0084 for Raji, p<0.0001 for Daudi) and rituximab (p=0.0004 for Daudi). Detailed percentages of survival and p-values are shown in figure 3.2. Of note, co-culturing tumour and stromal cells for 20 hr and treating them for 4 hr with GA101 led to a similar extent of increase in survival (data not shown). Shorter co-culture times were also investigated (i.e. 2 hr and 4 hr total co-culture); however, a protective effect was not observed in these cases, suggesting that the presence of stroma was not able to affect tumour cells’ viability during this shortened timeframe.

Once confirmed that GA101 had the greatest ability to induce PCD, the experiment was repeated using the human stromal cell line HS-5, to make sure that the same levels of protection from GA101-induced PCD were observed with different stromal cells. HS-5 was able to protect Raji and Daudi cells from GA101-induced PCD (p<0.0001), with protection only slightly less than that obtained with M2-10B4 (figure 3.3). Detailed percentages of survival and p-values are shown in figure 3.3.

To confirm that the protection observed from GA101-mediated PCD was specific to stromal cells, and not observed with any other adherent cell lines, tumour cells were cultured for 24 hours on a confluent layer of DU145 cells (human prostate cancer) and treated with GA101. In these conditions, no differences in survival percentages were observed between tumour cells cultured on plastic and tumour cells cultured on a DU145 layer, demonstrating that the protective effect observed is indeed specific to stromal cells (data not shown).

74 A R a ji, P C D

1 0 0 R a ji

8 0 * * * * + M 2 1 0 B 4 l

a * * * * v

i 6 0

u s

4 0 %

2 0

0 N T H e r A M E R T X T O S G A 1 0 1

B D a u d i, P C D

1 0 0 * * * * D a u d i

8 0 * * * * + M 2 1 0 B 4 l

a * * * * v

i 6 0

u s

4 0 %

2 0

0 N T H e r A M E R T X T O S G A 1 0 1

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 92.600 0.830 84.217 0.934 0.0231 83.267 1.428 81.817 0.442 0.9721 Her 93.783 0.843 83.217 1.307 0.0021 83.667 1.293 81.417 0.700 0.8101 AME 92.050 1.030 82.900 0.513 0.0104 81.217 1.598 81.633 0.292 >0.9999 RTX 87.283 0.958 80.683 1.188 0.1211 68.550 1.759 78.967 1.006 <0.0001 TOS 46.233 3.260 60.183 2.752 <0.0001 43.017 1.129 65.950 1.524 <0.0001 GA101 27.550 3.444 52.850 3.007 <0.0001 28.283 1.854 56.350 1.887 <0.0001

Figure 3.2. Stromal cells protect Raji (A) and Daudi (B) cells from anti-CD20 mAb-induced programmed cell death. Raji (A) or Daudi (B) cells were cultured without (green bars) or with (red bars) M2-10B4 stromal cells at concentration 5:1 respectively. After 1 hour of co-culture, cells were incubated for an additional 23 hours with either type-I anti-CD20 mAbs AME-133v (AME) or rituximab (RTX) or type-II mAbs tositumumab (TOS) or GA101 (10 μg/ml). Herceptin (Her) was used as a negative control. Cells were then removed from the wells and stained for viability with 7-AAD/Annexin V. M2-10B4 were gated out by positivity to PKH67. % of survival of Raji and Daudi cells was measured as % of PKH67- cells which were 7-AAD-/Annexin V-. Data are the mean ± SEM of 3 independent experiments. **** p<0.0001, measured by using a two-way Anova test.

A B R a ji, P C D D a u d i, P C D

1 0 0 1 0 0 R a ji D a u d i * * * *

8 0 + H S -5 8 0 * * * * + H S -5

a v

6 0 v 6 0

s s

4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 N T G A 1 0 1

RAJI + HS-5 DAUDI + HS-5

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 89.167 1.019 85.250 1.433 0.1570 81.933 0.516 78.317 0.782 0.4001 GA101 45.350 0.909 64.783 2.284 <0.0001 43.933 2.256 60.383 3.280 <0.0001

Figure 3.3. Human stromal cells HS-5 protect Raji (A) and Daudi (B) cells from GA101-induced programmed cell death. Raji (A) or Daudi (B) cells were cultured without (green bars) or with (red bars) HS-5 stromal cells. After 1 hour of co-culture, cells were incubated for an additional 23 hours with GA101 (10 μg/ml). Cells were then removed from the wells and stained for viability with 7-AAD/Annexin V. HS-5 were gated out by positivity to PKH67. % of survival of Raji and Daudi cells was measured as % of PKH67- cells which were 7-AAD-/Annexin V-. Data are the mean ± SEM of 3 independent experiments. **** p<0.0001, measured by using a two-way Anova test.

76 3.2. Role of the CXCR-4/CXCL-12 pathway in stromal-tumour cell interactions

The contact and interactions between stromal and tumour cells have previously been shown to be mediated by the CXCR-4/CXCL-12 signalling axis, with CXCR-4-expressing tumour cells migrating towards the CXCL-12 chemokine released by stromal cells. The blockade of CXCR-4 on tumour B cells inhibited the migration, thus abrogating stroma- mediated protection from type-I antibody rituximab [19,20]. Therefore, the potential role of the CXCR-4/CXCL-12 signalling axis was investigated by first confirming the expression of the receptor CXCR-4 on the tumour cell surfaces. The receptor CXCR-7 has been recently characterised as another possible player in the CXCL-12-mediated signalling pathways [178]; therefore, CXCR-7 expression levels were also measured in the study. Raji and Daudi cells were analysed for surface expression of CXCR-4 and CXCR-7 by flow cytometry. Raji cells expressed high levels of CXCR-4 (MFI: 315.2 vs 3.700 for unstained cells), while the expression was still high but slightly lower for Daudi cells (174.8 vs 4.673 for unstained cells). The levels were similar for CXCR-7 expression in Raji (51.42 vs 4.883) and in Daudi (46.37 vs 6.250) (figure 3.4).

C X C R -4 e x p r e s s io n C X C R -7 e x p r e s s io n

4 0 0 1 5 0 u n s ta in e d u n s ta in e d a n ti-C X C R -4 a n ti-C X C R -7 3 0 0

1 0 0

I F

F 2 0 0

M M

5 0 1 0 0

0 0 R a ji D a u d i R a ji D a u d i

live cells live live cells live

CXCR-4 CXCR-7

Unstained Anti-CXCR-4 Unstained Anti-CXCR-7

MFI ± SEM MFI ± SEM MFI ± SEM MFI ± SEM

Raji 3.700 0.133 315.167 6.675 4.833 0.524 51.417 6.060 Daudi 4.673 0.665 174.833 25.881 6.250 0.609 46.367 4.812

Figure 3.4. CXCR-4 and CXCR-7 are highly expressed on Raji and Daudi cell surface. A) Raji and Daudi were incubated with anti-CXCR-4-APC or anti-CXCR-7-APC antibodies (50 μg/ml), then analysed by flow cytometry. Green bars show unlabelled cells, red bars show labelled cells. Percentage of positivity to the antibodies was measured as % of APC+ cells. Data are the mean ± SEM of the MFI of 3 independent experiments. B) The image shows a representative histogram of 3 independent experiments for each condition.

78 3.2.1. CXCR-4-dependent tumour cell migration

To confirm that the interaction between CXCR-4 and its ligand CXCL-12 mediates the gradient-dependent chemotaxis of tumour cells toward the stromal cells, a transwell migration assay was performed. Firstly, CXCR-4-expressing tumour cells were exposed to various concentration of the CXCL-12 chemokine and their ability to migrate was determined. The inserts of 24-well transwell plates were coated with fibronectin (50 μg/ml); Daudi cells were then seeded on the upper compartment. Mouse SDF-1α (CXCL- 12) chemokine was added in the bottom compartment at either 30 ng/ml or 50 ng/ml and cells were incubated for either 2 or 4 hours. After 2 or 4 hours, cells that had migrated in the bottom compartments were counted. After 2 hours, cells had migrated toward the SDF- 1α concentration, with the higher levels of migration observed for the higher concentration of chemokine. Cells were almost absent in those wells where CXCL-12 was not present (3416 cells counted), but the number of cells was significantly higher in the presence of 30 ng/ml SDF-1α (11819, p=0.0357) or 50 ng/ml SDF-1α (16074, p=0.0039) (figure 3.5 A). By 4 hours migration had risen further, with few cells migrating where CXCL-12 was not present (7193), but the number of cells migrating was higher in the presence of 30 ng/ml SDF-1α (29339, p=0.0235) and even higher for 50 ng/ml SDF-1α (43477, p=0.0013) (figure 3.5 B).

79 A B D a u d i m ig ra tio n a fte r 2 h rs D a u d i m ig ra tio n a fte r 4 h rs

* * 6 0 0 0 0 6 0 0 0 0 *

* * t

n 4 0 0 0 0 t 4 0 0 0 0

u *

e e

C 2 0 0 0 0

0 0 n o S D F -1  3 0 n g /m l 5 0 n g /m l n o S D F -1  3 0 n g /m l 5 0 n g /m l

S D F -1  S D F -1 

Daudi no SDF-1α 30 ng/ml 50 ng/ml migration Mean ± SEM Mean ± SEM p-value vs Mean ± SEM p-value vs after no SDF-1α no SDF-1α 2 hrs 3416 1028 11819 2487 0.0357 16074 2456 0.0235 4 hrs 7193 1240 29339 6849 0.0039 43477 5362 0.0013

Figure 3.5. Daudi cells migrate through a transwell membrane in the presence of SDF-1α. Daudi cells were plated in the upper compartment of 24-well transwell plates and exposed to different concentrations of the chemokine SDF-1α (added in the bottom compartment). After either 2 (A) or 4 (B) hours, cells in the bottom compartment were collected and counted by flow cytometry, after the addition of counting beads. Data are the mean ± SEM of 4 replicates in 1 independent experiment. * p<0.05, ** p<0.01, measured by using one-way Anova.

80 To investigate whether the CXCR-4 antagonist plerixafor was able to block CXCL-12- mediated migration, the assay was repeated and plerixafor was used to pre-treat the tumour cells at 10 μM. Daudi cells were pre-treated with plerixafor for 30 minutes and then plated into the upper compartment and the chemokine SDF-1α was added into the wells at 50 ng/ml. After 4 hours, cells were counted by flow cytometry. As expected, cells were not detected in those wells where CXCL-12 was not added (number of cells: 885.1), but the number of non-treated cells was higher in the presence of the chemokine (7924, p<0.0001). However, when the cells were pre-treated with plerixafor, only a low number of cells migrated in the bottom compartment (2551, p<0.0001 vs migration in the presence of 50 ng/ml chemokine), confirming that plerixafor is able to block B-cell chemotaxis toward CXCL-12 and that such migration is CXCR-4 dependent (figure 3.6). These results are in agreement with previously published studies [168].

81 D a u d i m ig ra tio n a fte r 4 h rs

1 5 0 0 0 * * * * * * * *

t 1 0 0 0 0

n n s

l e

C 5 0 0 0

0 n o S D F -1  S D F -1  P X F S D F -1  + P X F 5 0 n g /m l

Daudi migration after 4 hrs

Mean ± SEM p-value vs no p-value vs p-value vs SDF-1α SDF-1α 50 PXF ng/ml

no SDF-1α 885.1 88.56 - <0.0001 0.9995

SDF-1α 7924 799.1 <0.0001 - <0.0001 50 ng/ml

PXF 810.3 283.5 0.9995 <0.0001 -

SDF-1α + PXF 2551 420.8 0.1146 <0.0001 0.0954

Figure 3.6. Daudi cells migration in response to SDF-1α is inhibited by the CXCR-4 antagonist plerixafor. Daudi cells were pre-treated with plerixafor (10 μM, “PXF”) for 30 minutes, then plated in the upper compartment of 24-well transwell plates and exposed to 50 ng/ml SDF-1α (added in the bottom compartment). After 4 hours, cells in the bottom compartment were collected and counted by flow cytometry, after the addition of counting beads. Data are the mean ± SEM of 4 replicates in 1 independent experiment. * p<0.05, ** p<0.01, *** p<0.001, ns=non-significant, measured by using one-way Anova.

82 To confirm that tumour cell migration also occurred in the presence of stromal cells expressing the chemokine CXCL-12, M2-10B4 (2.5x104 cells/well) were seeded onto the bottom compartment of 24-well transwell plates for 72 hours. Inserts were coated with fibronectin (50 μg/ml); then, Raji and Daudi (1.25x105 cells/well) were added on the top compartment after being pre-treated with plerixafor (10 μM). After 4 hours, cells that had migrated towards the bottom chamber were counted by flow cytometry. Tumour cells were able to migrate towards the stromal layer (12640 migrated Raji cells in non-treated group vs 34121 on stroma, p<0.0001; 13270 migrated Daudi cells in non-treated group vs 25115 on stroma, p=0.0012). This was in agreement with what had been previously observed in [15]. Furthermore, pre-treatment with plerixafor blocked such an effect, demonstrating that migration is dependent on the CXCR-4 receptor on tumour cell surface (34121 on stroma vs 6059 on stroma after addition of plerixafor, p<0.0001, for Raji; 25115 on stroma vs 5963 on stroma after addition of plerixafor, p<0.0001, for Daudi) (figure 3.7). To confirm that migration was dependent on the CXCL-12 chemokine, rather than any other cytokines that could have been bound by CXCR-4, the experiment was repeated, this time treating the stromal cells with the anti-CXCL-12 antibody. Again, treatment with the anti-CXCL- 12 antibody blocked tumour cell migration towards the stromal layer (21414 on stroma vs 6615 on stroma after addition of anti-CXCL-12 antibody, p=0.0035, for Raji; 31528 on stroma vs 6606 on stroma after addition of anti-CXCL-12 antibody, p=0.0063, for Daudi) (figure 3.8). This confirmed that stromal cells express the chemokine CXCL-12, and tumour cell migration towards the stromal cells is dependent on the CXCR-4/CXCL-12 axis.

83 A R a ji m ig ra tio n - 4 h rs B D a u d i m ig ra tio n - 4 h rs

* * * * * * * * * * * * * * * * 4 0 0 0 0 4 0 0 0 0

3 0 0 0 0 3 0 0 0 0

o o

c 2 0 0 0 0

C C 1 0 0 0 0 1 0 0 0 0

0 0 N T P X F + M 2 1 0 B 4 P X F + M 2 1 0 B 4 N T P X F + M 2 1 0 B 4 P X F + M 2 1 0 B 4

Raji migration after 4 hrs Daudi migration after 4 hrs

± p-value p-value p-value vs ± p-value p-value p-value vs Mean SEM vs NT vs PXF + M210B4 Mean SEM vs NT vs PXF + M210B4

NT 12640 1402 - 0.6437 <0.0001 13270 780 - 0.9880 0.0012

PXF 10523 1239 0.6437 - <0.0001 13881 980.6 0.9880 - 0.0017

+ 34121 1253 <0.0001 <0.0001 - 25115 1172 0.0012 0.0017 - M210B4 PXF + 6059 1073 0.0240 0.1288 <0.0001 5963 2099 0.0215 0.0140 <0.0001 M210B4

Figure 3.7. Tumour cell migration toward a layer of stromal cells is blocked upon CXCR-4 inhibition. Raji (A) and Daudi (B) were pre-treated with plerixafor (10 μM, “P10”) for 30 minutes, then plated in the upper compartment of 24-well transwell plates with or without M2-10B4 stromal cells (in the bottom compartment). After 4 hours, cells in the bottom compartment were collected and counted by flow cytometry, after the addition of counting beads. Data are the mean ± SEM of 4 replicates in 1 independent experiment. * p<0.05, ** p<0.01, **** p<0.0001, measured by using one-way Anova.

84 A R a ji m ig ra tio n - 4 h s B D a u d i m ig ra tio n - 4 h s

n s n s * 5 0 0 0 0 * * * * 5 0 0 0 0 * *

4 0 0 0 0 4 0 0 0 0

t n

3 0 0 0 0 n 3 0 0 0 0

l l 2 0 0 0 0 l

e 2 0 0 0 0

C C

1 0 0 0 0 1 0 0 0 0

0 0 N T a n ti-S D F + M 2 1 0 B 4 a n tiS D F N T a n ti-S D F + M 2 1 0 B 4 a n tiS D F + M 2 1 0 B 4 + M 2 1 0 B 4

Raji migration after 4 hrs Daudi migration after 4 hrs

± p-value p-value p-value vs ± p-value p-value p-value vs Mean SEM vs NT vs anti- + M210B4 Mean SEM vs NT vs anti- + SDF SDF M210B4

NT 6350 840.7 - 0.9951 0.0032 12753 2054 - 0.9969 0.0295

Anti-SDF 5684 2271 0.9951 - 0.0024 13800 2280 0.9969 - 0.0389

+ M210B4 21414 2295 0.0032 0.0024 - 31528 6723 0.0295 0.0389 -

Anti-SDF 6615 2221 0.9997 0.9868 0.0035 6606 603.2 0.6592 0.5480 0.0063 + M210B4

Figure 3.8. Tumour cell migration toward a layer of stromal cells is blocked upon CXCL-12 inhibition. Raji (A) and Daudi (B) were plated in the upper compartment of 24-well transwell plates with or without M2-10B4 stromal cells, and anti-CXCL-12 antibody was added at 10 μg/ml in the bottom compartment. After 4 hours, cells in the bottom compartment were collected and counted by flow cytometry, after the addition of counting beads. Data are the mean ± SEM of 4 replicates in 1 independent experiment. * p<0.05, ** p<0.01, ns=non-significant, measured by using one-way Anova.

85 3.2.2. Effects of CXCR-4/CXCL-12 blockade on GA101-induced cell death

Given the dependence of tumour cell migration towards M2-10B4 on the CXCR-4/CXCL- 12 signalling axis, it was postulated that this pathway could have a role in the stroma- mediated protection of Raji and Daudi from GA101-induced PCD. To test this hypothesis that stroma mediated tumour cell protection via CXCR-4, the established co-culture system of stromal and tumour cells was used. Before being seeded onto the plates, tumour cells were pre-treated with the CXCR-4 antagonist plerixafor (AMD-3100) at 10 μM for 30 minutes and then added to the stromal layer. After 1 hour of co-culture with M2-10B4, cells were incubated with GA101 for 23 additional hours. Cells were then analysed by flow cytometry to determine whether the blockade of CXCR-4 had enhanced GA101 efficacy in the presence of stromal cells. The stromal cells were able to prevent GA101-induced killing in cells that were not pre-treated with plerixafor (survival: 54.37 vs 40.18 in the absence of stroma, p<0.0001 in Raji; 54.73 vs 35.87 in the absence of stroma, p=0.001 in Daudi). However, the combination of plerixafor with GA101 did not alter the level of protection when tumour cells were co-cultured with stromal cells (survival %: 56.77 vs 43.37 in the absence of stroma, p=0.0004 in Raji; 53.27 vs 36.72 in the absence of stroma, p=0.0044 in Daudi) (figure 3.9).

86 R a ji, P C D D a u d i, P C D

1 0 0 1 0 0 R a ji D a u d i

8 0 + M 2 1 0 B 4 8 0 + M 2 1 0 B 4

**** *** ** **

l a 6 0 a

v 6 0

u s 4 0 s

4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 P X F P X F + G A 1 0 1 N T G A 1 0 1 P X F P X F + G A 1 0 1

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 91.383 0.513 85.983 1.919 0.1766 79.133 1.960 76.767 2.093 0.9787 GA101 40.183 1.737 54.367 2.454 <0.0001 35.867 3.185 54.733 3.441 0.0010 PXF 91.933 0.709 84.050 2.826 0.0191 77.283 3.281 75.867 2.097 0.9969 PXF+GA101 45.367 1.364 56.767 2.131 0.0004 36.717 4.965 53.267 4.308 0.0044

Figure 3.9. Blockade of CXCR-4 on cell surface does not abrogate stromal-mediated protection from GA101-induced PCD. Tumour cells were pre-treated with plerixafor for 30 minutes at 10 μM, then seeded onto the stromal layer for 24 hours. GA101 (10 μg/ml) was added after 1 hour of co-culture. Viability was measured by flow cytometry. % of survival was measured as % of PKH67- cells which were 7-AAD- /Annexin V-. Data are the mean ± SEM of two duplicates in 3 independent experiments. ** p<0.01, *** p<0.001, **** p<0.0001, measured by using a two-way Anova test.

87 A previous report had demonstrated that the cell surface receptor CXCR-7 was able to interact with CXCL-12 (SDF-1α), and its functions in the signalling pathway were enhanced in cells with CXCR-4 deletion [179]. Therefore, the co-inhibition of CXCR-4 and CXCR-7 was investigated as a means to abrogate stromal-mediated protection. The experiment was repeated using, instead of plerixafor, an anti-SDF-1α antibody, in order to inhibit any interactions between CXCL-12 and CXCR-4/-7. Despite the combined blockade of CXCR-4/CXCL-12 and CXCR-7/CXCL-12 interactions, Raji cell survival was not decreased when cells were treated with GA101 and anti-CXCL-12 antibody in the presence of stromal cells (49.42% vs 19.67% without stroma, p<0.0001), compared to the treatment with GA101 only (49.5% vs 24.17% without stroma, p<0.0001). In the same way, Daudi cell survival was not significantly decreased when cells were treated with the anti-CXCL-12 antibody (50.37% in combination, with stroma, vs 22.8% without stroma, p<0.0001) (figure 3.10).

88 R a ji, P C D D a u d i, P C D

R a ji D a u d i 1 0 0 1 0 0 + M 2 1 0 B 4 + M 2 1 0 B 4

8 0 8 0 l

l * * * * a

* * * * * * * * a * * * *

v i

6 0 i 6 0

4 0 4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 a n ti-S D F a n ti-S D F N T G A 1 0 1 a n ti-S D F a n ti-S D F + G A 1 0 1 + G A 1 0 1

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 93.375 0.904 84.275 0.649 0.0314 88.100 2.249 84.050 1.477 0.3829 GA101 24.175 3.066 49.500 3.014 <0.0001 24.725 1.698 55.775 2.255 <0.0001 Anti-SDF 93.725 0.375 84.375 0.333 0.0261 88.325 1.441 84.100 1.496 0.3429 + GA101 19.675 2.779 49.425 3.437 <0.0001 22.800 1.064 50.375 1.920 <0.0001

Figure 3.10. Blockade of CXCL-12/CXCR-4 and CXCL-12/CXCR-7 interactions does not abrogate stromal-mediated protection from GA101-induced PCD. M2-10B4 cells were treated with anti-SDF-1α antibody (10 μg/ml), then Raji and Daudi cells were seeded onto the stromal layer for 24 hours. After 1 hour of co-culture, cells were treated with GA101 (10 μg/ml), either alone or in combination with anti-CXCL-12 antibody (“+ GA101”). Viability was measured by flow cytometry. % of survival was measured as % of PKH67- cells which were 7-AAD-/AnnV-. Data are the mean ± SEM of two replicates in 2 independent experiments. **** p<0.0001, measured by using a two-way Anova test.

89 3.3. Role of stromal cell contact versus stroma-released soluble factors

Given that the CXCR4/CXCL-12 pathway did not appear to be involved in the stromal cell protection from anti-CD20 mAb induced PCD, it was next investigated whether protection was stroma-tumour cell contact-dependent or independent.

3.3.1. GA101-induced PCD in the presence of medium conditioned by stromal cells.

To further characterise the mechanisms by which stromal cells mediate protection of tumour cells from anti-CD20 mAb induced PCD, medium conditioned by stromal cells was collected and used to culture Raji and Daudi cells. Conditioned medium (CM) was collected in two different ways: in the first method, stromal cells were plated for 72 hours in 24-well plates to allow the formation of a confluent layer, then cells were washed and medium was replaced by fresh RPMI; after 24 hours, medium was collected, filtered and used to seed tumour cells. Tumour cells were seeded in CM for 24 hours (figure 3.11). After one hour from the beginning of culture in CM, cells were treated with GA101, because it was previously shown to be the most effective mAb at inducing PCD (see 3.1). Cells were removed from the wells and cell viability was measured by flow cytometry. Interestingly, stromal-CM was not able to protect Raji and Daudi cells from GA101- induced cell death (p=0.99, Raji; p=0.18, Daudi). In the second method, stromal cells were plated for 72 hours, then tumour cells were added for a further 24 hours; the medium (conditioned by both tumour and stromal cells) was then collected, filtered and added to tumour cells in the presence of GA101 for 24 hours (figure 3.12). This was done because tumour and stromal cells can influence each other’s behaviour, and the presence of tumour cells in the micro-environment might lead to the release of soluble factors which would in turn protect tumour cells from killing. Again, tumour/stromal-CM was not able to protect Raji and Daudi cells from GA101-induced cell death. There was no significant difference between the levels of cell death of tumour cells cultured in RPMI and in CM (p=0.87, Raji; >0.99, Daudi).

These studies suggested that the presence of (and the contact with) stromal cells is required for the achievement of protection of tumour cells from GA101-induced PCD.

90 R a ji, P C D D a u d i, P C D

1 0 0 1 0 0 R P M I R P M I

8 0 + S -C M 8 0 + S -C M

l a

n s a n s v

6 0 v 6 0

s s

4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 N T G A 1 0 1

Raji, RPMI Raji + S-CM Daudi, RPMI Daudi + S-CM

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 93.750 0.537 94.750 0.673 0.9091 87.550 0.689 87.817 0.568 0.9900 GA101 39.350 2.436 39.483 2.505 0.9983 37.517 1.775 41.167 2.188 0.1830

Figure 3.11. Stromal cell-CM does not protect Raji and Daudi cells from GA101-induced PCD. Raji and Daudi cells were seeded for 24 hours in either RPMI (green bars) or stromal cell-conditioned medium (S-CM, red bars). After 1 hour, GA101 (10 μg/ml) was added. Viability was measured by flow cytometry, staining tumour cells with 7-AAD/AnnexinV. % of survival was measured as % of 7-AAD-/Annexin V- cells. Data are the mean ± SEM of two duplicates in 3 independent experiments. ns = non-significant, measured by using a two-way Anova test.

91 R a ji, P C D D a u d i, P C D

1 0 0 1 0 0 R P M I R P M I

8 0 + T /S -C M 8 0 + T /S -C M l

l n s a

n s a

6 0 6 0

u s 4 0 s

4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 N T G A 1 0 1

Raji, RPMI Raji + T/S-CM Daudi, RPMI Daudi + T/S-CM

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 87.000 2.454 86.500 3.492 0.9971 77.300 3.481 81.600 3.093 0.7892 GA101 33.217 5.939 36.633 7.357 0.8750 37.533 6.048 37.617 6.104 >0.9999

Figure 3.12. Tumour/stromal cell-CM does not protect Raji and Daudi cells from GA101-induced PCD. Stromal cells were cultured for 72 hours before adding Raji and Daudi cells, and the co-culture system was maintained for 24 additional hours. After 24 hours, either RPMI (green bars) or media conditioned by both tumour and stromal cells (TS-CM) (red bars) were used to seed new Raji and Daudi cells. After 1 hour tumour cells were treated with GA101 (10 μg/ml) and cultured for 24 hours. Viability was measured by flow cytometry, staining tumour cells with 7-AAD/Annexin V. % of survival was measured as % of 7-AAD- /Annexin V- cells. Data are the mean ± SEM of two duplicates in 3 independent experiments. ns = non- significant, measured by using a two-way Anova test.

92 3.3.2. Non-contact co-culture of stromal cells and tumour cells

To determine whether the stroma-mediated protection of B-cell lymphoma cells was dependent on the contact of tumour cells with the stromal cells, transwell plates with inserts of 0.4 μm pore size were used. This specific size was chosen to allow the passage of soluble factors and cytokines, but keep the tumour cells physically separated from the stroma. The established co-culture system previously shown was used. After 24 hours of non-contact co-culture, tumour cells in the upper compartment were collected and analysed by flow cytometry. At the same time, a control assay was set up, where the same number of cells was seeded in normal 24-well plates, allowing the contact between stromal and tumour cells. This was analysed and used as a positive control for the stroma-mediated protection (figure 3.13). No difference in survival was seen between Raji cells treated with GA101 alone versus cells that were separated from stromal cells via a 0.4 μm membrane (43.48% vs 40.6%, p=0.5684, figure 3.13 A, upper panel). A similar pattern was seen with Daudi cells (44.15% for cells alone vs 42.2% for cells separated from stromal cells via a 0.4 μm membrane, p=0.8329, figure 3.13 B, upper panel). As seen previously, stromal cells in contact with Raji and Daudi cells protected from mAb-induced PCD (figure 3.13, bottom panel).

93 A B

R a ji, P C D D a u d i, P C D

1 0 0 1 0 0 R a ji D a u d i

8 0 + M 2 1 0 B 4 8 0 + M 2 1 0 B 4

l a

a n s n s

v i

N o n -c o n ta c t i 6 0 6 0

v r

tra n s w e ll r

a s s a y 4 0 4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 N T G A 1 0 1

1 0 0 1 0 0 R a ji D a u d i * * * * * * * *

8 0 + M 2 1 0 B 4 8 0 + M 2 1 0 B 4

v i

D ire c t 6 0 i 6 0

r u

c o n ta c t u

4 0 4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 N T G A 1 0 1

Raji + M2-10B4 Daudi + M2-10B4 Non-contact assay Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 89.467 1.960 91.517 1.457 0.7478 79.650 3.171 80.733 2.853 0.9447 GA101 43.483 2.814 40.600 1.936 0.5684 44.150 1.637 42.200 2.159 0.8329

Raji + M2-10B4 Daudi + M2-10B4 Direct contact Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 90.383 1.734 82.250 1.348 0.0079 80.367 2.878 74.400 2.426 0.2306 GA101 37.950 2.627 68.583 0.862 <0.0001 38.117 2.990 64.250 2.080 <0.0001

Figure 3.13. Stromal cells protection of tumour cells is lost in non-contact conditions. Stromal cells were cultured for 72 hours at the bottom of 24-well transwell plates before adding Raji (A) and Daudi (B) cells in the upper compartment, to impede the contact between the two cell lines. GA101 (10 μg/ml) was added after 1 hour of non-contact co-culture to the upper compartments. The non-contact co-culture system was maintained for 23 additional hours. Viability was measured by flow cytometry, staining tumour cells with 7- AAD/Annexin V. % of survival was measured as % of 7-AAD-/Annexin V- cells. Data are the mean ± SEM of two duplicates in 3 independent experiments. ns=non-significant, **** p<0.0001, measured by using a two-way Anova test.

94 3.4. Role of the VLA-4/VCAM-1 pathway in stromal-tumour cell interactions

The integrin VLA-4 (CD49d) had been shown to be one of the most commonly expressed receptors on tumour cell surfaces, and to have a crucial role in modulating stromal-cell adhesion and signalling pathways [150]. Given the contact-dependent nature of stromal- dependent protection of B-cell lymphoma cells from anti-CD20 mAbs, it was postulated that the interaction between VLA-4 and its ligand VCAM-1 could mediate this protection. To determine whether Raji and Daudi cell lines express the integrin VLA-4 on the cell surface, tumour cells were stained with anti-VLA-4 antibody. Cells were then analysed by flow cytometry. Raji cells expressed high levels of VLA-4 on their surfaces (291.5 vs 2.205 for unlabelled cells). A similar level of expression was observed for Daudi (264.5 vs 1.440 for unlabelled cells). This suggested that the VLA-4/VCAM-1 signalling pathway could potentially have a role in the contact between stromal-tumour cell and in the stromal- mediated tumour cell protection. (figure 3.14).

V L A 4 e x p re s s io n

4 0 0 u n s ta in e d a n ti-V L A -4 3 0 0

F 2 0 0 M

1 0 0

0 R a ji D a u d i

Unstained Anti-VLA-4

MFI ± SEM MFI ± SEM

Raji 2.205 0.4150 291.5 5.500 Daudi 1.440 0.0500 264.5 2.500

Figure 3.14. VLA-4 is expressed on Raji and Daudi cell surface. A) Raji and Daudi were labelled with anti-VLA-4-PE antibody (50 μg/ml.), then analysed by flow cytometry. Data are the mean ± SEM of the MFI of two duplicates. B) shows a representative histogram of three samples for each condition. The X-axis shows the expression of VLA-4 on the cells, the Y-axis shows the number of live cells.

96 3.4.1. Effects of VLA-4 blockade on GA101-induced cell death

The integrin VLA-4 had been shown to be involved in mediating the contact between tumour and stromal cells [180], and its blockade, both on its own or combined with treatment with CXCR-4 antagonists, led to the abrogation of resistance to chemotherapeutic agents in mantle cell lymphoma [164]. To understand whether its inhibition could prevent the stromal-mediated protection of B-cell lymphoma cells from GA101-induced PCD, Raji and Daudi cells were pre-treated with an anti-VLA-4 neutralising antibody for 30 minutes. After 30 minutes, cells were seeded onto the established co-culture system for 24 hours with GA101 added after 1 hour as appropriate. Cells were then analysed by flow cytometry. In Raji cells, the addition of the anti-VLA-4 neutralising antibody did not increase GA101 efficacy, which remained impaired in the presence of stromal cells (% of survival: 45.75 for GA101 plus stromal cells vs 39.28 with VLA-4 blockade, p=0.99, figure 3.15 A). The same results were obtained with Daudi cells, with no statistical significance observed between cells treated with GA101 alone and cells treated with GA101+anti-VLA-4 antibody in the presence of stroma (47.08 vs 43.28, p>0.99, figure 3.15 B).

97 R a ji, P C D D a u d i, P C D

R a ji D a u d i 1 0 0 1 0 0 + M 2 -1 0 B 4 + M 2 -1 0 B 4

8 0 8 0 * * *

l * * *

l * * * a 6 0 * * a

v 6 0

u s 4 0 s

4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 a n ti-V L A 4 a n ti-V L A 4 N T G A 1 0 1 a n ti-V L A 4 a n ti-V L A 4 + G A 1 0 1 + G A 1 0 1

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 84.780 2.193 75.700 2.166 0.3413 75.900 2.941 71.950 1.512 0.8587 GA101 22.460 4.838 45.750 3.879 0.0002 28.367 2.220 47.083 3.786 0.0007 Anti-VLA4 80.850 3.531 78.567 3.249 0.9864 83.450 1.783 71.817 2.139 0.0540 + GA101 20.367 3.876 39.283 4.244 0.0027 23.700 3.179 43.283 5.788 0.0004

Figure 3.15. Blockade of VLA-4/VCAM-1 interactions does not impair stromal-mediated protection from GA101-induced PCD. Raji (A) and Daudi (B) cells were pre-treated with anti-VLA-4 antibody (10 μg/ml) for 30 minutes, then seeded onto the stromal layer for 24 hours. After 1 hour of co-culture, cells were treated with GA101 (10 μg/ml), either alone or in combination with anti-VLA-4 antibody (“+ GA101”). Viability was measured by flow cytometry. % of survival was measured as % of PKH67- cells which were 7- AAD-/Annexin V-. Data are the mean ± SEM of two duplicates in 3 independent experiment. ** p <0.01, *** p<0.001, measured using a two-way Anova test.

98 3.5. Effect of mAb pre-treatment on stroma-mediated protection

GA101 induces PCD by provoking actin reorganisation and cellular aggregation, termed homotypic adhesion, followed by lysosomal-membrane permeabilization, release of ROS and cell death [87]. After treatment of tumour cells with GA101, homotypic adhesion was visible under the microscope as both Raji and Daudi formed clumps. However, this phenomenon was not observed in the presence of stromal cells, suggesting that the mechanism is somehow prevented. Conversely, cells appeared attached to the stromal layer (shown in figures 3.16 B and 3.17 B, middle panels).

To better understand the effect that co-culture with stromal cells has on the ability of GA101 to execute homotypic adhesion, it was asked whether the presence of the stromal layer could not only prevent, but reverse the process of cellular aggregation started upon GA101 treatment. To address this question, tumour cells were pre-treated with GA101 for 2 hours in tubes, to ensure that the homotypic adhesion process had started before tumour cells entered in contact with the stroma. After 2 hours, cells were carefully poured onto the stromal layer, avoiding the destruction of cellular aggregates. To confirm that pre- treatment with GA101 had led to homotypic adhesion before the pouring of tumour cells onto the stromal layer, cells were analysed under a low-light Zeiss microscope at timepoints 0 hrs (immediately after pouring treated cells onto the stromal layer, figures 3.16 A and 3.17 A) and 24 hrs (after 24 hours from the beginning of the co-culture, figures 3.16 B and 3.17 B). It was clear that during the 24 hours of co-culture the tumour cells that had undergone homotypic adhesion prior to pouring onto stromal cells appeared to reverse this process and spread out onto the stromal layer (figure 3.16 B and 3.17 B, bottom panel), suggesting an active remodelling of the tumour cells’ actin cytoskeleton in the presence of stroma.

99 NT 2-hour pre-treatment

Tumour cells alone

100 μm

+ stromal stromal cells +

Tumour

B NT GA101 2-hour pre-treatment

Tumour cells alone

+ stromal stromal cells +

Tumour

Figure 3.16. Representative images of Raji cells pre-treated with GA101 and then poured onto stroma. Raji cells were treated with GA101 (10 μg/ml) for 2 hours in tubes, then poured on top of the stromal layer. Raji cells (A, B) were imaged (low-light Zeiss microscope, 10x) immediately after the pouring (0 hr time- point, A; upper panel: on plastic; bottom panel: on stroma) or 24 hours after pouring (24 hr time-point, B; upper panel: on plastic; bottom panel: on stroma). Whilst cells on plastic were still clumped after 24 hours from pouring, in the presence of stroma Raji cells seemed spread out and detached from each other. Images are representative of two duplicates in 3 independent experiments.

100

A NT 2-hour pre-treatment

Tumour cells alone Tumour cells 100 μm

+ stromal stromal cells +

Tumour

B NT GA101 2-hour pre-treatment

Tumour cells alone

+ stromal stromal cells + Tumour

Figure 3.17. Representative images of Daudi cells pre-treated with GA101 and then poured onto stroma. Daudi cells were treated with GA101 (10 μg/ml) for 2 hours in tubes, then poured on top of the stromal layer. Daudi cells (A, B) were imaged (low-light Zeiss microscope, 10x) immediately after the pouring (0 hr time-point, A; upper panel: on plastic; bottom panel: on stroma) or 24 hours after pouring (24 hr time-point, B; upper panel: on plastic; bottom panel: on stroma). Whilst cells on plastic were still clumped after 24 hours from pouring, in the presence of stroma Daudi cells seemed spread out and detached from each other. Images are representative of two duplicates in 3 independent experiments.

101 To further characterise the ability of stromal cells to reverse GA101-induced homotypic adhesion, a time-lapse experiment was performed. Stromal cells were cultured in 35-mm poly-D-lysine-coated culture dishes until confluency. Raji cells were pre-treated for 2 hours with GA101 (10 µg/ml) in 1.5 ml tubes and, once cell aggregates had formed, poured onto the stromal layer. The dishes were then imaged for 24 hours under a low-light Zeiss microscope. Images were taken every 30 minutes for each different field of view, which was centred on a B-cell aggregate, in order to observe the conformational changes in the presence of stromal cells.

Figure 3.18 and figure 3.19 show stills from the time-lapse after 0.5 hr (A), 3 hr (B), 6 hr (C), 9 hr (D), 12 hr (E), 15 hr (F), 18 hr (G), 21 hr (H) and 24 hr (I) from the pouring onto dishes. Whilst, at 0.5 hr (3.18 A and 3.19 A), a B-cell aggregate is clearly visible in the images, at the 12-hour time-point (3.18 E and 3.19 E) the aggregate starts separating, and appears spread out on the underlying stromal layer by 24 hours (3.18 I and 3.19 I).

102

A – 30’ B – 3 hr C – 6 hr

D – 9 hr E – 12 hr F – 15 hr

G – 18 hr H – 21 hr I – 24 hr

Figure 3.18. Representative images of Raji-GFP-actin cells pre-treated with GA101 and then poured onto stroma. Raji-GFP-actin cells were treated with GA101 (10 μg/ml) for 2 hours in 1.5 ml tubes, then poured on top of the stromal layer, which had been grown on a 35-mm glass-bottom culture dish. Cells were imaged (low-light Zeiss microscope, 20x) for a period of 24 hours, and pictures were taken every 30 minutes starting at 30’ after the pouring. The figure shows stills taken approximately every three hours. Images are representative of a field of view out of 5 fields taken in 2 independent experiments.

103

100 μm A – 30’ B – 3 hr C – 6 hr

D – 9 hr E – 12 hr F – 15 hr

G – 18 hr H – 21 hr I – 24 hr

Figure 3.19. Representative images of Raji cells pre-treated with GA101 and then poured onto stroma. Raji cells were treated with GA101 (10 μg/ml) for 2 hours in 1.5 ml tubes, then poured on top of the stromal layer, which had been grown on a flat-bottom 24-well plate. Cells were imaged (low-light Zeiss microscope, 10x) for a period of 24 hours, and pictures were taken every 30 minutes starting at 30’ after the pouring. The figure shows stills taken approximately every three hours. Images are representative of a field of view out of 3 fields taken in 2 independent experiments.

104 To further characterise this process in the presence of stroma, Raji and Daudi cells were pre-incubated with GA101 for 2 hours, before being seeded onto the 24-well plates. The seeding was performed by carefully pouring the cells from tubes into the plates, to ensure that the clumps provoked by GA101 activity were formed before the cells entered in contact with the stroma and were not destroyed when pouring the cells. Cells were then analysed by flow cytometry after 24 hours from the seeding. Interestingly, there was not a significant difference in terms of stromal-mediated protection between cells pre-treated with GA101 and then poured into the plates and the cells treated with GA101 after 1 hour from the beginning of the co-culture time as described previously. In Raji cells (A), the percentage of survival in the presence of stromal cells when cells were normally treated with GA101 was 2-fold higher than in the absence of stromal cells, and the same level of protection was observed with 2 hours of pre-incubation with GA101 (% of survival in the presence of stroma: 39.71 for GA101 vs 48.35 for pre-incubation, p=0.6352) (figure 3.20 A).

In Daudi cells (B), the percentage of survival in the presence of stromal cells when cells were normally treated with GA101 was 59.87%, and such protection only slightly decreased for the 2 hour pre-incubation (54.89%) – but was still statistically significant compared to the survival in the absence of stromal cells (54.89% with stroma vs 39.53%, p=0.0005) (figure 3.20 B).

This experiment suggested that contact with stroma is able to reverse the homotypic adhesion process, therefore blocking PCD and increasing tumour cells survival after GA101 treatment.

105 A R a ji, P C D B D a u d i, P C D

D a u d i 1 0 0 R a ji 1 0 0

+ M 2 1 0 B 4 + M 2 1 0 B 4 8 0 8 0 * * * * * * *

l * * *

l a

6 0 a

v 6 0 v

i * * *

u u

s 4 0 s

4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 2 h r s N T G A 1 0 1 2 h r s

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 90.233 2.190 79.167 2.411 0.0591 82.183 2.307 77.817 1.593 0.5431 GA101 20.917 2.919 39.717 1.400 0.0007 30.267 2.340 59.867 3.408 <0.0001 2 hrs 27.950 1.661 48.350 6.091 0.0003 39.533 2.532 54.883 2.590 0.0005

Figure 3.20. Pre-incubation of tumour cells with GA101 followed by pouring cells onto the stromal layer does not reduce stroma-mediated protection from PCD. Raji (A) or Daudi (B) cells were pre- incubated with GA101 (10 μg/ml) for 2 hours, and then poured from tubes into the plates. Cells that entered in contact with stromal cells after being pre-treated and poured (“2 hrs”) did not show a reduced protection from GA101-induced PCD in the presence of stromal cells (red bars). Viability was measured by flow cytometry, staining tumour cells with 7-AAD/Annexin V. % of survival was measured as % of PKH67- cells which were 7-AAD-/Annexin V-. Data are the mean ± SEM of two duplicates in 3 independent experiment. *** p<0.001, **** p<0.0001, measured by using a two-way Anova test.

106 3.6. Effect of co-culture on tumour cell death after removal of contact

To understand whether the increased survival conferred by direct contact with stromal cells lasted even after removal of contact, or conversely whether protection was lost upon detachment of tumour cells from stroma, a fluorescence-based sorting was performed. Raji and Daudi cells were co-cultured for 24 hours with M2-10B4 cells, which had been stained with a membrane-labelling dye. Cells were then harvested from the plates and sorted by using a BD FACSAria II or BD Influx cell sorter. Once the two cell populations were separated, tumour cells (purity = virtually 100%, representative plots in figure 3.21) were collected and plated on plastic for various time-points (either 0, 4, 16 or 20 hours) before being treated for 4 hours with GA101, as shown in the diagram below:

After the 4-hour treatment, cell survival was analysed by flow cytometry. Interestingly, Raji and Daudi cell survival seemed to be greater for cells that had been previously in contact with stromal cells (“M210B4-conditioned”) than for cells that had been only cultured on plastic (figure 3.22). This was especially true for the shorter time-points post sorting, with significant differences after treatment with GA101 between cells cultured on plastic and M2-10B4-conditioned cells (0 hr + 4 hr GA101: p<0.0001 for Raji, p=0.0002 for Daudi; 4 hr + 4 hr GA101: p=0.0051 for Raji). Protective effect tended to lessen as time post stromal cell contact increased, with p=0.0131 for Raji and p=0.0129 for Daudi at 16 hr + 4 hr GA101 time-point, p>0.05 for both cell lines at 20 hr + 4 hr GA101 time- point.

These results suggested that protection from GA101-induced PCD conferred by stromal cells lasts even after removal of contact but gradually disappear with time. Such an effect might mean that there are specific signalling pathways initiated upon contact with stroma, which once activated mediate increased survival for a prolonged time before these pathways are switched off.

107

Figure 3.21. Representative histograms showing absence of contaminating stromal cells. M2-10B4 stromal cells were stained with the membrane-labelling dye PHK67 and plated until confluency. Raji tumour cells were cultured either on plastic or on stroma for 24 hours. Cell populations were then separated by FACS sorting and tumour cells only (PKH67-) were collected and used for further analyses. An aliquot from a sample containing PKH67- Raji cells was taken and analysed by flow cytometry to determine the purity of the sort and exclude contamination by M2-10B4 (PKH67+). Left panels show PKH67+ (A) and PKH67- (B) populations in the sample before FACS sorting, right panels show the remaining Raji cells (PKH67-) after separation from M2-10B4 through FACS sorting (C: PKH67+ population after FACS sorting; D: PKH67- population after FACS sorting).

108 A R a ji

1 0 0 R a ji **** ** 8 0 M 2 1 0 B 4 -

l c o n d itio n e d

a n s v

i 6 0

v *

u s

4 0 %

2 0

0 N T 0 h r 4 h r 1 6 h r 2 0 h r p o s t-s o rt p o s t-s o rt p o s t-s o rt p o s t-s o rt

+ 4 h r G A 1 0 1 tre a tm e n t

B D a u d i

1 0 0 n s D a u d i *** 8 0 M 2 1 0 B 4 -

l c o n d itio n e d a n s v *

i 6 0

u s

4 0 %

2 0

0 N T 0 h r 4 h r 1 6 h r 2 0 h r p o s t-s o rt p o s t-s o rt p o s t-s o rt p o s t-s o rt

+ 4 h r G A 1 0 1 tre a tm e n t

RAJI M2-10B4-cond. DAUDI M2-10B4-cond.

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 89.788 0.751 89.575 0.979 >0.9999 81.463 1.135 81.075 1.643 >0.9999 0 hr + 4 hr 56.263 3.468 73.500 1.784 <0.0001 56.513 2.836 70.500 0.655 0.0002 4 hr + 4 hr 61.600 1.812 74.000 1.653 0.0051 63.500 5.346 73.575 2.727 0.1223 16 hr + 4 hr 33.500 2.154 42.700 1.268 0.0131 31.850 1.757 43.183 3.897 0.0129 20 hr + 4 hr 40.867 2.139 46.000 1.219 0.3554 38.817 1.577 44.450 3.325 0.4787

Figure 3.22. Stroma-mediated protective effect lasts after removal of contact. Raji (A) or Daudi (B) cells were co-cultured with the stromal line M2-10B4 (previously stained with PKH67) for 24 hours, the separated by FACS sorting. Tumour cells were collected, seeded on plastic and treated for 4 hours with GA101 (10 μg/ml) at either 0, 4, 16 or 20 hours post-sort. Of note, % of PKH67+ cells after sort was always < than 2%. Viability was measured by flow cytometry, staining tumour cells with 7-AAD/Annexin V. % of survival was measured as % of 7-AAD-/Annexin V- cells. Data are the mean ± SEM of two duplicates in 2 (4 hr post-sort) or 3 (all other time-points) independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns = non-significant, measured by using a two-way Anova test.

109 3.7. Effect of co-culture on actin remodelling after removal of contact

To investigate whether the process of reorganisation of the actin cytoskeleton normally seen with GA101-induced homotypic adhesion was affected by tumour cell contact with stromal cells, Raji cells expressing GFP-labelled actin were used (“Raji-GFP-actin”, generated as described in [84]). Stromal cells were stained with the membrane-labelling red dye PKH26 and Raji-GFP-actin cells were cultured on the stromal layer for 24 hours. Cells were separated by using the cell sorters previously mentioned, and Raji-GFP-actin cells (GFP+/PKH26- population) were collected, and treated for 4 hours with GA101 immediately after sort. Cells were then poured onto slides, fixed, stained with DAPI and imaged on a gSTED confocal nanoscope in order to detect actin cytoskeletal changes. While cell aggregates were easily visible in Raji-GFP-actin cells cultured on plastic and treated with GA101 for 4 hours (figure 3.23 B), and actin appeared re-localised on the cell-cell junctions as described previously [87] (figure 3.23 B, arrows), the homotypic adhesion was not visible in cells treated with GA101 that had previously been cultured with M2-10B4. Actin appeared located around the nuclei in a round-shaped conformation (figure 3.23 C), resembling the non-treated cytoskeletal structure (figure 3.23 A).

110 A Raji-GFP-actin non-treated (not co-cultured) – 4 hrs after sorting

GFP GFP

DAPI DAPI DAPI

B Raji-GFP-actin + GA101 (not co-cultured) – 4 hrs after sorting and treatment

GFP GFP

DAPI DAPI DAPI DAPI

C Raji-GFP-actin + GA101, M210B4-conditioned – 4 hrs after sorting and treatment

GFP GFP

DAPI DAPI DAPI DAPI

Figure 3.23. Representative images of actin remodelling in Raji cells after treatment. Raji-GFP-actin cells were either cultured on plastic or with M2-10B4 for 24 hours, then sorted by FACS and treated with GA101 (10 μg/ml) for 4 hours. A) Raji-GFP-actin cells were cultured on plastic, then plated for additional 4 hours, without being treated. Actin is found in the cytoplasm, around the nucleus, forming round-shaped rings. B) Raji-GFP-actin cells were cultured on plastic, then plated and treated with GA101 (10 μg/ml) for 4 hours. The actin seems to form protrusions that link the cells to each other (arrows). C) Raji-GFP-actin cells were co-cultured with M2-10B4 for 24 hours, then sorted and treated alone for 4 hours with GA101. Protrusions are less evident and actin seems to be distributed around the nucleus, in round-shaped rings. Images (taken by a gSTED confocal nanoscope, 63x) are representative of two duplicates in 2 independent experiments.

111 3.8. Comparison between culture on plastic, vs stroma, vs fibronectin

To investigate whether the protective effect observed after treatment with GA101 required the presence of stromal cells, or whether it could also be mediated by attachment to different elements of the tumour microenvironment, the extracellular matrix (ECM) component fibronectin was employed. Plates were coated with 10 μg/ml (equal to 5 μg/cm2) human fibronectin, and tumour cells were seeded onto either plastic, or stroma, or fibronectin-coated wells for 24 hours. Tumour cells were either left untreated, or were pre- treated with GA101 (10 μg/ml) for 2 hours, or were treated as normal with GA101 after 1 hour from the beginning of co-culture. This was done to address not only if the attachment to fibronectin was able to protect tumour cells from PCD, but also if its presence could reverse the homotypic adhesion that was initiated before contact. After 24 hours, cells were collected and survival was measured by flow cytometry. Cells were protected when in contact with M2-10B4 from both normal treatment and pre-treatment with GA101 (p- values for Raji<0.0001 in both conditions, p-values for Daudi<0.0001 for normal treatment with GA101, p=0.0007 for pre-treatment). However, contact with fibronectin did not lead to any survival advantage, for Daudi, in any of the conditions observed (survival percentages and p-values are shown in details in figure 3.24). Raji only showed a higher survival with fibronectin after normal treatment with GA101 (32.3% on plastic vs 43.2% on fibronectin, p=0.0196), but not after the 2-hour pre-treatment (29.02% on plastic vs 32.15% on fibronectin, p=0.70) (figure 3.24). This experiment suggested that in general contact with the ECM component fibronectin cannot mediate a biologically relevant protective effect from PCD.

112 R a ji, P C D D a u d i, P C D R a ji D a u d i + M 2 1 0 B 4 1 0 0 1 0 0 + M 2 1 0 B 4 * + F N n s n s n s + F N 8 0 **** 8 0 ****

*** ***

l a

6 0 a 6 0

u s 4 0 s

4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 2 h rs N T G A 1 0 1 2 h rs

RAJI + M2-10B4 + FN

Mean ± SEM Mean ± SEM p-value vs Raji Mean ± SEM p-value vs Raji

NT 90.933 1.014 85.200 0.895 0.3107 89.333 0.994 0.9106 GA101 32.300 3.165 61.300 1.672 <0.0001 43.200 0.532 0.0196 2 hrs 29.017 4.010 49.933 4.875 <0.0001 32.150 3.451 0.7001

DAUDI + M2-10B4 + FN

Mean ± SEM Mean ± SEM p-value vs Daudi Mean ± SEM p-value vs Daudi

NT 80.533 1.870 78.433 1.026 0.8490 78.517 1.085 0.8598 GA101 34.817 4.298 60.283 1.063 <0.0001 35.420 1.630 0.9877 2 hrs 38.083 3.982 53.483 3.198 0.0007 38.183 3.444 0.9996

Figure 3.24. Attachment to the ECM component fibronectin does not mediate relevant protection from GA101-induced PCD. Raji and Daudi cells were cultured for 24 hours on either plastic (green bars), or stroma (M2-10B4, red bars), or fibronectin (FN)-coated wells (blue bars). Cells were either treated with GA101 after 1 hour from the beginning of the co-culture (“GA101”) or pre-treated for 2 hours before addition onto the plate (“2 hrs”). After 24 hours, viability was measured by flow cytometry, staining tumour cells with 7-AAD/Annexin V. % of survival was measured as % of PKH67- cells which were 7-AAD- /Annexin V-. Data are the mean ± SEM of two duplicates in 3 independent experiments. * p<0.05, *** p<0.001, **** p<0.0001, ns = non-significant, measured by using a two-way Anova test.

113 To determine whether the failure in mediating an increased survival correlated with a lack of actin reorganisation after culture on fibronectin, and therefore the presence of B cell aggregates that did not spread out on the fibronectin layer, pictures were taken with a low- light microscope after 24 hours of contact of GA101 pre-treated tumour cells with either plastic, or stroma, or fibronectin (figure 3.25). As expected, despite the presence of a number of cells that had attached to the fibronectin coated wells, cell aggregates were still easily visible after 24 hours, suggesting that actin remodelling which occurs after co- culture on stroma correlates with a survival advantage.

114

100 μm

Figure 3.25. Representative images of tumour cells pre-treated with GA101 and cultured on plastic, stroma or fibronectin. Cells were treated with GA101 (10 μg/ml) for 2 hours in tubes, then poured on plastic (left-hand side), or stroma (middle), or fibronectin-coated wells (right-hand side). Raji (upper panel) and Daudi (lower panel) were imaged (LowLight Zeiss microscope, 10x) 24 hours after pouring. Whilst cells on plastic (“alone”) were still clumped after 24 hours from pouring, in the presence of stroma (“on M210B4”) both Raji and Daudi cells seemed spread out and detached from each other. On fibronectin – coated wells, (“on FN”), despite the presence of a number of cells that seemed stuck to the well, B cell aggregates were clearly visible for both the cell lines. Images are representative of two duplicates in 2 independent experiments.

115 In an attempt to improve the adhesion of tumour cells to fibronectin and exclude the possibility that the homotypic adhesion process was still detectable only because of the high number of cells that had not adhered, the experiment was repeated after coating the wells with 20 μg/ml (equal to 10 μg/cm2) human fibronectin. Again, tumour cells were either pre-treated with GA101 and poured onto the wells (“2 hrs”, figure 3.26), or added to the wells for an hour and then treated with GA101 (“GA101”, figure 3.26). Survival percentages in cells on plastic, on stroma or on fibronectin were measured 24 hours later by flow cytometry. Pictures of the wells were taken by using an EVOS digital microscope (10 X) at the 24-hour endpoint (figure 3.27).

The attachment of both Raji and Daudi cells to fibronectin is clearly visible after 24 hours from the beginning of the culture, the pictures taken revealed that tumour cells on fibronectin do not separate from the aggregates formed after both treatment and pre- treatment with GA101. This process is observed in the presence of M2-10B4 stromal cells (figure 3.27). In correlation with what was seen by microscopy, there is not a survival advantage for Daudi cells in any of the treatment conditions observed. In Raji cells, as observed with the lowest concentration of fibronectin, there is a higher survival after normal treatment with GA101 (10.74% on plastic vs 22.7% on fibronectin, p=0.0003), but not after the 2-hour pre-treatment (7.753% on plastic vs 12.84% on fibronectin, p=0.1396). Detailed means and p-values are shown in figure 3.26.

Of note, Raji cells that were treated with GA101 after 1 hour from the beginning of culture on the fibronectin layer did not display a high number of B-cell aggregates, whereas Daudi cells seemed to execute homotypic adhesion at a greater extent. Such an observation, taken with the significant increase in survival in Raji but not Daudi cells, further suggests that the ability of a substrate to impede or decrease GA101-induced homotypic adhesion correlates with higher tumour cell survival (figure 3.28).

116 R a ji, P C D R a ji D a u d i, P C D D a u d i + M 2 1 0 B 4 + M 2 1 0 B 4 1 0 0 1 0 0 + F N + F N * * * n s n s 8 0 n s 8 0

l * * * *

* * * * l a

6 0 a * * * *

v 6 0

i v

* * * * i

u u

s 4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 2 h rs N T G A 1 0 1 2 h rs

RAJI + M2-10B4 + FN

Mean ± SEM Mean ± SEM p-value vs Raji Mean ± SEM p-value vs Raji

NT 93.700 0.191 71.650 2.185 <0.0001 89.300 0.636 0.2209 GA101 10.740 1.572 49.325 2.589 <0.0001 22.700 1.739 0.0003 2 hrs 7.753 0.940 31.425 3.604 <0.0001 12.838 1.413 0.1396

DAUDI + M2-10B4 + FN

Mean ± SEM Mean ± SEM p-value vs Daudi Mean ± SEM p-value vs Daudi

NT 90.100 0.505 78.250 0.768 0.0181 82.775 1.661 0.1788 GA101 12.550 2.385 47.200 4.306 <0.0001 13.275 1.858 0.9817 2 hrs 17.043 4.565 38.375 4.727 <0.0001 13.713 2.357 0.6826

Figure 3.26. A higher concentration of the ECM component fibronectin does not increase protection of tumour cells from GA101-induced PCD. Raji and Daudi cells were cultured for 24 hours on either plastic (green bars), or stroma (red bars), or wells coated with 10 µg/cm2 human fibronectin (blue bars). Cells were either treated with GA101 after 1 hour from the beginning of the co-culture (“GA101”) or pre-treated for 2 hours before addition onto the plate (“2 hrs”). After 24 hours, viability was measured by flow cytometry, staining tumour cells with 7-AAD/Annexin V. % of survival was measured as % of PKH67- cells which were 7-AAD-/Annexin V-. Data are the mean ± SEM of two duplicates in 2 independent experiments. *** p<0.001, **** p<0.0001, ns = non-significant, measured by using a two-way Anova test.

117

Figure 3.27. Representative images of tumour cells pre-treated with GA101 and cultured on plastic, stroma or fibronectin (10 µg/cm2). Cells were treated with GA101 (10 μg/ml) for 2 hours in tubes, then poured on plastic (left-hand side), or stroma (middle), or fibronectin-coated wells (right-hand side). Raji (upper panel) and Daudi (lower panel) were imaged (EVOS digital microscope, 10x) 24 hours after pouring. Whilst cells on plastic (“alone”) were still clumped after 24 hours from pouring, in the presence of stroma (“on M210B4”) both Raji and Daudi cells seemed spread out and detached from each other. On fibronectin- coated wells, (“on FN”), despite the presence of a number of cells that seemed stuck to the well, B cell aggregates were clearly visible for both the cell lines. Images are representative of two duplicates in 2 independent experiments.

118

Figure 3.28. Representative images of tumour cells treated with GA101 on fibronectin (10 µg/cm2). Raji (left-hand side) and Daudi (right-hand side) cells were plated on fibronectin-coated wells for 1 hour, before the addition of GA101 (10 μg/ml) for further 23 hours. GA101-treated cells (bottom panel) and non-treated controls (upper panel) were imaged at the end of the 24-hour assay (EVOS digital microscope, 10x). Not many B-cell aggregates are observed in Raji cells after GA101 treatment, while they can be easily detected in Daudi cells. Images are representative of two duplicates in 2 independent experiments.

119 3.9. Discussion

The experiments performed in this chapter strongly suggest that stromal cells are able to mediate protection from GA101-induced PCD. This protective effect appears to be dependent on direct contact between tumour and stromal cells, rather than on soluble factors released into the tumour microenvironment. In fact, neither culture in stroma- conditioned media nor culture on micropore inserts separating tumour cells from stroma, but ensuring exchange of media, recapitulated the increase in survival observed in conditions of direct contact. Moreover, adhesion to other microenvironmental elements, such as the extracellular matrix component fibronectin, did not induce the same protective effect. Such contact-mediated protection was shown to be sustained over 16 to 20 hours after removal of contact, revealing that the signalling axes activated by stromal cells are maintained over an extended time period.

Compelling evidence [19,20,166,168] suggested that the stroma-mediated protection from anti-CD20 antibody therapy could be dependent on the interactions between the chemokine CXCL-12, which stromal cells constitutively release in the environment, and its cognate receptor CXCR-4, highly expressed on the surface of malignant B cells [133]. The role of this signalling axis in the experimental system was therefore analysed. However, despite the migratory abilities of Raji and Daudi tumour cells towards the stromal layer indeed being dependent on CXCR-4-mediated interactions with M2-10B4-released soluble factors, blockade of such interactions did not lead to the abrogation of the protective effect.

In order to further investigate other causes of the observed increased survival after GA101 treatment, over and above the cell-cell contact, the potential role played by the combination of soluble factors was investigated. The assays performed led to the observation that not only was protection not dependent on soluble factors, but also that direct contact between stromal and tumour cells was required to achieve protection from GA101-induced killing. These results are consistent with some published findings: for instance, Edelmann et al. showed that the murine line M2-10B4 was able to support the growth of CLL cells over time when in direct contact, but such an effect was almost entirely lost if the two populations were grown in transwell plates (i.e. separated by a membrane insert) [181]. Such a loss of protection in non-contact conditions was similarly observed by another group, again using the M2-10B4 stromal line and CLL samples from patients [117]. On the other hand, these results are in contrast with those published in other studies, where soluble factors were able to recapitulate, at least partly, the protective effect observed in conditions of direct contact [150,177,182]. The mechanisms of protection

120 executed by stroma, therefore, could differ on the basis of both stromal cell line employed and type of B-cell malignancy analysed.

Intriguingly, microscopy experiments showed that the presence of stromal cells impedes the homotypic adhesion of B cells. Since GA101-induced PCD involves formation of B cell aggregates through a process of reorganisation and remodelling of the actin cytoskeleton towards the cell-cell junction points [86], the ability of stromal cells to interfere with such a process could be responsible for the observed decreased efficacy of the antibody. Importantly, the absence of homotypic adhesion in the presence of stromal cells could not be mirrored in fibronectin-coated plates, sustaining the possibility that the ability of stroma to interfere with this important step of the execution of PCD could play a role in mediating increased survival from GA101. However, additional microscopy experiments are required, with the aim of determining whether there are differences in actin cytoskeleton reorganisation in the presence of stromal cells, compared to in tumour cells previously in contact with stroma. The visualisation of stroma, however, is made difficult by the fact that the membrane of stromal cells would need to be stained over a 5- day period, and many commercially available long-term dyes for live imaging do not ensure a uniform staining for such a long timeframe and after many cell divisions. A more appropriate option would be to transfect stromal cells with a fluorescently-labelled vector, in order to generate a stable line. This technique, although time-consuming, could potentially help circumnavigate the issues linked with the use of a long-term dye for live imaging.

Another important step in the execution of GA101-induced PCD is the generation of reactive oxygen species (ROS), provoked by lysosomal membrane permeabilisation and release of lysosomal content into the cytoplasm which follows B cell homotypic adhesion [87]. The presence of the stromal microenvironment has been shown to be able to affect the ability of tumour cells to respond to oxidative stress in several different models. In a lung adenocarcinoma cell line, for instance, H2O2- and paclitaxel-induced production of ROS was found to be reduced in the presence of mesenchymal stromal cells, ultimately leading to increased tumour cell survival [183]. More recently, a study by Liu et al. reported that the long-term co-culture between BMSCs and ALL cell lines dramatically increased production of anti-oxidant agents in the latter [184]. If a similar mechanism occurred in the analysed system, such a reduced susceptibility to ROS could explain the impaired ability of GA101 to induce PCD. However, more work is required in order to investigate such a hypothesis and be able to determine whether A) there is a reduction in

121 levels of ROS in the presence of stromal cells; B) such reduction occurs in conditions of direct contact between stromal and tumour cells and therefore could be mediating the contact-dependent protection; C) an exogenous increase in ROS could restore GA101- induced PCD in the presence of stroma and in turn abrogate the protective effect. A more in-depth analysis on the signalling pathways that occur after tumour-stroma co-culture could help further understanding the role played by the microenvironment in protecting from GA101 – and are further explored in chapter 6.

122 4. Stroma-mediated protection of tumour cells from anti-CD20 mAb-induced antibody-dependent cellular phagocytosis (ADCP)

To assess the degree of ADCP induced by GA101 treatment and to understand how the presence of stromal cells could potentially interfere with the antibody tumour cell kill, the co-culture system previously established has been used. Stromal cells were labelled with the membrane-labelling dye PKH67 and seeded on 96-well plates until confluency (approx. 72 hours). Tumour cells were labelled with the membrane-labelling dye PKH26 and then added to the culture for 24 hours. After 24 hours, PBMCs were isolated from buffy coats and different sets of immune effector cells were separated using specific isolation kits. Immune cells were then added to the co-culture and cells were treated with GA101 for different times depending on the set of immune cells chosen.

At the end of the assay, cells were collected and stained for CD11b, a surface marker of phagocytic immune cells (i.e. monocytes, macrophages, neutrophils), or for CD14/CD16, surface markers of monocyte subclasses. Samples were analysed by flow cytometry. The percentage of immune effector cells that had engulfed tumour cells was measured as % CD11b+ cells which were also PKH26+ – after gating out the PKH67+ cells. Dead cells and associated debris were gated out by virtue of their forward/side scatter values as represented in the FlowJo plot shown in figure 4.1.

123

Figure 4.1. Representative image of the gating strategy used in FlowJo. The first plot (left-hand side) shows the gating strategy used to separate tumour and immune cells (inside the gate) from stromal cells based on the negativity for PKH67 (X-axis) prior to analysis of phagocytosis. The second plot (middle) shows the gating of immune cells based on CD11b (Y-axis) positivity, versus tumour cells (PKH26+, on X- axis) outside the gate. The third plot (right-hand side) shows immune cells positivity for PKH26 (X-axis) and CD11b (Y-axis). The plots are representative of a non-treated sample (control) which was used to determine the PKH26+ gate within the monocyte (CD11b+) population.

124 4.1. Stroma-mediated protection from monocyte-mediated ADCP

Monocytes are a subset of immune effector cells that are activated upon binding of the Fc region of monoclonal antibodies, and execute ADCP by engulfing target cells. Whether the presence of stroma could protect tumour cells from the phagocytic activity of monocytes was unknown. To address this question, monocytes were isolated from PBMCs and added to the stroma-tumour co-culture, which had been established 24 hours earlier. Cells were treated with GA101 at either 0.1 or 10 μg/ml. The three-way culture was kept for a further two hours, after which cells were collected from the wells by vigorous pipetting, stained for the surface marker CD11b and analysed by flow cytometry. Even amounts of GA101 as small as 0.1 μg/ml induced high levels of ADCP (% of monocytes that engulfed tumour cells: 47.39 for Raji, 49.71 for Daudi) (figure 4.2). Interestingly, the presence of stroma strikingly reduced the ability of monocytes to mediate phagocytosis (16.72% for Raji, 17.52% for Daudi, both p<0.0001). The same pattern was observed with the higher dose of GA101 (p<0.0001 for both Raji and Daudi).

125 A R a ji, A D C P (h u m a n m o n o c y te s ) E : T 1 : 1

1 0 0

R a ji

+ 6

2 8 0 + M 2 1 0 B 4 H

K * * * *

P * * * *

6 0

t y

c 4 0

n o

2 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

B D a u d i, A D C P (h u m a n m o n o c y te s ) E : T 1 : 1

1 0 0

D a u d i

+ 6

2 8 0 + M 2 1 0 B 4

H * * * * * * * *

K P

6 0

t y

c 4 0

n o

2 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 8.743 1.745 1.144 0.122 0.2221 7.944 1.544 1.472 0.279 0.5292 0.1 μg/ml 47.488 5.215 16.719 1.592 <0.0001 49.711 5.768 15.297 2.066 <0.0001 10 μg/ml 47.644 4.948 17.522 1.359 <0.0001 51.678 6.169 17.221 2.014 <0.0001

Figure 4.2. Stromal cells decrease the ability of monocytes to phagocyte tumour cells. Raji (A) and Daudi (B) were seeded onto the stromal layer for 24 hours. Human monocytes (effector to target ratio 1:1) were added in the culture and cells were treated with GA101 (0.1 and 10 μg/ml). After two hours, cells were collected and stained with the anti-CD11b-APC antibody at 1 μg/ml. The % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Of note, the unspecific reduction in phagocytosis observed in the non-treated group is not statistically significant (p=0.24 for Raji, 0.66 for Daudi). Data are the mean ± SEM of three replicates in 3 independent experiments. **** p<0.0001, measured by using a two-way Anova test.

126 To ensure that the protective effect lasted for longer than 2 hours of three-way culture with monocytes, the assay was repeated and cells were collected and analysed after either 2 hours (used as a control assay), 4 hours, 8 hours or 24 hours from addition of monocytes and treatment. On plastic, tumour cells were efficiently phagocytosed in the 2 hr, 4 hr and 8 hr time-points in the presence of GA101. At 24 hr, however, high levels of phagocytosis were observed in non-treated cells (figures 4.3, 4.4 D and 4.5 D), suggesting that an increasing non-specific ADCP occurs over this time period. Stroma-mediated protection from ADCP was maintained at similar levels in 4 hr (figures 4.4 B and 4.5 B) and 8 hr (figures 4.4 C and 4.5 C) co-cultures as the standard 2 hr co-culture (figures 4.4 A and 4.5 A). (detailed p-values are shown in figures 4.3 and 4.4). Importantly, the degree of ADCP in the presence of stromal cells at 24 hours was reduced to a level that is lower than that of non-specific phagocytosis in non-treated samples at 24 hours (e.g. in Raji, fold changes of treated samples in the presence of M210B4 over non-treated control on plastic were 0.405 (GA101 0.1 µg/ml) and 0.394 (GA101 10 µg/ml); in Daudi, fold changes over control were 0.549 (GA101 0.1 µg/ml) and 0.522 (GA101 10 µg/ml), suggesting a strong protective effect from ADCP even at 24 hours.

This experiment demonstrated that the protective effect is not transient, but lasts over a 24- hour period, although is not specific to inhibition of anti-CD20 mAb mediated ADCP but phagocytosis in general.

127 A R a ji, A D C P o n p la s tic E : T 1 : 1

1 0 0 * * * *

+ 6

2 8 0 * * * * G A 1 0 1 0 .1 u g /m l H

K * * * * G A 1 0 1 1 0 u g /m l P

6 0 * * * *

t y

c 4 0

n o

2 0

% 0 2 4 8 2 4

H o u rs

RAJI on NT GA101 0.1 µg/ml GA101 10 µg/ml plastic Mean ± SEM Mean ± SEM p-value vs Mean ± SEM p-value vs NT NT 2 5.173 0.675 37.767 0.763 <0.0001 40.883 0.445 <0.0001 4 10.382 0.931 43.267 1.054 <0.0001 46.333 0.965 <0.0001 8 16.378 0.833 46.178 2.003 <0.0001 46.733 2.775 <0.0001 24 43.456 3.080 64.189 4.719 <0.0001 64.900 5.466 <0.0001

Figure 4.3. Monocyte-mediated ADCP of tumour cells cultured on plastic increases over time. Raji cells were seeded on plastic for 24 hours. Human monocytes (effector to target ratio 1:1) were added in the culture and cells were treated with GA101 (0.1 and 10 μg/ml). After either 2, 4, 8 or 24 hours, cells were collected and stained with the anti-CD11b-APC antibody at 1 μg/ml. The graph shows the percentage of phagocytosis of Raji cells on plastic over time in different conditions (NT, GA101 0.1 μg/ml and GA101 10 μg/ml). % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+. Data are the mean ± SEM of three replicates in 2 (2 hrs, 4 hrs) or 3 (8 hrs, 24 hrs) independent experiments. **** p<0.0001, measured by using a two-way Anova test.

128 R a ji, A D C P (h u m a n m o n o c y te s ) - 4 h r s A R a ji, A D C P (h u m a n m o n o c y te s ) - 2 h rs B E : T 1 : 1 E : T 1 : 1

8 1 5 R a ji R a ji * * * * * * * * * * * * * * *

l + M 2 1 0 B 4 e + M 2 1 0 B 4 l

e 6

t t

n 1 0

h c

c 4

e v

5 o

F o o F 2

0 0 N T G A 1 0 1 G A 1 0 1 N T G A 1 0 1 G A 1 0 1 0 .1 u g /m l 1 0 u g /m l 0 .1 u g /m l 1 0 u g /m l C D R a ji, A D C P (h u m a n m o n o c y te s ) - 8 h rs R a ji, A D C P (h u m a n m o n o c y te s ) - 2 4 h rs E : T 1 : 1 E : T 1 : 1

5 * * * * 2 .0 * * * * * * * * R a ji * * * * R a ji

4 + M 2 1 0 B 4 * * * * + M 2 1 0 B 4

l e

e 1 .5

n a

3 n

c c

c 1 .0

d d

l * *

e 2

F o o F 0 .5 1

0 0 .0 N T G A 1 0 1 G A 1 0 1 N T G A 1 0 1 G A 1 0 1 0 .1 u g /m l 1 0 u g /m l 0 .1 u g /m l 1 0 u g /m l

NT RAJI + M2-10B4 0.1 RAJI + M2-10B4 Mean ± SEM Mean ± SEM p-value µg/ml Mean ± SEM Mean ± SEM p-value 2 hr 1.000 0.056 0.388 0.034 >0.9999 2 hr 7.871 1.003 3.841 0.395 0.0002 4 hr 1.000 0.027 0.185 0.030 0.1335 4 hr 4.357 0.453 1.625 0.111 <0.0001 8 hr 1.000 0.027 0.160 0.035 0.0015 8 hr 2.892 0.227 0.927 0.088 <0.0001 24 hr 1.000 0.038 0.078 0.025 <0.0001 24 hr 1.473 0.033 0.405 0.072 <0.0001

10 RAJI + M2-10B4 µg/ml Mean ± SEM Mean ± SEM p-value

2 hr 8.452 0.958 4.033 0.408 <0.0001 4 hr 4.665 0.482 1.756 0.066 <0.0001 8 hr 2.954 0.295 0.931 0.088 <0.0001 24 hr 1.478 0.048 0.394 0.079 <0.0001

Figure 4.4. Stromal cells’ ability to decrease monocyte-mediated phagocytosis of Raji cells lasts over time. Raji cells were seeded onto the stromal layer for 24 hours. Human monocytes (effector to target ratio 1:1) were added in the culture and cells were treated with GA101. After either 2 (A), 4 (B), 8 (C) or 24 (D) hours, cells were collected and stained with anti-CD11b-APC antibody (1 μg/ml). % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 2 (2 hrs, 4 hrs) or 3 (8 hrs, 24 hrs) independent experiments. Fold change is the percentage of ADCP obtained for each condition divided by control (Raji non-treated on plastic). *** p<0.001, **** p<0.0001, measured by using a two-way Anova test.

129 A B D a u d i, A D C P (h u m a n m o n o c y te s ) - 2 h rs D a u d i, A D C P (h u m a n m o n o c y te s ) - 4 h rs E : T 1 : 1 E : T 1 : 1 * * * * 1 0 * * * 6 D a u d i

* * * * * * D a u d i l e 8

o + M 2 1 0 B 4 l

g + M 2 1 0 B 4

n a

6 n 4

4 r

o v

o 2

F o 2 F

0 N T G A 1 0 1 G A 1 0 1 0 0 .1 u g /m l 1 0 u g /m l N T G A 1 0 1 G A 1 0 1 0 .1 u g /m l 1 0 u g /m l C D D a u d i, A D C P (h u m a n m o n o c y te s ) - 8 h rs D a u d i, A D C P (h u m a n m o n o c y te s ) - 2 4 h rs E : T 1 : 1 E : T 1 : 1

5 * * * 2 .5 * * * * D a u d i * * * *

* * * * D a u d i l

4 + M 2 1 0 B 4 l 2 .0 + M 2 1 0 B 4

n a

3 a 1 .5 * * * *

r r

d *

d l

e 2 l

e 1 .0

F o

F o 1 0 .5

0 0 .0 N T G A 1 0 1 G A 1 0 1 N T G A 1 0 1 G A 1 0 1 0 .1 u g /m l 1 0 u g /m l 0 .1 u g /m l 1 0 u g /m l

NT DAUDI + M2-10B4 0.1 DAUDI + M2-10B4 Mean ± SEM Mean ± SEM p-value µg/ml Mean ± SEM Mean ± SEM p-value 2 hr 1.000 0.055 0.439 0.049 0.9540 2 hr 5.262 0.479 2.960 0.381 0.0007 4 hr 1.000 0.047 0.258 0.027 0.1831 4 hr 3.547 0.358 1.857 0.176 0.0003 8 hr 1.000 0.030 0.190 0.025 0.0268 8 hr 2.318 0.299 0.877 0.154 <0.0001 24 hr 1.000 0.013 0.142 0.044 <0.0001 24 hr 1.625 0.111 0.549 0.091 <0.0001

10 DAUDI + M2-10B4

µg/ml Mean ± SEM Mean ± SEM p-value 2 hr 5.844 0.651 3.367 0.337 0.0003 4 hr 3.958 0.478 1.947 0.214 <0.0001

8 hr 2.380 0.340 1.037 0.186 0.0001 24 hr 1.593 0.083 0.522 0.096 <0.0001

Figure 4.5. Stromal cells’ ability to decrease monocyte-mediated phagocytosis of Daudi cells lasts over time. Daudi cells were seeded onto the stromal layer for 24 hours. Human monocytes (effector to target ratio 1:1) were added in the culture and cells were treated with GA101. After either 2 (A), 4 (B), 8 (C) or 24 (D) hours, cells were collected and stained with anti-CD11b-APC antibody (1 μg/ml). % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 2 (2 hrs, 4 hrs) or 3 (8 hrs, 24 hrs) independent experiments. Fold change is the percentage of ADCP obtained for each condition divided by control (Raji non-treated on plastic). *** p<0.001, **** p<0.0001, measured by using a two-way Anova test.

130 4.1.1. Influence of length of tumour/stroma co-culture times on protection from monocyte-mediated ADCP

To understand whether stromal cells interfere with ADCP by directly affecting the monocytes’ ability to perform phagocytosis, or by virtue of a protective effect exerted on tumour cells, the assay was repeated using different co-culture times. Tumour cells were cultured on stroma for either 0 hours (no co-culture time prior to addition of monocytes and treatment) 30 minutes, 1 hour, 2 hours, 4 hours or 24 hours (control assay), before adding monocytes and treating cells with GA101. After 2 hours of phagocytosis, cells were collected and analysed by flow cytometry. Interestingly, no protection was observed from monocyte-mediated ADCP when both target tumour cells and effector cells were added at the same time (0-hour co-culture). The same result was obtained after 30 minutes of tumour/stroma co-culture before the addition of monocytes and treatment. A protective effect, albeit a small effect, was observed when effector cells were added after 1 hour of tumour/stroma co-culture, and this protection increased with time and reached its maximum at the 24-hour time-point (control assay). Figures 4.6 and 4.7 show the degree of ADCP observed in Raji and Daudi cells, respectively, in non-treated cells (“NT”), cells treated with 0.1 µg/ml GA101 and cells treated with 10 µg/ml GA101 at each different contact time (“0” = no contact before addition of monocytes; “0.5” = 30 minutes of contact, “1” = 1 hour of contact, “2” = 2 hours, “4” = 4 hours, “24” = 24 hours, control assay). Therefore, this experiment demonstrated that a pre-contact between stromal and tumour cells of at least 1 hour is needed for a protective effect to be observed.

Of note, a statistically significant reduction in non-specific ADCP was observed in non- treated cells, and in Raji cells this was also observed when no contact between stromal and tumour cells was allowed before the addition of monocytes (figure 4.6) – but not in Daudi cells (figure 4.7). Detailed means ± SEM and p-values are shown in figure 4.6 and 4.7.

131 R a ji, A D C P (h u m a n m o n o c y te s ) R a ji E : T 1 : 1 + M 2 1 0 B 4

1 0 0

+ *** n s n s **** 6 n s n s n s **** * ****

2 8 0

K P **** ****

s 6 0

t y

c 4 0

n o 2 0 **** **** **** m *

* **** % 0 0 0 .5 1 2 4 2 4 0 0 .5 1 2 4 2 4 0 0 .5 1 2 4 2 4 H o u rs H ours H o u rs

N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

NT RAJI + M2-10B4 0.1 RAJI + M2-10B4 Mean ± Mean ± p-value µg/ml Mean ± Mean ± p-value SEM SEM SEM SEM 0 hr 15.717 0.699 7.425 0.248 <0.0001 0 hr 75.533 0.444 74.300 1.169 0.9998 0.5 hr 6.398 0.383 3.912 0.455 0.0205 0.5 hr 78.167 1.286 74.433 1.302 0.8639 1 hr 5.705 0.390 2.775 0.529 0.0284 1 hr 75.317 2.528 67.850 1.430 0.3999 2 hr 8.891 0.429 3.353 0.582 <0.0001 2 hr 82.375 1.582 65.813 2.317 0.0001 4 hr 11.869 1.092 3.256 0.659 <0.0001 4 hr 76.900 2.680 60.056 5.123 <0.0001 24 hr 12.260 0.625 3.924 0.793 <0.0001 24 hr 49.300 3.785 25.620 0.560 <0.0001

10 RAJI + M2-10B4 µg/ml Mean ± Mean ± p-value SEM SEM 0 hr 75.367 0.986 72.300 1.934 0.9109 0.5 hr 76.678 0.806 72.211 0.997 0.4279 1 hr 75.150 1.883 65.783 0.981 0.0254

2 hr 82.125 1.511 62.288 0.914 <0.0001 4 hr 77.033 2.725 61.467 3.171 <0.0001 24 hr 50.500 3.963 26.480 1.491 <0.0001

Figure 4.6. Protection from monocyte-mediated ADCP in Raji cells is only observed after a pre-contact time of at least 1 hour. Raji cells were seeded onto the stromal layer for either 0, 0.5, 1, 2, 4 or 24 hours (control assay), before adding monocytes (effector to target ratio 1:1) and treating cells with GA101 (0.1 and 10 μg/ml) for a further 2 hours. Cells were collected and stained with the anti-CD11b-APC antibody at 1 μg/ml. % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 2 (1 hr, 24 hr) or 3 (all other time-points) independent experiments. ns=non-significant, * p<0.05, *** p<0.001, **** p<0.0001, measured by using a two-way Anova test.

132 D a u d i D a u d i, A D C P (h u m a n m o n o c y te s ) + M 2 1 0 B 4 E : T 1 : 1

+ 1 0 0 6 n s **** 2 n s n s n s ** **** n s n s **** H 8 0 K ****

P ****

s 6 0

y c

o 4 0 n

o **** n s ** * **** ***

m 2 0

% 0 0 0 .5 1 2 4 2 4 0 0 .5 1 2 4 2 4 0 0 .5 1 2 4 2 4

H o u rs H ours H o u rs

N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

NT DAUDI + M2-10B4 0.1 DAUDI + M2-10B4 Mean ± Mean ± p-value µg/ml Mean ± Mean ± p-value SEM SEM SEM SEM 0 hr 5.790 1.105 4.090 0.644 0.9471 0 hr 68.367 1.338 67.550 1.796 >0.9999 0.5 hr 10.584 1.956 5.293 0.272 0.0106 0.5 hr 78.511 1.782 76.022 1.879 0.9923 1 hr 9.352 2.046 3.055 0.126 0.0140 1 hr 78.667 3.690 70.950 2.569 0.5920 2 hr 12.969 1.730 4.285 0.924 <0.0001 2 hr 84.363 2.214 69.263 1.810 0.0071 4 hr 13.061 1.458 3.522 0.720 <0.0001 4 hr 79.267 2.922 57.056 5.293 <0.0001 24 hr 13.480 0.619 4.478 0.672 0.0004 24 hr 57.800 7.366 30.560 2.521 <0.0001

10 DAUDI + M2-10B4 µg/ml Mean ± Mean ± p-value SEM SEM 0 hr 67.100 1.539 64.633 2.282 0.9947 0.5 hr 77.567 1.614 72.422 1.515 0.6733 1 hr 78.083 3.048 68.167 1.823 0.1800 2 hr 82.463 2.671 63.288 1.385 <0.0001 4 hr 77.989 3.431 57.700 3.793 <0.0001 24 hr 57.620 6.948 33.960 2.458 <0.0001

Figure 4.7. Protection from monocyte-mediated ADCP in Daudi cells is only observed after a pre- contact time of at least 1 hour. Daudi cells were seeded onto the stromal layer for either 0, 0.5, 1, 2, 4 or 24 hours (control assay), before adding monocytes (effector to target ratio 1:1) and treating cells with GA101 (0.1 and 10 μg/ml) for a further 2 hours. Cells were collected and stained with the anti-CD11b-APC antibody at 1 μg/ml. % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 2 (1 hr, 24 hr) or 3 (all other time-points) independent experiments. ns=non-significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, measured by using a two-way Anova test.

133 4.1.2. Role of contact with stromal cells vs soluble factors

To establish the role that soluble factors released by stroma have in generating protection from monocyte-mediated ADCP, stromal cells were cultured in plates for 72 hours and the conditioned media (S-CM) generated was collected and filtered. Tumour cells were labelled with the PKH26 dye and cultured in either normal RPMI, or S-CM, for 24 hours, then monocytes were added to the cultured and cells were treated with GA101 (0.1 or 10 μg/ml) for 2 hours. After 2 hours, cells were labelled with an anti-CD11b antibody, in order to be able to distinguish monocytes from tumour cells, and analysed by flow cytometry. As shown in figure 4.8, culturing tumour cells in S-CM did not lead to any protective effects. The percentage of monocytes (plotted as fold change) that had phagocyted tumour cells was alike, whether cells had been treated while in RPMI or in S- CM (p>0.05 for all conditions tested).

To investigate whether the stromal cells could have released soluble factors able to mediate protection only after a 24-hour co-culture with tumour cells, and that this media, conditioned by both tumour and stromal cells (T/S-CM), could have led to protection, Raji and Daudi were cultured on the stromal layer for 24 hours. The media thus generated were collected, filtered and used to culture tumour cells. After 24 hours of culture in either RPMI or T/S-CM, monocytes were added to the cultures and cells were treated with GA101. Cells were then labelled with anti-CD11b antibody and analysed by flow cytometry. Again, the presence of T/S-CM did not decrease the degree of phagocytosis induced by the treatment (figure 4.9). There were no significant differences between the percentages of monocytes that had phagocyted tumour cells while in T/S-CM, compared to in normal RPMI (p-values for all conditions tested are shown in figure 4.9). This suggested that, as observed for PCD, the presence of stroma is required to achieve protection from monocyte-mediated ADCP.

134 A R a ji, A D C P (h u m a n m o n o c y te s ) E : T 1 : 1

1 0 R a ji n s

8 n s + S -C M

r t

n 6

d 4

F o 2

0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l B D a u d i, A D C P (h u m a n m o n o c y te s ) E : T 1 : 1

1 0 D a u d i

8 n s + S -C M

l n s

r t

n 6

d 4

F o 2

0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 1.000 0.026 0.775 0.069 0.9949 1.000 0.028 0.845 0.050 0.9811 0.1 μg/ml 5.518 0.914 5.719 1.001 0.9964 4.827 0.243 5.749 0.583 0.1413 10 μg/ml 5.077 0.662 5.700 0.940 0.9083 4.876 0.247 5.237 0.381 0.8144

Figure 4.8. Culture of tumour cells in S-CM does not decrease monocyte-mediated ADCP. Stromal cells were seeded into plates for 72 hours, then media (S-CM) were collected and filtered. Raji (A) and Daudi (B) were plated in either RPMI (green bars) or S-CM (red bars) for 24 hours. Human monocytes (effector to target ratio 1:1) were then added to the culture and cells were treated with GA101 (0.1 and 10 μg/ml). After two hours, cells were collected and stained with the anti-CD11b-APC antibody at 1 μg/ml. % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+. Data are plotted as fold change over control (non-treated cells in RPMI). Data are the mean ± SEM of three replicates in 2 independent experiments. Fold change is the percentage of ADCP obtained for each condition divided by control (Raji/Daudi non-treated in normal RPMI). ns=non-significant, measured by using a two-way Anova test.

135 A R a ji, A D C P (m o n o c y te -m e d ia te d ) E : T 1 : 1

1 0 0

R a ji

+ 6

2 8 0 + T /S -C M H n s

K n s P

6 0

t y

c 4 0

n o

2 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

B D a u d i, A D C P (m o n o c y te -m e d ia te d ) E : T 1 : 1

1 0 0

D a u d i

+ 6

2 8 0 + T /S -C M H

K n s P

n s

6 0

t y

c 4 0

n o

2 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 11.448 1.845 10.933 1.525 0.9895 6.850 1.106 7.458 1.276 0.9995 0.1 μg/ml 47.717 1.020 47.533 0.954 0.9995 36.800 4.237 41.550 5.658 0.8180 10 μg/ml 48.650 0.898 46.817 1.266 0.6932 41.100 5.642 40.833 4.760 >0.9999

Figure 4.9. Culture of tumour cells in T/S-CM does not decrease monocyte-mediated ADCP. Tumour cells were co-cultured with stromal cells for 24 hours, then media conditioned by both cell populations (T/S- CM) were collected and filtered. Raji (A) and Daudi (B) were plated in either RPMI (green bars) or T/S-CM (red bars) for 24 hours. Human monocytes (effector to target ratio 1:1) were added to the culture and cells were treated with GA101 (0.1 and 10 μg/ml). After two hours, cells were collected and stained with the anti- CD11b-APC antibody at 1 μg/ml. % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+. Data are the mean ± SEM of three replicates in 2 independent experiments. ns=non-significant, measured by using a two-way Anova test.

136 4.1.3. Effect of attachment on the ECM component fibronectin on ADCP

To rule out the hypothesis that the presence of the stroma could render the phagocytic process more difficult to perform because of the physical obstruction produced by the attachment of target tumour cells to a substrate, the assay was performed in the presence of fibronectin (FN). The attachment of tumour cells on fibronectin would physically mimic the contact with stroma, without necessarily recapitulating the biological effects of contact on signalling pathways. Cells were either seeded on plastic, or on FN-coated wells (5 µg/cm2), or on stroma for 24 hours. Monocytes were then isolated from PBMCs and added to the culture, and cells were treated with GA101 for 2 additional hours. The three-way culture obtained was analysed by flow cytometry. The attachment of tumour cells to FN did not lead to a decrease in the degree of monocyte-mediated ADCP (figure 4.10). Instead, the percentage of monocytes that had phagocytosed target cells cultured on FN was as high as the one observed for cells cultured on plastic (e.g. 63.717% on plastic vs 55.683% on FN for GA101 10 µg/ml in Raji, p=0.0975). A protective effect was only observed when target cells were treated in the presence of stromal cells (e.g. 63.717% on plastic vs 31.650% on stroma for GA101 10 µg/ml in Raji, p<0.0001). These findings suggested that the decreased ADCP is not provoked merely by attachment to a substrate, but that stromal cells are required in order to observe a protective effect.

137 A R a ji, A D C P (h u m a n m o n o c y te s ) B D a u d i, A D C P (h u m a n m o n o c y te s ) E : T 1 : 1 E : T 1 : 1

1 0 0 n s 1 0 0 n s n s + n s

R a ji + D a u d i

6 2

8 0 + M 2 1 0 B 4 2 8 0 + M 2 1 0 B 4 H

* * * * H * * * * K

* * * * K * * * * P

+ F N P + F N

6 0 6 0

y c

4 0 c 4 0

o o 2 0 *

m 2 0

% % 0 0 N T G A 1 0 1 G A 1 0 1 N T G A 1 0 1 G A 1 0 1 0 .1 u g /m l 1 0 u g /m l 0 .1 u g /m l 1 0 u g /m l

RAJI + M2-10B4 + FN

Mean ± SEM Mean ± SEM p-value vs Raji Mean ± SEM p-value vs Raji

NT 12.057 1.585 3.215 0.862 0.0624 10.980 1.489 0.9552 0.1 μg/ml 62.083 0.915 32.300 6.316 <0.0001 59.967 1.046 0.8386 10 μg/ml 63.717 1.571 31.650 4.559 <0.0001 55.683 1.018 0.0975

DAUDI + M2-10B4 + FN

Mean ± SEM Mean ± SEM p-value vs Daudi Mean ± SEM p-value vs Daudi

NT 11.307 1.255 2.962 0.890 0.0114 10.872 1.039 0.9857 0.1 μg/ml 62.933 1.370 24.783 3.962 <0.0001 60.733 1.230 0.6954 10 μg/ml 65.550 1.044 30.833 3.556 <0.0001 59.950 0.959 0.1120

Figure 4.10. Culture of tumour cells on fibronectin does not lead to decreased monocyte-mediated ADCP. Raji (A) and Daudi (B) were cultured either on plastic (green bars), or on M2-10B4 (red bars), or on fibronectin (FN, blue bars) for 24 hours. Human monocytes (effector to target ratio 1:1) were added to the culture and cells were treated with GA101 (0.1 and 10 μg/ml). After two hours, cells were collected and stained with the anti-CD11b-APC antibody at 1 μg/ml. % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 2 independent experiments. ns=non-significant, * p<0.05, **** p<0.0001, measured by using a two-way Anova test.

138 4.1.4. Role of CD47/CD172a (SIRP-α) signalling pathway in stroma-mediated protection from monocyte-mediated ADCP

The CD47 molecule is highly expressed in malignant B lymphocytes, and interacts with the inhibitory receptor CD172a (SIRP-α), expressed on antigen-presenting cells, to down- regulate phagocytosis [185]. The CD47/CD172a pathway has been also shown to play a role in bone homeostasis in a study performed by Koskinen et al. [186]. This study highlighted that bone marrow stromal cells expressing a defective CD172a receptor were unable to support osteoblast differentiation and bone formation, due to impaired signalling between CD47 and CD172a.

Therefore, it was next investigated whether the activation of CD47 on B cells upon binding to CD172a expressed on stromal cells resulted in a reduction in the levels of ADCP observed in the current settings.

The ADCP assay was performed upon blockade of the CD172a molecule on the surface of stromal cells through the use of a neutralising antibody against mouse CD172a. To confirm the efficacy of the neutralising antibody to bind to and block its epitope on the CD172a molecule, stromal cells were treated for 24 hours with either 1 or 10 μg/ml anti-mouse CD172a antibody, then labelled for CD172a expression with an APC-conjugated antibody sharing the binding epitope with the neutralising antibody used for the treatment (figure 4.11). The use of the neutralising antibody completely abrogated the binding of the APC- conjugated antibody, confirming that the epitope is efficiently blocked after treatment.

139 C D 1 7 2 a e x p re s s io n M 2 -1 0 B 4

1 0 0 U n s ta in e d

8 0 Is o ty p e

A n ti-C D 1 7 2 a

6 0

I F

M 4 0

2 0

0 N T Ab 1 u g /m l Ab 1 0 u g /m l

Unstained Isotype Anti-CD172a

Mean ± SEM Mean ± SEM Mean ± SEM

NT 3.060 0.250 20.650 0.250 71.750 3.250 Ab 1 μg/ml 2.670 0.000 19.500 0.000 20.100 0.100 Ab 10 μg/ml 2.570 0.020 19.100 0.500 18.000 0.100

Figure 4.11. Anti-mouse CD172a antibody efficiently blocks its binding epitope on the CD172a molecule. M2-10B4 were treated for 24 hours with either 1 or 10 μg/ml anti-mouse CD172a neutralising antibody (“Ab 1μg/ml”, “Ab 10μg/ml” on the X-axis). Cells were then either left unlabelled (green bars), or labelled with the rat IgG1κ isotype control (red bars), or with an anti-mouse CD172a antibody which shared the binding epitope with the neutralising antibody (clone P84) (blue bars). Data are the mean of MFI of two replicates.

140 To investigate the effect of such blocking on the stroma-mediated protection from ADCP, M2-10B4 cells were pre-treated for 24 hours with 10 μg/ml anti-CD172a antibody. The antibody was maintained in the media during the 24-hour co-culture period. After addition of monocytes and treatment with GA101, cells were collected, labelled for CD11b and analysed by flow cytometry. The blockade of CD172a, however, did not induce an increase in ADCP in the presence of stromal cells (figure 4.12). There was no significant difference between the degree of ADCP observed in the presence of stroma upon treatment with anti- CD172a antibody and the ADCP observed in the presence of untreated stroma (detailed values are shown in figure 4.12).

141

A B R a ji, A D C P (m o n o c y te -m e d ia te d ) D a u d i, A D C P (m o n o c y te -m e d ia te d ) E :T 1 : 1 E :T 1 : 1

6 0 6 0

O n p la s tic

+ 6

6 + M 2 1 0 B 4 2

* * * * 2 H

* * * * H

K 4 0

K 4 0 * * * * + M 2 1 0 B 4 P

P * * * *

+ a n ti-C D 1 7 2 a s

n s s

e e

t n s

y c

c n s o 2 0 o 2 0 n s

n *

% % 0 0 N T G A 1 0 1 G A 1 0 1 N T G A 1 0 1 G A 1 0 1 0 .1 u g /m l 1 0 u g /m l 0 .1 u g /m l 1 0 u g /m l

RAJI + M2-10B4 + M2-10B4 + anti- CD172a

Mean ± SEM Mean ± SEM p-value Mean ± SEM p-value p-value vs + vs Raji vs Raji M2-10B4

NT 7.062 0.988 2.684 0.567 0.0375 2.214 0.498 0.0186 0.9893 0.1 μg/ml 31.960 1.420 17.240 1.154 <0.0001 16.100 1.118 <0.0001 0.8740 10 μg/ml 36.180 1.224 17.960 1.814 <0.0001 18.240 1.260 <0.0001 0.9977

DAUDI + M2-10B4 + M2-10B4 + anti-CD172a

Mean ± SEM Mean ± SEM p-value vs Mean ± SEM p-value vs p-value vs + Daudi Daudi M2-10B4

NT 5.594 0.404 2.486 0.746 0.1490 3.070 0.792 0.3485 0.9790 0.1 μg/ml 25.580 0.806 11.422 1.393 <0.0001 10.336 1.772 <0.0001 0.8653 10 μg/ml 27.400 0.583 13.980 1.448 <0.0001 14.320 1.102 <0.0001 0.9949

Figure 4.12. Blockade of CD172a fails to abrogate protection from monocyte-mediated ADCP. M2- 10B4 were pre-treated for 24 hours with the anti-mouse CD172a antibody (10 μg/ml), before the addition of Raji (A) and Daudi (B) in the culture. The antibody was maintained in the media during the 24-hour co- culture with tumour cells. Cells were then incubated with GA101 at either 0.1 or 10 μg/ml for 2 hours, after the addition of monocytes (E:T 1:1). After two hours, cells were collected and labelled with the anti-CD11b- APC antibody at 1 μg/ml. % of monocytes that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 2 independent experiments. ns=non-significant, * p<0.05, **** p<0.0001, measured by using a two-way Anova test.

142 4.1.5. Effect of stroma on ADCP mediated by different subset of monocytes

Based on the differential expression of the surface markers CD14 and CD16, monocytes can be subdivided into three distinct populations: classical monocytes, expressing high (Hi) CD14, but no (-) CD16; non-classical monocytes, expressing Hi CD16, low (Lo) CD14; intermediate monocytes, expressing Hi CD14, intermediate (Int) CD16 [187]. It was questioned whether the use of different population of monocytes as effector cells could affect the stroma-mediated protection from ADCP. To address this question, monocytes were isolated from PBMCs and labelled with anti-CD14 and anti-CD16 antibody. A representative plot showing the three different subsets of monocytes is shown in figure 4.13.

The classical population (CD14Hi/CD16-) constituted the great majority (approx. 95%) of monocytes. Non-classical monocytes (CD14Lo/CD16Hi) and intermediate monocytes (CD14Hi/CD16Int) accounted for approx. 5% of monocytes. To understand the contribution of each of the subsets of monocytes to the degree of ADCP observed, the assay was performed as described above, and at the end of the experiment cells were labelled with anti-CD14 and anti-CD16 antibodies, in order to measure the percentages of monocyte subsets phagocytosing tumour cells. However, the extremely low number of CD16+ monocytes in the samples only allowed for the analysis of classical monocyte-mediated ADCP. The CD14Hi/CD16- monocytes seemed to recapitulate what was observed in general with the CD11b+ population: the ADCP induced by GA101 was strongly reduced in the presence of stromal cells (figure 4.14) with the percentage phagocytosis halved in the presence of stroma, compared to treatment on plastic (e.g. 32.24% vs 58.74% for GA101 0.1 μg/ml, p<0.0001in Raji).

143

Figure 4.13. Representative plot showing three different subsets of monocytes based on CD14/CD16 expression. Monocytes were isolated from PBMCs and then labelled with anti-human CD14-APC (Y-axis) and anti-human CD16-PE-Cy7 (X-axis) (0.5 and 0.06 μg/ml, respectively). Three populations can be distinguished: classical monocytes (CD14Hi/CD16-), intermediate monocytes (CD14Hi/CD16Int) and non- classical monocytes (CD14Lo/CD16Hi). The great majority of monocytes belong to the classical population (approx. 93%), while the CD16+ monocytes make up only 5%.

144 A R a ji, A D C P (c la s s ic a l m o n o c y te s ) E : T 1 : 1

s 1 0 0 e

t R a ji

y c

o 8 0 + M 2 1 0 B 4

n * * * * * * * *

6 6 0

H D

K 4 0

+ 4

1 2 0

0 % N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

B D a u d i, A D C P (c la s s ic a l m o n o c y te s ) E : T 1 : 1

s 1 0 0 e

t D a u d i

y c

o 8 0 * * * * + M 2 1 0 B 4

n * * * *

6 6 0

H D

K 4 0

+ 4

1 2 0

0 % N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 8.663 1.231 2.168 0.113 0.1483 8.053 0.685 3.460 0.653 0.3188 0.1 μg/ml 58.744 2.201 32.238 3.651 <0.0001 61.813 2.596 34.456 2.587 <0.0001 10 μg/ml 59.189 1.718 36.478 3.394 <0.0001 63.122 2.572 37.938 2.469 <0.0001

Figure 4.14. Stromal cells strongly decrease ADCP mediated by the classical subset of monocytes. Raji (A) and Daudi (B) were seeded onto the stromal layer for 24 hours. Human monocytes (effector to target ratio 1:1) were added in the culture and cells were treated with GA101 (0.1 and 10 μg/ml). After two hours, cells were collected and stained with anti-CD14-APC and anti-CD16-PE-Cy7 antibodies at 0.5 and 0.12 μg/ml respectively. % of classical monocytes that had engulfed tumour cells was measured as % of CD14+/CD16- cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 3 independent experiments. **** p<0.0001, measured by using a two-way Anova test.

145 To determine whether a similar protective effect in the presence of stroma could be observed in non-classical and intermediate monocyte-mediated ADCP, a CD16+-specific isolation kit was used. This approach ensured a higher yield of the wanted cells (i.e. CD16+ monocytes), separating out the unwanted cells (CD14+ monocytes).

The CD16+-specific isolation kit employed included a positive selection of CD16+ cells; in other words, the CD16 molecules are bound by an anti-CD16 antibody and CD16- expressing cells are then collected. However, this could lead to the occupation of the CD16-binding epitopes and therefore to a weaker labelling efficacy when the CD16+ cells are subsequently labelled in order to be analysed in the assay. For this reason, a different staining approach was used. Non-classical monocytes were isolated from PBMCs using a human CD16+ monocyte isolation kit and then labelled with the CellVue Lavender membrane-labelling dye. Such an approach ensured the labelling of isolated monocytes regardless of the expression status of CD16. The ADCP assay was then performed and data analysed by flow cytometry. In contrast with what was observed for classical monocytes, the degree of ADCP mediated by non-classical monocytes was decreased in the presence of stromal cells by only 8% (GA101 0.1 µg/ml) and 10.6% (GA101 10 µg/ml) in Raji, 6.8% (GA101 0.1 µg/ml) and 6.3% (GA101 10 µg/ml) in Daudi (figure 4.15). The protective effect, therefore, appeared to be more pronounced against classical monocyte- mediated ADCP.

Of note, when looking at dot plots from the flow cytometric analysis, a population of contaminating classical monocytes (CD14+), as well as a population of non-monocytic cells (CD14-/CD16-) can be observed (figure 4.16). This suggests that the CellVue+ population might be including non-phagocytic cells, which would then account for a percentage of the single-positive cells – in other words, cells unable to perform ADCP would be analysed as non-classical monocytes that did not engulf tumour cells. Additionally around 10% of monocytes appeared to be classical monocytes. Therefore, a definitive conclusion as to whether non-classical monocytes are able to kill tumour cells more efficiently than their classical counterpart in the presence of stromal cells cannot be taken.

146 R a ji, A D C P (n o n -c la s s ic a l m o n o c y te s ) - 2 h rs E : T 1 : 1

8 0

s R a ji

e t

y + M 2 1 0 B 4

c 6 0 o

n * * *

+ * *

m 2

4 0

V l

l 2 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

D a u d i, A D C P (n o n -c la s s ic a l m o n o c y te s ) - 2 h rs E : T 1 : 1

8 0

s D a u d i

e t

y n s n s + M 2 1 0 B 4

c 6 0

m 2

4 0

V l

l 2 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 9.796 0.859 4.171 0.494 0.0554 14.684 2.689 6.566 1.300 0.3098 0.1 μg/ml 39.663 1.989 31.663 1.677 0.0036 47.063 3.640 40.238 4.170 0.4582 10 μg/ml 39.438 2.094 28.825 1.938 0.0001 45.925 3.880 39.588 4.716 0.5208

Figure 4.15. Stromal cells do not induce a strong protective effect from non-classical monocyte- mediated ADCP. Raji (A) and Daudi (B) were seeded onto the stromal layer for 24 hours. Human CD16+ monocytes were isolated from PMBCs through the use of a CD16-specific isolation kit and then labelled with CellVue Lavender general membrane-labelling dye. Cells were added in the culture (effector to target ratio 1:1) and wells were treated with GA101 (0.1 and 10 μg/ml) for two hours. % of non-classical monocytes that had engulfed tumour cells was measured as % of CellVue Lavender+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 3 independent experiments. ns=non- significant, ** p<0.01, *** p<0.001, measured by using a two-way Anova test.

147

Figure 4.16. Representative dot plots showing the percentage of CD14+ and CD16+ cells within the CellVue+-isolated cells. CD16+ monocytes were isolated from PBMCs through the use of a CD16+-specific isolation kit and then either left unlabelled (left panel) or labelled with an anti-human CD14-APC antibody (used at 0.5 µg/ml) and an anti-human CD16-PE-Cyanine7 antibody (used at 0.12 µg/ml) (right panel). Labelled samples showed the presence of four different phenotypes in the isolated population: a classical population which was CD14+ but CD16- (top left, approximately 9% of isolated cells), a non-classical population which was CD16+ but CD14- (bottom right, approximately 65% of isolated cells), an intermediated population which was CD14+/CD16+ (top right, approximately 1.7% of isolated cells) and a final population of cells which was CD14-/CD16- (bottom left, approximately 24% of isolated cells) and therefore could not be characterised as monocytes.

148 4.2. Stroma-mediated protection from macrophage-mediated ADCP

Macrophages, which directly differentiate from monocytes after their egression from blood vessels into tissues, are able to perform phagocytosis upon engagement by monoclonal antibodies. To investigate whether the degree of macrophage-mediated ADCP induced by GA101 could be affected by the presence of stroma, monocytes were isolated from PBMCs as previously described and cultured for a week in the presence of 50 ng/ml recombinant human macrophage-colony stimulating factor (M-CSF). After a week, flasks were checked under a light microscope to confirm they had developed into macrophages (defined by a spindle-like shape, compared to the round shape of monocytes). Macrophages were then added to the tumour-stroma co-culture and cells were treated for 2 hours with GA101 (0.1 and 10 μg/ml). Cells were collected, labelled with an anti-human CD11b antibody and analysed by flow cytometry. As observed with monocytes, stroma was able to strongly reduce the degree of macrophage-mediated ADCP (figure 4.17). The percentage of macrophages that had phagocytosed tumour cells halved in the presence of stromal cells (detailed percentages and p-values are shown in figure 4.17).

The experiment was also performed by using an established murine macrophage line, the RAW cell line. Cells were cultured with stromal and tumour cells and treated with GA101 (0.1 and 10 μg/ml) for 2 hours before being labelled with an anti-mouse CD11b and analysed by flow cytometry. Again, the ADCP degree induced by GA101 in the presence of macrophages was halved when tumour cells had been co-cultured with stroma (e.g. Raji: 16.4% on stroma vs 42.5% on plastic for GA101 0.1 μg/ml, p<0.0001; Daudi: 18.9% on stroma vs 46.6% on plastic for GA101 0.1 μg/ml, p=0.0011) (figure 4.18).

149 A R a ji, A D C P (h u m a n m a c ro p h a g e s ) E : T 1 : 1

5 * * * * * * * R a ji

l 4 + M 2 1 0 B 4

n n

a 3

d l

e 2 *

F o 1

0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

B D a u d i, A D C P (h u m a n m a c ro p h a g e s ) E : T 1 : 1 * * * * 5 * * * D a u d i

l 4 + M 2 1 0 B 4

n n

a 3

d l

e 2

F o 1

0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 1.000 0.034 0.218 0.033 0.0489 1.000 0.022 0.162 0.031 0.1141 0.1 μg/ml 3.703 0.416 1.952 0.331 0.0001 3.981 0.516 1.880 0.434 0.0003 10 μg/ml 4.182 0.265 2.159 0.259 <0.0001 4.306 0.362 2.395 0.293 <0.0001

Figure 4.17. Stromal cells mediate protection of tumour cells from human macrophage-mediated ADCP. Raji (A) and Daudi (B) were seeded onto the stromal layer for 24 hours. Human macrophages were grown by culturing monocytes (isolated from PBMCs on day 0) with 50 ng/ml M-CSF, added in the media on day 0 and day 4. Macrophages were then harvested and added in the culture (effector to target ratio 1:1) and cells were treated with GA101 (0.1 and 10 μg/ml). After two hours, cells were collected and stained with the anti-CD11b-APC antibody at 1 μg/ml. % of macrophages that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 3 independent experiments. Fold change is the percentage of ADCP obtained for each condition divided by control (Raji/Daudi non-treated in normal RPMI). * p<0.05, *** p<0.001, **** p<0.0001, measured by using a two-way Anova test.

150 A R a ji, A D C P (R A W m a c ro p h a g e s ) E : T 1 : 1

1 0 0 R a ji

+ 8 0 + M 2 1 0 B 4

2 H

K 6 0 * * * * * * * *

W 4 0

% 2 0

0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

B D a u d i, A D C P (R A W m a c ro p h a g e s ) E : T 1 : 1

1 0 0 D a u d i

+ 8 0 + M 2 1 0 B 4 6

2 * *

H *

K 6 0

W 4 0

% 2 0

0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 13.454 2.652 4.358 1.220 0.1223 14.463 2.778 5.072 1.432 0.4887 0.1 μg/ml 42.467 4.187 16.397 2.721 <0.0001 46.622 7.080 18.952 4.779 0.0011 10 μg/ml 35.467 4.085 13.824 2.653 <0.0001 39.611 7.349 18.022 4.494 0.0133

Figure 4.18. Stromal cells mediate protection of tumour cells from murine macrophage-mediated ADCP. Raji (A) and Daudi (B) were seeded onto the stromal layer for 24 hours. Murine RAW macrophages were then added in the culture (effector to target ratio 1:1) and cells were treated with GA101 (0.1 and 10 μg/ml). After two hours, cells were collected and stained with an anti-mouse CD11b-APC antibody at 2 μg/ml. % of macrophages that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 3 independent experiments. * p<0.05, ** p<0.01, **** p<0.0001, measured by using a two-way Anova test.

151 4.3. Stroma-mediated protection from neutrophil-mediated ADCP

Another subset of immune cells that harbours phagocytic capacities are neutrophils. To understand whether neutrophil-mediated ADCP could be impaired by co-culturing tumour cells with stromal cells, neutrophils were isolated from PBMCs and then used to perform an ADCP assay. Tumour cells were co-cultured with stromal cells for 24 hours, then neutrophils were added to the culture for additional 24 hours. Cells were treated with GA101 at either 0.1 or 10 μg/ml for 24 hours (treatment started after the addition of neutrophils in the culture). Cells were then collected and analysed by flow cytometry, after labelling the immune cells with an anti-CD11b antibody. The degree of ADCP induced by neutrophils was not as high as that which was observed with monocytes and macrophages (figure 4.19). For Raji, the percentage of neutrophils that phagocyted tumour cells was 15.9% for 0.1 μg/ml GA101, 31.9% for 10 μg/ml GA101. For Daudi, percentages were 17.1% for 0.1 μg/ml GA101, 31.5% for 10 μg/ml GA101. The presence of stromal cells, therefore, led to a reduction in phagocytosis, with differences between culture on stroma and culture on plastic which were, however, smaller than what was observed for monocyte- and macrophage-mediated ADCP (phagocytosis on stroma: 12.9% for 0.1 μg/ml GA101 vs 15.9% on plastic, p=0.3333 for Raji; 13.6% for 0.1 μg/ml GA101 vs 17.1% on plastic, p=0.1048 for Daudi; similar significant results were seen with 10 μg/ml GA101).

152 A R a ji, A D C P (h u m a n n e u tro p h ils ) - 2 4 h rs E : T 1 : 1

6 0

+ R a ji 6

2 + M 2 1 0 B 4 H

K *

4 0

l i

h n s

o r

t 2 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

B D a u d i, A D C P (h u m a n n e u tro p h ils ) - 2 4 h rs E : T 1 : 1

6 0

+ D a u d i 6

2 + M 2 1 0 B 4 H

K 4 0 *

l i

h n s

o r

t 2 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 5.248 0.543 2.568 0.540 0.4195 3.644 0.570 2.611 0.408 0.8970 0.1 μg/ml 15.867 1.370 12.904 1.258 0.3333 17.133 0.899 13.603 1.274 0.1048 10 μg/ml 31.911 1.846 26.578 1.851 0.0218 31.489 0.970 26.467 2.046 0.0106

Figure 4.19. Stromal cells mediate protection of tumour cells from human neutrophil-mediated ADCP. Raji (A) and Daudi (B) were seeded onto the stromal layer for 24 hours. Neutrophils were isolated from PBMCs and added in the culture (effector to target ratio 1:1), cells were then treated with GA101 (0.1 and 10 μg/ml). After additional 24 hours, cells were collected and stained with the anti CD11b-APC antibody at 1 μg/ml. % of macrophages that had engulfed tumour cells was measured as % of CD11b+ cells which were PKH26+, after gating out the PKH67- cells. Data are the mean ± SEM of three replicates in 3 independent experiments. ns=non-significant, * p<0.05, measured by using a two-way Anova test.

153 4.4. Discussion

Taken together, the results obtained suggest that the stromal environment is able to mediate tumour cell protection from GA101-induced ADCP. This is the first time such an effect has been studied: in fact, reduction in the degree of anti-CD20 antibody-induced killing was only shown for direct cell death [21], CDC [166] and ADCC [167]. Therefore, this observation constitutes an important finding in terms of clinical implications. Protection from ADCP is dependent on direct contact with stroma rather than on soluble factors released upon co-culture. Just as observed for PCD, the mere adhesion between tumour cells and a substrate such as fibronectin does not lead to a decreased susceptibility to phagocytosis, revealing that more complex mechanisms of protection could take place.

The signalling axes which are potentially responsible for the observed protective effect seem to be initiated shortly after contact between tumour and stromal cells, since a reduced percentage of death in the presence of stroma is already visible in tumour cells after as little as 2 hours of contact. Importantly, a non-specific reduction in phagocytosis is also observed – and this also occurs after short times of co-culture between stromal and tumour cells. Such an observation, therefore, indicates that the presence of stroma protects tumour cells from phagocytes independently of the therapeutic regime. Rather than an acquired resistance, thus, protection is actively achieved through interactions between stromal and tumour components.

Many reports have highlighted the ability of mesenchymal stromal cells to interact with and affect the activity of immune cells – and particularly the innate immune components, of which phagocytic cells are part (reviewed in [188]). In this context, it would be important to investigate whether tumour cells are able to escape monocyte-mediated phagocytosis even when monocytes are added after removal of contact with stromal cells. Performing this experiment could help to understand whether stromal cells are affecting immune cells, rather than tumour cells. Stroma-mediated changes in the phagocyte population could then hamper phagocytosis, leading to the observed protective effect. Nevertheless, a direct effect on the tumour cell population might still be mediated by the stromal microenvironment. Further studies are therefore warranted to address this question.

It is well-known that the cell surface could display molecules, called “don’t eat me” signals, whose activation leads to reduced ability of phagocytic cells to engulf target cells [189]. CD47, as described in 4.5.1, acts as a “don’t eat me” signal by binding to its cognate receptor CD172a on the surface of phagocytes. As CD172a appeared to be also expressed

154 on stromal cells [186], its blockade has been interrogated to understand whether the binding by CD172a on stromal cells could up-regulate CD47 expression, thus reducing ADCP efficacy. However, treatment with an anti-CD172a antibody did not abrogate protection from ADCP. Importantly, several other receptors have been observed to be able to mediate the same effect and classified as “don’t eat me” signals: for instance, expression of the molecule CD31 on live cells prevents the attachment of phagocytes to target cells, thus impeding phagocytosis [190]. One could therefore speculate that, upon contact with stroma, tumour cells activate signalling pathways which ultimately increase CD31 surface expression. This could then act as a “don’t eat me” signal and impede the interaction between phagocytic cells and their targets. Exploring this field would be interesting and could potentially provide researchers with novel insights on the ability of the stromal microenvironment to affect tumour cells in the context of ADCP.

The monocyte population comprises three main subclasses, namely the classical (CD14Hi/CD16-), non-classical (CD14Lo/CD16Hi) and intermediate (CD14Hi/CD16Int) subsets [187]. Gene expression profiling and principal component analysis revealed that the intermediate and non-classical subsets share more similarities in terms of gene expression and cluster separately from the classical subset [187]. In terms of functional features, each different subset plays specific roles in eliminating foreign particles in the body [191]. Classical monocytes, for instance, seem to be able to induce greater phagocytosis than non-classical and intermediate [192]. Non-classical monocytes, on the other hand, efficiently respond to viruses [193]. Intermediate monocytes, as revealed by transcriptomic analyses, tend to differentially express antigen presentation-, inflammation- and angiogenesis-related genes [194]. These studies delineate that the three monocyte subsets can perform a very variegate repertoire of functions. In the context of cancer and ADCP, however, less is known. Classical monocytes are suggested to mediate better phagocytosis; however, contact of stroma to tumour cells appeared to be able to strongly reduce the killing efficacy of such a subtype. Although efforts have been made to try and determine whether the stroma could specifically affect the non-classical and intermediate subsets, technical issues have meant it was not possible to answer such a question. Therefore, more work is needed to further explore the abilities of each subset in regards to ADCP and how the stromal microenvironment could affect these.

Interestingly, it was observed that while the phagocytic ability of both human and murine macrophages was strongly reduced in the presence of stromal cells, the same did not occur when neutrophils were employed. In fact, despite the fact that a significant reduction in

155 phagocytosis was still observed, contact with stroma did not induce a biologically relevant decrease in neutrophil-mediated ADCP. This indicates that there have to be differences in the mechanisms leading to ADCP of tumour cells in monocytes and neutrophils, which would be differently affected by contact with stromal cells. For example, a different set of molecules could mediate interactions of tumour cells with monocytes and neutrophils, and only the expression/functionality of those exploited by neutrophils could remain unaffected upon contact with stroma. At the same time, the reduced ability of stromal cells to protect from neutrophil-mediated ADCP suggests that such effector cells could be further explored in order to obtain more efficient therapeutic strategies for B-cell lymphoma patients.

In summary, this chapter highlighted that stromal cells are able to affect GA101-induced ADCP, reducing monocyte- and macrophage-mediated phagocytosis. Several different questions remain unaddressed: 1) whether protection is dependent on the engagement of “don’t eat me” signals by stroma; 2) whether different subsets of monocytes could be differently affected by the stromal microenvironment; 3) whether the different ability of stroma to protect from neutrophil- and monocyte-mediated ADCP could be further exploited to understand the mechanisms at the basis of the stroma-mediated protection from GA101-induced phagocytosis. Therefore, this chapter highlighted a field of research that should be further exploited and that might have in future great potential in terms of improvement of anti-CD20 monoclonal antibody efficacy.

156 5. Stroma-mediated protection of tumour cells from other mechanisms of action of anti-CD20 mAbs

Anti-CD20 mAbs are able to induce cell death through two additional mechanisms of action, namely CDC and ADCC. Whilst CDC is mainly brought about by type-I antibodies, by virtue of their ability to provoke reorganisation of the cell membrane into lipid rafts (see 1.3.1), ADCC is equally promoted by both type-I and type-II antibodies with higher levels of ADCC by glycoengineered antibodies. For both the modes of action, assays aimed at determining the ability of stroma to mediate protection from killing were performed, and where such a protective effect was observed, the potential mechanisms leading to protection were studied mirroring what was done for PCD and ADCP.

5.1. Stroma-mediated protection of tumour cells from anti-CD20 mAb- induced antibody-dependent cellular cytotoxicity (ADCC)

To measure the degree of ADCC induced by GA101 treatment and understand how stroma could interfere with the antibody’s ability to recruit NK cells and promote cytotoxicity, an indirect assay has been used. Such an assay aims at assessing the degree of ADCC through the measurement of interferon (IFN)-γ that is produced and released by NK cells upon activation [195]. This assay was performed both in the absence and in the presence of stromal cells, to be able to understand the effect of co-culture on GA101 therapeutic efficacy.

5.1.1. Effect of culture with stromal cell on GA101-induced ADCC measured as NK cell activation

In this assay, the effect of the presence of stroma on GA101-mediated NK cell production of IFN-γ was measured by using the co-culture system previously established. Stromal cells were labelled with the membrane-labelling dye PKH67 and seeded on 96-well plates until confluent (approx. 72 hours). Tumour cells were then added to the culture for 24 hours. NK cells, used as effector cells, were isolated from PBMCs by using a NK cell- specific isolation kit. NK cells were treated with the GolgiStop protein transport inhibitor Brefeldin A to ensure that IFN-γ was retained in the cytoplasm rather than released into the

157 surrounding environment. Cells were then added to the co-culture and treated with GA101, at either 0.1 or 10 µg/ml for 4 hours (unless otherwise stated).

At the end of the assay, cells were harvested and labelled for CD56, a surface marker of lymphoid cells. After the labelling, cells were fixed and permeabilised, in order to intracellularly label the cytokine IFN-γ (see 2.6.4). The production of IFN-γ is indicative of NK cell activity [195,196], and therefore indirectly measures the level of NK cell-mediated ADCC. Samples were then analysed by flow cytometry. Percentage of IFN-γ produced by NK cells was measured as % of IFN-γ+ NK cells (positive to both IFN-γ and CD56) – after gating out the PKH67+ cells. Dead cells and debris were excluded from the analysis based on their forward/side scatter values. A FlowJo plot representing the gating strategy that has been used is shown in figure 5.1.

158

Figure 5.1. Representative image of the gating strategy used in FlowJo. The first plot (left-hand side) shows the gating strategy used to separate tumour and NK cells (inside the gate) from stromal cells (outside) based on the negativity for PKH67 (X-axis) prior to analysis of IFN-γ expression. The second plot (middle) shows the gating of NK cells based on CD56 (Y-axis) positivity, versus tumour cells (unstained) outside the gate. The third plot (right-hand side) shows NK cells positivity for IFN-γ (X-axis). The plots are representative of a non-treated sample (control) which was used to determine the IFN-γ gate within the NK cell (CD56+) population.

159 GA101 treatment was able to induce a strong degree of IFN-γ production by NK cells compared to non-treated controls, at both 0.1 (29.4% in Raji, 30.24% in Daudi) and 10 µg/ml (28.94% in Raji, 28.84% in Daudi – all p<0.0001) (figure 5.2). However, in the presence of stromal cells the amount of IFN-γ detected significantly decreased compared to cells cultured on plastic, and such a pattern was observed for both cell lines with 0.1 µg/ml GA101 (12.06% vs 29.4% on plastic, p<0.0001 for Raji, 16.30% vs 30.24% on plastic, p<0.0001 for Daudi) and 10 µg/ml GA101 (12.81% vs 28.94% on plastic, p<0.0001 for Raji, 16.82% vs 28.84% on plastic, p<0.0001 for Daudi). This experiment highlighted the ability of stromal cells to reduce NK cells’ activity, and subsequently the degree of ADCC induced by GA101.

160 A R a ji, A D C C (N K c e lls ) E :T 1 : 1 R a ji

s + M 2 1 0 B 4

l 5 0

K 4 0 * * * * * * * *

v 3 0

m 2 0

a g

N 1 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

B D a u d i, A D C C (N K c e lls ) E :T 1 : 1 D a u d i

s + M 2 1 0 B 4

l 5 0

K 4 0 * * * *

N * * * *

v 3 0

m 2 0

a g

N 1 0

% 0 N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 4.870 0.206 2.408 0.928 0.5920 5.362 0.456 3.149 0.987 0.6830 0.1 μg/ml 29.400 2.097 12.057 0.802 <0.0001 30.244 2.416 16.300 1.084 <0.0001 10 μg/ml 28.944 2.588 12.806 1.120 <0.0001 28.844 1.649 16.818 1.879 <0.0001

Figure 5.2. Stromal cells decrease NK cells’ activity in the presence of GA101. Raji (A) and Daudi (B) cells were cultured either on plastic (green bars) or in the presence of M2-10B4 stromal cells (red bars) for 24 hours. Human NK cells were isolated from PBMCs, treated with a GolgiStop protein transport inhibitor (Brefeldin A solution, used at 3 µg/ml) and then added to the co-culture (effector:target ratio = 1:1) for a further 4 hours. Cells were treated with GA101 at either 0.1 or 10 µg/ml at the same time. Cells were then harvested and stained with an anti-human CD56-APC antibody (at 0.5 µg/ml), before being fixed, permeabilised and intracellularly stained with an anti-human IFN-γ-PE antibody (at 0.5 µg/ml). % of NK cells that had produced IFN-γ was measured as % of CD56+ cells which were IFN-γ+, after gating out the PKH67+ cells. Data are the mean ± SEM of three replicates in 3 independent experiments. **** p<0.0001, measured by using a two-way Anova test.

161 5.1.1.1. Effect of stroma-conditioned media on NK cell activation

To assess whether the decrease in NK cell activity observed in the presence of stroma is mediated by soluble factors, or whether the presence of stromal cells is required to reduce NK cell activity, the indirect ADCC assay was performed in the presence of stroma- conditioned medium. M2-10B4 cells were cultured until confluency and medium was then collected. The collected S-CM was then used to culture Raji and Daudi tumour cells for 24 hours. Human NK cells were isolated by PBMCs through the use of a NK cell-specific kit and added to the wells at effector:target ratio 1:1. Cells were then treated with GA101 at either 0.1 or 10 µg/ml for 4 hours, and wells were harvested and analysed by flow cytometry, after being intracellularly labelled for IFN-γ.

There were no significant differences in NK cell activation with cells treated with GA101 in the presence of S-CM and cells treated in normal RPMI medium. The percentage of NK cells that had produced IFN-γ in the presence of S-CM did not change for either Raji or Daudi in any of the conditions tested (detailed values are shown in figure 5.3). This suggests that the impairment in NK cell activation depends on contact with stromal cells, rather than on soluble factors which are released in the medium.

162 A R a ji, A D C C (N K c e lls a c tiv a tio n )

E :T 1 : 1 s

l 3 0 l

e R P M I

K + S -C M

e 2 0

v n s +

n s

a m

m 1 0

% 0 N T G A 1 0 1 G A 1 0 1 0 .1 u g /m l 1 0 u g /m l

B D a u d i, A D C C (N K c e lls a c tiv a tio n )

E :T 1 : 1 s

l 3 0 l

e R P M I

K + S -C M

e 2 0

v +

n s n s

a m

m 1 0

% 0 N T G A 1 0 1 G A 1 0 1 0 .1 u g /m l 1 0 u g /m l

RAJI + S-CM DAUDI + S-CM

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 3.460 0.354 1.872 0.224 0.5694 1.948 0.407 1.257 0.139 0.9081 0.1 μg/ml 10.607 1.328 9.347 1.199 0.7307 9.470 0.876 8.970 1.090 0.9621 10 μg/ml 9.608 0.897 7.728 1.095 0.4294 9.072 1.048 7.212 0.813 0.3028

Figure 5.3. Culture and treatment in S-CM does not impair the ability of GA101 to activate NK cells. M2-10B4 cells were cultured in 96-well plates until confluency, then the S-CM was collected and used to culture Raji (A) and Daudi (B) cells for 24 hours. NK cells were isolated from PBMCs, treated with 3 µg/ml protein transport inhibitor Brefeldin A and added to the culture (E:T ratio = 1:1). Wells were treated with GA101 at either 0.1 or 10 µg/ml for 4 hours. Cells were then harvested and stained with an anti-human CD56-APC antibody (at 0.5 µg/ml), before being fixed, permeabilised and intracellularly stained with an anti-human IFN-γ-PE antibody (at 0.5 µg/ml). % of NK cells that had produced IFN-γ was measured as % of CD56+ cells which were IFN-γ+. Data are the mean ± SEM of three replicates in 2 independent experiments. ns = non-significant, measured by using a two-way Anova test.

163 5.1.1.2. Effect of different tumour-stroma contact times on NK cell activity

As previously observed, the presence of stromal cells in co-culture with tumour cells is able to decrease the NK cell-mediated production of IFN-γ upon treatment with GA101. To understand whether the NK-cell impaired activity required a minimum contact time between stroma and tumour cells, the assay was repeated, with NK cells added to the co- culture after different tumour-stroma contact times.

M2-10B4 stromal cells were seeded on wells until confluency. Then, Raji and Daudi tumour cells were plated onto the stromal layer. Human NK cells were isolated from PBMCs and added to the co-culture either immediately after plating tumour cells (0 hr co- culture time-point), or after 2 hours, 4 hours or 24 hours (control assay). Cells were treated with GA101 (at either 0.1 or 10 µg/ml) for 4 additional hours. Wells were then harvested and cells were labelled extracellularly for CD56, before fixing and permeabilising them to allow the intracellular staining of IFN-γ.

Interestingly, the stroma-mediated protective effect from NK cell activation did not seem to diminish with shorter contact times (figure 5.4 and 5.5). Specifically, for the 24 hr time- point, the percentage of IFN-γ-positive NK cells was reduced in the presence of stromal cells from 26% to 13.65% for Raji cells treated with 10 µg/ml GA101 (p<0.0001). Similarly, in Daudi cells the percentage of IFN-γ-positive NK cells was reduced from 30.64% to 16.58% after treatment with 10 µg/ml GA101 (p<0.0001). However, the same reduction upon stromal contact was observed for the 2 hr and 4 hr time-points in both Raji and Daudi cells. A similar pattern, although not always statistically significant, was also observed for the 0 hr time-point (details means and p-values are shown in figure 5.4 for Raji, figure 5.5 for Daudi).

164 R a ji, A D C C (N K c e lls a c tiv a tio n ) R a ji E :T 1 : 1 + M 2 1 0 B 4

e 4 0 v

+ * * * * * * * * a * * * * * * * * * *

m 3 0 n s

a g

N 2 0

l e

c 1 0

% 0 0 h r 2 h r 4 h r 2 4 h r 0 h r 2 h r 4 h r 2 4 h r 0 h r 2 h r 4 h r 2 4 h r

N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

NT RAJI + M2-10B4 0.1 RAJI + M2-10B4 Mean ± SEM Mean ± SEM p-value µg/ml Mean ± SEM Mean ± SEM p-value 0 hr 3.053 0.529 1.242 0.097 0.9050 0 hr 18.550 3.298 11.840 2.127 0.2064 2 hr 2.985 0.438 1.368 0.220 0.9337 2 hr 24.133 3.325 13.547 1.622 0.0042 4 hr 2.977 0.450 1.087 0.262 0.9306 4 hr 24.150 3.373 12.950 2.198 0.0088 24 hr 2.915 0.664 1.608 0.558 0.8667 24 hr 27.800 0.835 14.050 1.511 <0.0001

10 RAJI + M2-10B4 µg/ml Mean ± SEM Mean ± SEM p-value 0 hr 22.617 3.623 12.622 1.723 0.0066 2 hr 23.717 3.356 12.063 1.418 0.0016 4 hr 25.550 3.893 12.553 2.077 0.0022

24 hr 26.000 2.307 13.650 1.111 <0.0001

Figure 5.4. Stroma-mediated decrease of NK cell activation is independent of contact times between stromal and Raji cells. Raji cells were co-cultured on the stromal layer for either 0, 2 4 or 24 hrs (control assay). Human NK cells were isolated from PBMCs and treated with the protein transport inhibitor Brefeldin A (3 µg/ml), before adding them to the co-culture and treating the cells with GA101 (either 0.1 or 10 µg/ml) for 4 hours. Cells were then harvested and stained with an anti-human CD56-APC antibody (at 0.5 µg/ml), before being fixed, permeabilised and intracellularly stained with an anti-human IFN-γ-PE antibody (at 0.5 µg/ml). % of NK cells that had produced IFN-γ was measured as % of CD56+ cells which were IFN-γ+, after gating out the PKH67+ cells. Data are the mean ± SEM of two replicates in 2 (24 hr) or 3 (0 hr, 2 hr, 4 hr) independent experiments. ns = non-significant, ** p<0.01, **** p<0.0001, measured by using a two-way Anova test.

165 D a u d i, A D C C (N K c e lls a c tiv a tio n ) D a u d i

E :T 1 : 1 + M 2 1 0 B 4 e

v * * * *

+ 4 0 * * * *

a * * * * * * * * * * * * m * * * n s * *

m 3 0

N F

I 2 0

e c

1 0

% 0 0 h r 2 h r 4 h r 2 4 h r 0 h r 2 h r 4 h r 2 4 h r 0 h r 2 h r 4 h r 2 4 h r

N T G A 1 0 1 0 .1 u g /m l G A 1 0 1 1 0 u g /m l

NT DAUDI + M2-10B4 0.1 DAUDI + M2-10B4 Mean ± SEM Mean ± SEM p-value µg/ml Mean ± SEM Mean ± SEM p-value 0 hr 3.072 0.496 1.432 0.377 0.9204 0 hr 21.375 3.471 14.125 1.957 0.1366 2 hr 3.913 0.213 1.865 0.295 0.8408 2 hr 26.900 3.166 15.110 1.701 0.0005 4 hr 3.714 0.380 1.463 0.076 0.5344 4 hr 31.514 1.904 16.271 1.342 <0.0001 24 hr 3.982 0.556 1.972 0.108 0.0804 24 hr 30.940 0.601 15.880 0.752 <0.0001

10 DAUDI + M2-10B4 µg/ml Mean ± SEM Mean ± SEM p-value 0 hr 24.883 3.337 14.827 1.799 0.0047 2 hr 27.650 2.547 13.997 1.653 <0.0001

4 hr 31.229 1.552 14.866 1.411 <0.0001

24 hr 30.640 0.582 16.580 0.784 <0.0001

Figure 5.5. Stroma-mediated decrease of NK cell activation is independent of contact times between stromal and Daudi cells. Daudi cells were co-cultured on the stromal layer for either 0, 2 4 or 24 hrs (control assay). Human NK cells were isolated from PBMCs and treated with the protein transport inhibitor Brefeldin A (3 µg/ml), before adding them to the co-culture and treating the cells with GA101 (either 0.1 or 10 µg/ml) for 4 hours. Cells were then harvested and stained with an anti-human CD56-APC antibody (at 0.5 µg/ml), before being fixed, permeabilised and intracellularly stained with an anti-human IFN-γ-PE antibody (at 0.5 µg/ml). % of NK cells that had produced IFN-γ was measured as % of CD56+ cells which were IFN- γ+, after gating out the PKH67+ cells. Data are the mean ± SEM of two replicates in 2 (24 hr) or 3 (0 hr, 2 hr, 4 hr) independent experiments. ns = non-significant, ** p<0.01, *** p<0.001, **** p<0.0001, measured using a two-way Anova test.

166 5.1.1.3. Effect of attachment on the ECM component fibronectin on NK cell activity

To investigate whether contact of tumour cells with the ECM component fibronectin could mediate a reduction in IFN-γ production by NK cells, and thus a decreased ability of NK cells to kill target cells, the wells of 96-well plates were coated with human fibronectin (used at 5 µg/cm2). Raji and Daudi tumour cells were then plated for 24 hours either on plastic, or on fibronectin-coated wells, or on the stromal layer. Human NK cells isolated from PBMCs were treated with the protein transport inhibitor Brefeldin A, then added at an effector:target ratio of 1:1. Wells were then treated with GA101 (either 0.1 or 10 µg/ml) for 4 additional hours. At the end of the assay, cells were collected and labelled extracellularly for CD56, before fixing and permeabilising them to allow the intracellular staining of IFN-γ.

In line with what was observed with the PCD and ADCP mechanisms of action, adhesion to fibronectin failed to protect tumour cells from ADCC (or more precisely, failed to reduce activation of NK cells in the presence of tumour cells upon GA101 treatment) (figure 5.6). IFN-γ production, measured as fold change in percentage of NK cells positive to an anti-human IFN-γ antibody, was reduced from 2.035 on plastic to 0.915 on stromal cells in Raji cells treated with 10 µg/ml GA101 (p=0.0005) and from 2.474 on plastic to 1.351 on stromal cells in Daudi cells treated with 10 µg/ml GA101 (p=0.0069), but no decrease was observed in the presence of fibronectin. In fact, there was no significant difference between tumour cells treated on plastic vs tumour cells treated on fibronectin (2.035 on plastic vs 2.354 on fibronectin in Raji treated with 10 µg/ml GA101, p=0.3020; 2.474 on plastic vs 2.483 on fibronectin in Daudi treated with 10 µg/ml GA101, p>0.9999). Detailed means and p-values are shown in figure 5.6.

167

A B

R a ji, A D C C (N K c e lls a c tiv a tio n ) D a u d i, A D C C (N K c e lls a c tiv a tio n ) E :T 1 : 1 E :T 1 : 1 R a ji n s D a u d i 4 + M 2 1 0 B 4 4 n s + M 2 1 0 B 4 n s n s

+ F N * * * + F N l

3 l 3

o o

g * * *

g r

* * r

o h

c 2 c

c 2

F o o 1 F 1

0 0 N T G A 1 0 1 G A 1 0 1 N T G A 1 0 1 G A 1 0 1 0 .1 u g /m l 1 0 u g /m l 0 .1 u g /m l 1 0 u g /m l

RAJI + M2-10B4 + FN

Mean ± SEM Mean ± SEM p-value vs Raji Mean ± SEM p-value vs Raji

NT 1.000 0.198 0.347 0.103 0.0618 0.773 0.113 0.6293 0.1 μg/ml 1.982 0.087 1.003 0.107 0.0023 2.464 0.095 0.0586 10 μg/ml 2.035 0.122 0.915 0.065 0.0005 2.354 0.229 0.3020

DAUDI + M2-10B4 + FN

Mean ± SEM Mean ± SEM p-value vs Raji Mean ± SEM p-value vs Raji

NT 1.000 0.026 0.565 0.200 0.4953 1.151 0.079 0.9114 0.1 μg/ml 2.509 0.264 1.538 0.181 0.0213 2.705 0.280 0.8291 10 μg/ml 2.474 0.204 1.351 0.078 0.0069 2.483 0.102 >0.9999

Figure 5.6. Adhesion of tumour cells to fibronectin does not reduce NK cell activation upon GA101 treatment. Raji (A) and Daudi (B) cells were cultured either on plastic (green bars) or in the presence of M2- 10B4 stromal cells (red bars) or on fibronectin-coated wells (blue bars) for 24 hours. Human NK cells were isolated from PBMCs, treated with a GolgiStop protein transport inhibitor (Brefeldin A solution, used at 3 µg/ml) and then added to the mono- and co-culture (effector:target ratio = 1:1). Cells were treated with GA101 at either 0.1 or 10 µg/ml for 4 additional hours. Cells were then harvested and stained with an anti- human CD56-APC antibody (at 0.5 µg/ml), before being fixed, permeabilised and intracellularly stained with an anti-human IFN-γ-PE antibody (at 0.5 µg/ml). % of NK cells that had produced IFN-γ was measured as % of CD56+ cells which were IFN-γ+, after gating out the PKH67+ cells. Data are the mean ± SEM of two replicates in 2 independent experiments. **** p<0.0001, measured using a two-way Anova test.

168 5.2. Stroma-mediated protection of tumour cells from anti-CD20 mAb- induced complement-dependent cytotoxicity (CDC)

Since stromal cells could protect tumour cells from anti-CD20 mAb-induced PCD and ADCP, it was interesting to understand if the same results could have been observed for mAb-induced CDC. Since type-I anti-CD20 mAbs are known to induce high degrees of CDC in the presence of human serum, whereas only low levels are provoked by type-II mAbs [113], the type-I mAb rituximab was chosen for this assay. To investigate if the presence of stromal cells could decrease the death of tumour cells when cultured in the presence of complement, the established co-culture system was used. However, since M2- 10B4 stromal cells appeared to be sensitive to human serum (figure 5.7), human fibroblastic stromal cells HS-5 were used instead.

After seeding Raji and Daudi cells onto the plates, human serum obtained from healthy donors was added to the wells at concentrations stated and cells were then treated with rituximab for 24 hours. As expected, there was a high degree of tumour cell death in the presence of complement in Raji cells treated with rituximab on plastic (survival %: 24.27 with 5% serum, 14.4 with 10% serum, 9 with 20% serum, figure 5.8 A). However, a low but appreciable degree of protection from death was present in cells co-cultured with HS-5 stromal cells (survival %: 35.73 with 5% serum, p=0.0089 vs cells treated on plastic, 25.02 with 10% serum, p=0.0174 vs cells treated on plastic, 18.85 with 20% serum, p=0.0328 vs cells treated on plastic). Daudi cells, on the other hand, appeared to be less sensitive to complement, with a general lower susceptibility to rituximab-mediated CDC (figure 5.8 B). In the presence of stromal cells, moreover, there was only a small but non-significant increase in viability (percentage of survival: with addition of 5% serum, 67.05 on plastic vs 72.02 in the presence of stromal cells, p=0.6188; with addition of 10% serum, 53.70 vs 60.72 in the presence of stromal cells, p=0.2575; with addition of 20% serum, 40.55 vs 48.03 in the presence of stromal cells, p=0.1996).

169 M 2 1 0 B 4 v ia b ility

1 0 0

8 0

a v

i 6 0

u s

4 0 %

2 0

0 N T R T X + 0 .5 + 1 + 2 .5 + 5 + 1 0

S e r u m c o n c e n tr a tio n (% to ta l m e d ia )

Figure 5.7. Representative graph showing M2-10B4 sensitivity to human serum. M2-10B4 cells were cultured for 24 hours in the presence of either complete RPMI media (“NT”), or media + rituximab (10 µg/ml, “RTX”), or media + rituximab + human serum at either 0.5%, 1%, 2.5%, 5%, 10% of the total volume (1 ml/well). Cells were then analysed by flow cytometry. Viability was measured by staining tumour cells with 7-AAD/AnnexinV. % of survival was measured as % of cells which were 7-AAD-/AnnexinV-. The experiment was performed in duplicates.

170 A R a ji, C D C

1 0 0 R a ji

8 0 + H S -5

a v

i 6 0

v r

u * * s 4 0 *

% *

2 0

0 N T R T X + 5 % + 1 0 % + 2 0 % B D a u d i, C D C

1 0 0 n s D a u d i

8 0 n s + H S -5 l

a n s v

i 6 0

u s

4 0 %

2 0

0 N T R T X + 5 % + 1 0 % + 2 0 %

RAJI + HS-5 DAUDI + HS-5

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 92.767 0.909 87.050 1.046 0.4295 88.067 0.340 82.800 1.080 0.5595 RTX 82.817 2.388 82.383 2.696 >0.9999 73.100 5.055 78.233 2.253 0.5859 + 5% 24.267 4.997 35.733 2.725 0.0089 67.050 2.686 72.017 1.384 0.6188 + 10% 14.367 3.082 25.017 1.256 0.0174 53.700 1.710 60.717 0.888 0.2575 + 20% 9.012 0.862 18.850 1.123 0.0328 40.550 3.747 48.033 2.549 0.1996

Figure 5.8. Stromal cells protect tumour cells from rituximab-induced CDC. The degree of CDC in Raji and Daudi after treatment with rituximab (10 μg/ml) and serum (5%, 10% or 20%) was assessed in the absence (green bars) and in the presence (red bars) of HS-5. Cells, freshly isolated human serum and rituximab were added to the stromal cell-coated plates after 72 hours of single culture of stromal cells. After 24 additional hours, cells were collected and analysed by flow cytometry. Viability was measured by staining tumour cells with 7-AAD/AnnexinV. % of survival was measured as % of PKH67- cells which were 7-AAD- /AnnexinV-. Data are the mean ± SEM of two duplicates in 3 independent experiments. * p<0.05, ** p<0.01, ns=p>0.05, measured using a two-way Anova test.

171 5.2.1. Role of CXCR-4/CXCL-12 axis in stroma-mediated protection from rituximab-induced CDC

Previous literature suggests that the stroma-mediated protection from rituximab-induced CDC could depend on the CXCR-4/CXCL-12 axis. In a study published by Buchner et al, co-culture of B-CLL cells with stromal cells appeared to protect tumour cells from rituximab- and alemtuzumab-induced CDC [166]. Such a protective effect was abrogated upon treatment of cells with the CXCR-4 inhibitor TN14003. Therefore, the role of CXCR-4/CXCL-12 signalling pathway was analysed in the context of type-I antibody- induced CDC.

This assay was only performed with Raji cells, as the presence of stromal cells did not lead to a higher survival in Daudi cells. Raji cells were pre-treated for 1 hour with plerixafor (10 µM), cultured either on plastic or on HS-5 stromal cells and treated with rituximab on its own, or rituximab plus serum (5, 10 or 20%). Cells were then harvested and viability was measured by flow cytometry. The degree of CDC, however, did not significantly diminish in the presence of stromal cells, on the contrary of what was observed in figure 5.8 (figure 5.9). This discrepancy could be due to a different ability of each different donor’s serum to induce CDC. Therefore, it was not possible to determine whether, in those cases where the presence of stroma does mediate protection, the blockade of CXCR- 4 can overcome this effect. Detailed fold changes and p-values are shown in figure 5.9.

172

R a ji, C D C R a ji 1 .5

+ H S -5

o g

r 1 .0 n s t

n n s

a o

h n s n s n s c

c n s

e v

o 0 .5

F o

0 .0 N T R T X + P X F + 5 % + 5 % + 1 0 % + 1 0 % + 2 0 % + 2 0 % + P X F + P X F + P X F

RAJI + HS-5

Mean ± SEM Mean ± SEM p-value

NT 1.000 0.002 0.973 0.004 >0.9999 RTX 0.885 0.014 0.925 0.011 >0.9999 +PXF 0.878 0.012 0.931 0.008 0.9995 + 5% 0.562 0.046 0.607 0.056 0.9999 + 5% +PXF 0.539 0.036 0.605 0.046 0.9975 + 10% 0.367 0.072 0.436 0.091 0.9969 + 10% +PXF 0.319 0.065 0.400 0.082 0.9893 + 20% 0.322 0.095 0.350 0.106 >0.9999 + 20% +PXF 0.332 0.111 0.364 0.120 >0.9999

Figure 5.9. Inhibition of CXCR-4 does not sensitise Raji cells to death by CDC and does not reduce survival mediated by stromal cells. Raji cells were pre-treated for an hour with 10 µM plerixafor and cultured either on plastic or on HS-5 stromal cells for 24 hours. At the beginning of co-culture, cells were treated with rituximab and freshly isolated human serum (5, 10 or 20% of total media). 24 hours later, cells were harvested and analysed by flow cytometry. Viability was measured by staining tumour cells with 7- AAD/AnnexinV. % of survival was measured as % of PKH67- cells which were 7-AAD-/AnnV-. Data are the mean ± SEM of two duplicates in 2 independent experiments. Fold change is the percentage of CDC obtained for each condition divided by control (Raji non-treated in normal RPMI). ns=p>0.05, measured using a two- way Anova test.

173 5.3. Discussion

The results obtained showed that, in addition to protecting tumour cells from GA101- induced PCD and ADCP, the presence of stroma is able to reduce NK cell activation upon co-culture with antibody-opsonized tumour cells, measured as production and release of IFN-γ by NK cells. The modality by which stromal cells impair GA101 ability to activate NK cells’ targeting of tumour cells does not seem to be dependent on soluble factors, because the use of stroma-conditioned media did not recapitulate the same effect. This suggests the existence of similar mechanisms at the basis of protection from all the studied mechanisms of action of GA101. The absence of a reduced NK cell activation in the presence of fibronectin further supports this hypothesis.

The stroma-mediated reduction of NK cell activation can be observed at very early contact time-points. For instance, even immediately after the beginning of tumour and stromal cell contact the addition of NK cells and treatment with GA101 led to a significant reduction in NK cell activation. Whether such an outcome is due to the fact that stromal cells are able to initiate protective signalling pathways in less than the 4 hours which were used to measure NK cell activity, or whether stromal cells are able to affect NK cells directly, instead of tumour cells, and therefore such a protective effect does not depend on contact times, cannot be inferred from the experiments performed. A conclusive experiment would involve the culture of stromal and tumour cells for 24 hours, then separation of the populations by FACS sorting, addition of NK cells to the sorted tumour cells and treatment for 4 hours. If stromal cells are affecting the ability of NK cells to become activated, therefore, the protective effect would not be observed, as stromal cells and NK cells would not enter in contact with each other. Otherwise, a reduced production of IFN-γ would still be measured if protection was dependent on contact between stromal and tumour cells.

Importantly, the assays performed measure production of IFN-γ, and thus NK cell activation, but not direct killing of tumour cells by NK cells. The direct assessment of ADCC has been mainly performed, in the past, by labelling target cells with the radioactive 51Cr and measuring its release into the supernatant by dead tumour cells. This method, however, has been lately avoided by researchers because of the use of radioactive materials. One alternative technique used to measure NK cell-mediated tumour cell death is the calcein AM cell viability assay. Such an assay involves the incorporation of calcein AM into tumour cells and the detection of fluorescence emitted by the hydrolysed calcein, which would only be generated by live cells. However, the described assay could not be

174 successfully performed because of technical issues: in fact, dead tumour cells could release calcein AM before hydrolysation took place, and this efflux of calcein could cause the subsequent uptake by stromal cells. Live stromal cells would then incorporate calcein AM and proceed to its hydrolysation. When reading the results through a microplate reader, it would not be possible to distinguish the fluorescence emitted by tumour cells from the one emitted by stromal cells. This would complicate the picture and make an appropriate analysis not possible. Therefore, it could not be confirmed whether the reduced activation of NK cells actually correlated with a decreased killing of tumour cells in the experimental system analysed. A potential way to overcome this issue could involve the use of a FACS sorting to separate tumour and stromal cells after co-culture and the following assessment of positivity to calcein in tumour cells only (naïve vs stroma-conditioned). However, if no differences were detected, it could not be possible to understand whether this occurred because the removal of contact with stroma abrogates the protective effect, or because stroma does not actually impact on the efficacy of GA101 at inducing NK cell-mediated ADCC. More accurate techniques could in the future help to confirm whether the presence of a stromal microenvironment can impair GA101- and, more generally, mAb-induced ADCC.

As it has been thoroughly described in the literature, GA101 and type-II antibodies do not induce CDC, possibly because of a different modality of binding to the CD20 molecule that causes the lack of lipid raft formation [63] – and, in turn, a weakened ability to recruit the complement system component C1q at the cell membrane and induce CDC [58] (also see 1.3.1). Therefore, in order to investigate whether the presence of stroma could diminish the potency of CDC, the type-I antibody rituximab was employed. Treatment with rituximab did not lead to high percentages of cell death on its own; however, the addition of as little as 5% human serum strongly reduced cell viability in Raji tumour cells. Daudi cells showed a higher resistance to CDC, with significantly lower levels of survival only observed with a concentration of 20% human serum. Interestingly, however, a decrease in rituximab-induced CDC in the presence of stroma was only observed in Raji cells. In an attempt to understand whether the a higher CDC sensitivity could be restored by inhibiting the CXCR-4 receptor, as shown by [166], survival percentages in Raji cells were assessed after combining rituximab with 10 µM plerixafor. Such a strategy, however, did not lead to any further reduction in tumour cell viability, neither in the absence nor in the presence of the stromal microenvironment. Therefore, stroma-mediated protection from rituximab- induced CDC could not always be observed, possibly depending on the different donor’s

175 serum, and the addition of a CXCR-4 inhibitor to type-I anti-CD20 mAb therapy did not seem to improve killing efficacy in the experimental system analysed.

The experiments performed highlighted that contact between tumour and stromal cells is able to reduce the activation of NK cells, measured as production of IFN-γ, and could therefore affect GA101-induced ADCC. The characteristics of such a stroma-mediated protective effect resemble what was observed with PCD and ADCP, as direct contact appeared to be needed for the increased survival of tumour cells to be achieved, and contact with the ECM component fibronectin did not recapitulate the stroma-mediated protection. More studies are needed to 1) explore whether the presence of stroma has a direct impact on NK cell activation; 2) confirm that such a reduced ability to produce IFN- γ by NK cells is reflected by a decreased efficacy in NK cell-mediated ADCC by assessing direct cytotoxicity. This work could thus shed further light on the ways the stromal microenvironment impacts on GA101-induced modes of action, but also on how stroma might directly affect the different immune cell subsets, potentially revealing novel insights into the roles of the microenvironment in conferring resistance to antibody therapy.

176 6. Functional changes in tumour cells’ proteome after co-culture with stroma

The experiments performed and described in the previous chapters have demonstrated the importance of direct contact between stromal and tumour cells for protection from GA101- induced killing. Microscopical investigations also were suggestive that the stromal cells may play a part in remodelling of the actin cytoskeleton which has been established to be part of GA101-induced PCD. However further investigation is required to provide additional mechanistic data regarding the basis of the stroma-mediated protection. The hope is that such mechanistic data would provide further insights in order to allow therapeutic intervention to target pathways that might lead to a more efficient clearance of tumour cells using GA101 treatment in vivo.

Therefore, in order to identify the functional changes that occur in the tumour cell’s proteome when cells are co-cultured with stroma, a large-scale proteomic analysis was performed. The rationale for such an experiment is that, among the numerous signalling changes that co-culture with stroma induces, some of these pathway alterations may be responsible for the phenotypic changes in survival and actin remodelling experimentally observed.

6.1. Mass spectrometry-based proteomics

The introduction of mass spectrometry has revolutionised the field of proteomics, allowing scientists to perform large-scale analyses of proteins with more accuracy and reduced times. The basics of peptide digestion, fragmentation and identification, which are the essential steps of mass spectrometry-based proteomics, have been extensively reviewed in [197]. In recent years, many techniques have been introduced that made not only identification, but also quantification of protein content in cells by mass spectrometry possible. One such technique is SILAC (stable isotope labelling with amino acids in cell culture) [198,199]. SILAC is based on the concept that certain amino acids, known as essential amino acids, cannot be produced by cells and need to be taken up from the surrounding environment. Therefore, if a cell population is grown in a media containing an 13 15 isotopically-labelled essential amino acid, such as the heavier isotope of Lysine C6, N2

177 L-Lysine, each daughter cell will uptake this heavier form of Lysine. After 5 passages in the isotopically-labelled media, virtually every cell will have incorporated the labelled amino acids. Therefore, this will make possible the differentiation of peptides contained in a labelled population from the corresponding peptides contained in an un-labelled population on the basis of the different mass to charge ratio. The SILAC technique was used in the current study, in order to identify proteins which were differentially expressed in tumour cells cultured on stroma compared to tumour cells cultured on plastic.

6.1.1. SILAC experiment

Two different isotopically-labelled media were made as described in Materials and Methods, 2.12. Raji tumour cells were cultured for 5 passages in either medium RPMI, or heavy RPMI. A labelling efficiency test was performed to confirm the incorporation of the isotopically-labelled amino acids (L-Lysine and L-Arginine) in Raji cells (data not shown): this showed a virtually complete incorporation of the labelled amino acids in the cells – for the rest of this chapter, Raji cells will be called medium-Raji or heavy-Raji, depending on the isotope incorporated.

A co-culture assay was set up as previously described. M2-10B4 stromal cells were labelled with a membrane-labelling fluorescent dye and cultured until confluency. Then, heavy-Raji were added on the stromal layer, while medium-Raji were cultured on plastic, for 24 hours. During the 24 hours of co-culture, normal RPMI media was substitute for medium RPMI for medium-Raji and heavy RPMI for heavy-Raji. Cells were then harvested and M2-10B4 were separated from heavy-Raji by FACS sorting. Of note, an aliquot from each Raji population was taken and analysed by flow cytometry to confirm the purity of the sort, showing that the percentage of M2-10B4 stromal cells post-sort was lower than 1% (data not shown).

Proteins contained in each population were analysed by mass spectrometry (see 2.13). This results in the identification of peptide sequences derived from either medium- or heavy- Raji and their relative abundance. Such peptide fragments’ sequences are then matched to theoretically-predicted fragments by Peaks Protein Identification Software (see 2.13), in order to derive the corresponding protein IDs and p-values. The resulting p-values reflect the probability that the match obtained by Peaks is random (further information can be found on http://www.bioinfor.com/dbscoring-tutorial/ and in [173]). All protein IDs taken into consideration for further analysis have p<0.05.

178 6.2. In silico analysis of protein expression changes

Peaks is able to retrieve protein IDs from peptide sequences and provide an identification score (or significance score, equal to -10Log(P-value)) for each of them. The resulting dataset of proteins obtained from the mass spectrometry analysis, thus, was comprised of protein IDs, corresponding heavy to medium ratios, and significance score. This dataset was then subject to further analysis, in order to identify the most over-represented pathways (known as “enriched” pathways) of which the differentially expressed proteins identified are part. The aim of this enrichment analysis, therefore, was to associate all functionally related proteins to their possible functional clusters and thus provide a biological meaning to a large list of proteins.

This was done with the help of two tools, namely DAVID (Database for Annotation, Visualization and Integrated Discovery) and IPA (Ingenuity Pathway Analysis) – see 2.14.

Of note, this assay was performed twice. Protein IDs obtained in the first independent experiment were merged with protein IDs obtained in the second, and the resulting list of IDs, p-values and heavy to medium ratios was used in all following analyses.

6.2.1. DAVID (Database for Annotation, Visualization and Integrated Discovery)

DAVID (available at https://david.ncifcrf.gov/summary.jsp) provides users with a tool to upload proteins IDs and set a specific background, in order to associate proteins from the user’s dataset to biological terms or functional clusters that are enriched in the given proteins. For each of the pathways or clusters identified, DAVID provides an enrichment score and a p-value (described in 2.14 and 2.15).

In order to identify the most enriched pathways or clusters of pathways in the proteins that are differentially expressed in tumour cells cultured on stroma compared to tumour cells cultured on plastic, protein IDs that displayed a fold change >2 or <-2 and p<0.05 were taken into consideration. The significance calculated by Peaks was also considered when selecting the IDs. The background against which the enrichment analysis was performed was Homo Sapiens. A “functional annotation cluster” analysis was performed, with medium stringency (as per default), resulting in 195 clusters of biological terms enriched. As shown in figure 6.1, the most enriched cluster (enrichment score: 28.12) included terms such as “cadherin binding involved in cell-cell adhesion”, “cell-cell adherens junction” and “cell-cell adhesion”. The second most-enriched cluster included terms such as

179 “translational initiation”, “translation”, “ribosomal proteins” and “ribosome” (enrichment score: 25.32), followed by a third cluster including terms such as “mitochondrion” and “mitochondrial matrix” (enrichment score: 20.81). All these clusters had p<0.05. Other clusters followed, with enrichment scores higher than 6.5 (figure 6.2). Interestingly, the cluster including cell-cell adhesion-related terms was the highest enriched cluster, suggesting that molecules involved in cell adhesion might be involved in mediating contact between stromal and tumour cells – and therefore might be responsible for the observed contact-dependent protection from GA101.

180

Figure 6.1. The five most enriched functional clusters identified by DAVID. Protein IDs with p<0.05 and expression fold change >2 or <-2 were analysed with DAVID. A “functional annotation clustering” analysis was performed to visualise the most enriched clusters of pathways and gene terms. The figure shows the five most enriched clusters. For each cluster, an enrichment score and a p-value are calculated (red arrows). Protein IDs are merged from 2 independent experiments and duplicates are only taken into account once. Statistical analyses are performed by DAVID using a modified Fisher’s Exact test (see 2.15).

181

Figure 6.2. Functional clusters with enrichment score higher than 6.5 identified by DAVID. Protein IDs with p<0.05 and expression fold change >2 or <-2 were analysed with DAVID. A “functional annotation clustering” analysis was performed to visualise the most enriched clusters of pathways and gene terms. For each cluster, an enrichment score and a p-value are calculated (red arrows). The figure shows the some of the most enriched clusters, all with enrichment score higher than 6.5. Protein IDs are merged from 2 independent experiments and duplicates are only taken into account once. Statistical analyses are performed by DAVID using a modified Fisher’s Exact test (see 2.15).

182 6.2.2. IPA (Ingenuity Pathway Analysis)

The second software used to identify the most enriched biological terms and pathways in the protein dataset is IPA. Compared to DAVID, IPA has an additional function, which is the prediction of the directionality of an enriched pathway X based on the up-regulation or down-regulation of the user’s dataset of proteins. Therefore, IPA not only retrieves enriched pathways and corresponding p-values (described in 2.15), but also predicts whether a certain pathway will be activated or inhibited – taking into account both the user’s dataset of proteins’ fold changes and previously published studies describing the effect of a certain protein on a pathway X. This directionality can be numerically described as a Z-score, which is defined as the number of standard deviations from the mean of a normal distribution of activity edges – positive Z-scores and negative Z-scores will represent the number of standard deviations either above or below the mean that an activity edge is.

All protein IDs that had p<0.05, fold change >2 or <-2 and were considered significant by Peaks were taken into account. A core analysis in IPA includes several different analytical tools. Of these, canonical pathway analysis was considered the most useful in the current settings.

6.2.2.1. Canonical pathway analysis in IPA

Canonical pathway analysis displays the pathways that are enriched based on the protein IDs uploaded and predicts whether their activation status is changing, and in which direction, based on the fold change associated to each protein. Results are represented as bars, of which the length is a measure of their p-value (expressed as -log(p-value)) and the colour is a measure of their activation status (Z-score, orange for positive, blue for negative). As shown in figure 6.3, the most significantly enriched pathway was “eIF2 signalling”, where “eIF2” stands for “eukaryotic initiation factor 2” – a factor required in eukaryotic translation initiation [200]. Interestingly, amongst the 20 most significantly enriched pathways, three of them were related to translation initiation and translation (figure 6.3, green arrows). All of them were predicted to be activated (with Z-scores of 5.3 for “eIF2 signalling”, 1.5 for “regulation of eIF4 and p70S6K signalling”, 1.7 for “p70S6K signalling”).

Two pathways, namely “remodelling of epithelial adherens junctions” and “epithelial adherens junction signalling”, both related to cell-cell adhesion and adherens junctions, were also highly significant – however the Z-score was only slightly positive (0.6) for the

183 first, and undefined for the latter (figure 6.3, light orange arrows), therefore an overall directionality for this cluster cannot be inferred.

The “NRF2-mediated oxidative stress response” (figure 6.3, blue arrow) was the only pathway among the most significant 20 pathways in IPA that could be considered related to the mitochondrion; otherwise, no other pathways had relations with the “mitochondrion” cluster observed in DAVID. The Z-score (2.5) revealed the activated status of the pathway.

Intriguingly, several actin-related pathways could be found in the most significant 20 canonical pathways (figure 6.3, red arrows), supporting the hypothesis that actin remodelling could be involved in stroma-mediated protection from GA101-induced PCD. All of them had positive or slightly positive Z-scores.

The next 20 most significant pathways, from 21 to 40, included again several actin-related pathways (figure 6.4, red arrows), and “paxillin signalling”, which is part of the cell-cell adhesion-related pathways (figure 6.4, light orange arrows). Three pathways were linked to cell cycle regulation and cell cycle checkpoint (figure 6.4, light green arrows). Finally, the BCR signalling pathway, of which the PI3K/Akt/mTOR axis is part, was significantly up-regulated (“B cell receptor signalling”, Z-score: 1.8; figure 6.4, purple arrow). Both “mTOR signalling” and “PI3K signalling in B lymphocytes” were also present among the most enriched pathways (figure 6.3 and 6.4 respectively, purple arrows), and both were up-regulated based on their positive Z-scores (1.1 and 1, respectively).

Detailed p-values and Z-scores for each pathway are shown in figure 6.5.

184

Figure 6.3. Canonical pathway analysis in IPA showing the most significantly enriched pathways (1 to 20). Protein IDs with p<0.05 and expression fold change >2 or <-2 were analysed with IPA. The bar chart shows the 20 most enriched pathways. The height of each bar represents each pathway’s –log(P-value), the colour represents each pathway’s activation Z-score. Protein IDs are merged from 2 independent experiments and duplicates are resolved by taking the average of their expression fold change values. Statistical analyses are performed by IPA using the Right-Tailed Fisher’s Exact test. Of note, pathways are filtered to only show axes that are potentially relevant in the experimental settings, i.e. cytokine-dependent pathways were excluded as already found not to be responsible for the stroma-mediated protection from GA101-induced killing. Green arrows = translation-related cluster; purple arrow = BCR pathway-related cluster; light orange arrows = adherens junction-related cluster; blue arrow = oxidative stress-related cluster; red arrows = actin- related cluster.

185

Figure 6.4. Canonical pathway analysis in IPA showing the most significantly enriched pathways (21 to 40). Protein IDs with p<0.05 and expression fold change >2 or <-2 were analysed with IPA. The bar chart shows the most enriched pathways (21-40). The height of each bar represents each pathway’s –log(P-value), the colour represents each pathway’s activation Z-score. Protein IDs are merged from 2 independent experiments and duplicates are resolved by taking the average of their expression fold change values. Statistical analyses are performed by IPA using the Right-Tailed Fisher’s Exact test. Of note, pathways are filtered to only show axes that are potentially relevant in the experimental settings, i.e. cytokine-dependent pathways were excluded as already found not to be responsible for the stroma-mediated protection from GA101-induced killing. Red arrows = actin-related cluster; light green arrows = cell cycle-related cluster; purple arrows = BCR pathway-related cluster; light orange arrow = adherens junction-related cluster.

186

Figure 6.5. P-values and Z-scores for each canonical pathway analysed by IPA. The figure shows p- values (expressed as –log(P-value)), ratios and Z-scores for each of the canonical pathways analysed by IPA. –log(P-value)=1.3 corresponds to p=0.05; therefore, higher values of –log(P-value) correspond to p<0.05. Positive Z-scores (>0) represent predicted activation of the pathway, negative Z-scores (<0) represent inhibition. “#NUM!”=undefined Z-score. “Ratio” is the ratio between the number of protein IDs in the dataset that are part of a given pathway, and the number of total proteins known to be part of that given pathway. Statistical analyses are performed by IPA using the Right-Tailed Fisher’s Exact test, Z-scores are calculated by using specific algorithms as described in [201].

187 To try and determine the most biological meaningful pathway, a feature of the canonical pathway analysis tool in IPA, “overlapping pathway”, was employed. “Overlapping pathways” is able to generate a map of pathway interactions, where the most enriched signalling axes are linked to each other based on their shared molecules, i.e. molecules that are experimentally observed in the user’s dataset which are shared by of two or more pathways.

Figure 6.6 shows that, among the 20 most significantly enriched pathways, all share at least one protein – the amount of pathways and connections is such that any interpretation is hard to make. To augment the meaningfulness of the analysis, the settings were amended in order to only take into account the 20 most significantly enriched pathways that share at least 15 molecules (figure 6.7). This highlighted four main clusters of signalling axes: one including actin- and adhesion-related pathways (6.7 A), one including translation initiation and mTOR signalling (6.7 B), one that comprised three mitochondrion-related pathways (6.7 C) and a final one containing three signalling axes with no obvious connection (“protein ubiquitination pathway”, “aldosterone signalling in epithelial cells” and “NRF2- mediated oxidative stress response”, 6.7 D). Interestingly, this appeared to resemble the most enriched clusters observed in DAVID.

188

Figure 6.6. Overlapping pathway analysis performed by IPA showing the 20 most significantly enriched pathways, interconnected if shared proteins are present. Protein IDs with p<0.05 and expression fold change >2 or <-2 were analysed with IPA, “overlapping canonical pathway” tool. The figure shows the 20 most enriched pathways, which are represented connected by a line if a shared protein of the pathway is also present in the user’s dataset. The amount of connections does not allow any meaningful interpretation. Protein IDs are merged from 2 independent experiments and duplicates are resolved by taking the average of their expression fold change values. Of note, the figure includes the 20 most significantly enriched pathways, independently of filters previously applied – therefore, additional pathways are present compared to the list shown in the previous figures.

189

B B

Figure 6.7. Overlapping pathway analysis performed by IPA showing the 20 most significantly enriched pathways that share at least 15 proteins. Protein IDs with p<0.05 and expression fold change >2 or <-2 were analysed with IPA, “overlapping canonical pathway” tool. The figure shows the 20 most enriched pathways which are represented connected by a line if at least 15 shared proteins of the pathway are also present in the user’s dataset. Protein IDs are merged from 2 independent experiments and duplicates are resolved by taking the average of their expression fold change values. Of note, the figure includes the 20 most significantly enriched pathways, independently of filters previously applied – therefore, additional pathways are present compared to the list shown in the previous figures.

190 6.3. In vitro validation of enrichment analysis

The in silico analysis revealed a number of enriched pathways or clusters of pathways, some of which were present in the results from both the tools used, DAVID and IPA. In addition, IPA suggested predicted activation status for the enriched pathways and also suggested additional molecules which, despite being absent from the dataset, could lead to the protein expression changes experimentally observed.

These analyses on their own, however, often include a number of “false positives”: many experimentally observed proteins, in fact, could be a target of different upstream molecules, and therefore participate in different ways in the downstream signalling cascade of many several pathways. Therefore, the choice of the pathways or signalling axes to be validated in additional in vitro experiments was guided by a thorough crosscheck of the published literature – each pathway’s “reliability” was thus double-checked in the light of their links with terms such as “B cells”, “lymphoma”, “stroma” and “microenvironment”. The database PubMed (National Center for Biotechnology Information, Bethesda, USA) was used for this purpose.

The following sections describe the analysed pathways and the in vitro validation performed for each of them, providing the rationale for their choice and unravelling their role in the experimental system.

6.3.1. Role of the B-cell receptor (BCR) signalling pathway

The BCR signalling pathway, of which both the PI3K and mTOR signalling axes are part, resulted to be activated in the proteomic analysis with IPA. Given the finding that the increased tumour cell survival induced by the microenvironment is dependent on direct contact with stromal cells, established B cell survival pathways that might potentially play an important role in cell to cell contact were investigated. The spleen tyrosine kinase Syk, a key mediator of the BCR pathway, was found able to promote CLL tumour cell adhesion to M2-10B4, with its inhibition leading to impaired F-actin formation and attachment to stroma [202].

Another effector molecule downstream of the BCR pathway, the PI3K isoform PI3Kδ, had been recently analysed in the context of tumour-stroma cell adhesion and its blockade was shown to abrogate stroma-mediated protection in Hodgkin Lymphoma and CLL [203].

A third important effector of the BCR pathway is the BCR-associated kinase Bruton’s tyrosine kinase (BTK). The blockade of BTK through the use of the BTK inhibitor

191 ibrutinib in patients with CLL, either previously untreated or relapsed/refractory, led to durable responses in a phase-II trial [204]. In both in vitro and in vivo settings, treatment of CLL cells with ibrutinib impaired the ability of CLL cells to adhere to the ECM component fibronectin [205]. This suggested that BTK could play a potential role in the contact-dependent protection of tumour cells mediated by stroma.

To investigate the role of the BCR pathway and its components in the tumour-stroma system studied, blockade of Syk and of PI3Kδ were performed by using the active metabolite of the inhibitor fostamatinib (R406) or idelalisib (CAL-101) respectively. Tumour cells were pre-treated with either R406 at 4 μM or idelalisib at 10 μM for 30 minutes, prior to the 24-hour co-culture with M2-10B4, in the presence of GA101 (10 μg/ml). Cells were then collected and analysed by flow cytometry. However, inhibition of SYK or PI3Kδ did not prevent stromal cells from protecting against GA101-induced PCD, with increased survival still seen in the presence of stromal cells and inhibitors, suggesting that inhibiting SYK or PI3Kδ did not lead to an abrogation of protection (figure 6.8) (% of survival with R406: 51.07 vs 61.25 with stroma for Raji, p=0.0344; 60.02 vs 71.20 with stroma for Daudi, p=0.007. % of survival with idelalisib: 56.24 vs 70.65 with stroma for Raji, p=0.0008; 53.8 vs 68.35 with stroma for Daudi, p<0.0001). Interestingly, the degree of PCD induced by GA101 in combination with R406 was lower than the level observed with GA101 as a single treatment. Only a relatively small percentage of both Raji and Daudi remained viable when treated with GA101 (33.01% and 33.93%, respectively). However, addition of R406 or idelalisib increased cell survival, thus reducing GA101- induced PCD (51.07% and 56.23%, respectively for Raji and 60.02% and 53.8%, respectively for Daudi) (figure 6.8). Such an effect has also been observed in previously published studies, when the BCR pathway was blocked by the addition of ibrutinib [206], which was linked to a down-regulation of the surface level of the CD20 molecule upon BCR inhibition [207].

192 A R a ji, P C D

1 0 0 R a ji * * *

8 0 * + M 2 1 0 B 4 l

a * * * * v

i 6 0

u s

4 0 %

2 0

0 N T G A 1 0 1 R 4 0 6 G A + R 4 0 6 ID E G A + ID E

B D a u d i, P C D

1 0 0 D a u d i * * *

8 0 * * * * + M 2 1 0 B 4 l

a * * * * v

i 6 0

u s

4 0 %

2 0

0 N T G A 1 0 1 R 4 0 6 G A + R 4 0 6 ID E G A + ID E

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 90.700 0.926 77.550 1.241 0.0027 81.033 1.440 74.133 1.076 0.0764 GA101 33.013 1.417 51.675 3.219 <0.0001 33.933 0.945 53.383 2.332 <0.0001 R406 92.000 0.454 78.450 1.139 0.0018 86.167 1.243 79.350 0.789 0.0825 GA+R406 51.075 6.245 61.250 2.544 0.0344 60.017 2.295 71.200 0.985 0.0007 IDE 91.113 1.173 82.950 0.974 0.1449 80.517 2.634 78.717 2.130 0.9857 GA+IDE 56.238 2.747 70.650 2.438 0.0008 53.800 2.645 68.350 2.703 <0.0001

Figure 6.8. Blockade of Syk or PI3Kδ does not impair stromal-mediated protection from GA101- induced PCD. Raji (A) and Daudi (B) cells were pre-treated with either R406 at 4 μM or idelalisib at 10 μM for 30 minutes, then seeded onto the stromal layer for 24 hours. After 1 hour of co-culture, cells were treated with GA101 (10 μg/ml). Viability was measured by flow cytometry. % of survival was measured as % of PKH67- cells which were 7-AAD-/Annexin V-. Data are the mean ± SEM of two duplicates in 3 independent experiments. * p <0.05, *** p<0.001, **** p<0.0001, measured using a two-way Anova test.

193 The degree of PCD induced by GA101 in combination with the BTK inhibitor ibrutinib was then measured. Tumour cells were pre-treated with 10 µM ibrutinib for an hour and then plated on the stromal layer. After one hour from the beginning of the co-culture, GA101 (10 µg/ml) was added to the wells. 24 hours later, samples were harvested and cell death percentages were calculated by flow cytometry.

As observed with idelalisib and R406, in Daudi cells combination of GA101 and ibrutinib did not lead to abrogation of the protective effect mediated by stromal cells (figure 6.9 B). On the other hand, in Raji cells survival percentages for combination of GA101 with ibrutinib (“Ibru+GA101”) do not significantly differ between culture on plastic and culture on stroma (figure 6.9 A, p=0.1917). Such a result, however, is clearly due to the impaired efficacy of GA101 at inducing PCD in cells that are pre-treated with ibrutinib. Survival percentages in the combination-treated cells in the absence of stroma, in fact, are higher than what is observed for cells treated with GA101 only (60.15% with ibrutinib vs 22.85% without ibrutinib, for Raji; 42.47% with ibrutinib vs 19.67% without ibrutinib, for Daudi). As previously mentioned, the study conducted in 2014 by Bojarczuk et al. suggested that the blockade of the BCR pathway could interfere with CD20 expression on tumour cells. Therefore, surface levels of CD20 in Raji and Daudi after treatment with ibrutinib were then analysed.

194 A R a ji, P C D R a ji B D a u d i, P C D D a u d i

+ M 2 1 0 B 4 + M 2 1 0 B 4

1 0 0 1 0 0 n s * * * *

8 0 8 0 l

* * * * l a

a * * * *

v v

i 6 0

4 0 4 0

% %

2 0 2 0

0 0 N T G A 1 0 1 Ib ru Ib ru + G A 1 0 1 N T G A 1 0 1 Ib ru Ib ru + G A 1 0 1

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

NT 82.875 5.149 75.150 3.651 0.4547 88.625 0.085 79.025 0.986 0.0523 GA101 22.850 3.347 50.150 5.095 <0.0001 19.675 2.693 45.025 5.225 <0.0001 Ibru 89.050 1.469 86.225 2.450 0.9696 87.600 0.492 87.075 1.615 0.9998 Ibru+GA101 60.150 2.805 70.525 3.128 0.1917 42.475 2.831 70.025 2.288 <0.0001

Figure 6.9. Combination of GA101 with the BTK inhibitor ibrutinib does not abrogate stroma- mediated protection of tumour cells. Raji (A) and Daudi (B) cells were pre-treated with the BTK inhibitor ibrutinib at 10 μM for 1 hour, then seeded onto the stromal layer for 24 hours. After 1 hour of co-culture, cells were treated with GA101 (10 μg/ml). Viability was measured by flow cytometry. % of survival was measured as % of PKH67- cells which were 7-AAD-/Annexin V-. Of note, for Raji cells treated with GA101 in combination with ibrutinib (“Ibru+GA101”) survival in the presence of stromal cells does not significantly differ from survival on plastic; however, this appears to be due to a low degree of cell death induced by GA101 on plastic, rather than a reduction in the protective effect mediated by stromal cells. Data are the mean ± SEM of two duplicates in 2 independent experiments. ns = non-significant, **** p<0.0001, measured using a two-way Anova test.

195 6.3.2. Effect of blockade of the BCR pathway on CD20 expression

As observed with R406 and idelalisib, GA101 treatment induced a lower degree of cell death in cells on plastic when in combination with ibrutinib (see 6.3.1, figures 6.8 and 6.9). To understand whether such a result had been brought about by a reduction in CD20 surface expression, surface levels of the CD20 molecule in Raji and Daudi were measured by flow cytometry. Cells were pre-treated with ibrutinib, cultured either on plastic or on stroma for 24 hours, then harvested and stained with either anti-human CD20-APC antibody or mouse IgG2b, κ isotype control. The FACS analysis confirmed that cells pre- treated with ibrutinib expressed lower level of CD20 on their surface compared to non- treated cells (MFI relative to unstained controls for treated Raji: 69.23 vs 192.2 for non- treated cells, p<0.0001, figure 6.10; MFI relative to unstained controls for treated Daudi: 44.6 vs 86.3 for non-treated cells, p<0.0001, figure 6.11). In the presence of stromal cells, there was a further reduction in CD20 expression, for both non-treated cells (111.7 vs 192.2 without stroma, p<0.0001 for Raji; 46.2 vs 86.3 without stroma, p<0.0001 for Daudi) and ibrutinib-treated cells (46.9 vs 69.2 without stroma, p=0.0117 for Raji; 20.9 vs 44.6 without stroma, p<0.0001 for Daudi).

Although this experiment did not show that the decrease in GA101-induced cell death in ibrutinib-treated cells is due to a reduction in CD20 expression, it strongly suggested that such a lowered expression might be at the basis of the impaired ability of GA101 to kill tumour cells that were pre-treated with ibrutinib.

196

R a ji, C D 2 0 e x p re s s io n

2 5 0

* * * * R a ji s

l 2 0 0 + M 2 1 0 B 4

e o

v 1 5 0

l e

e *

r n

1 0 0

s M

n 5 0 u

0 N T Ib r u tin ib

RAJI + M2-10B4

Mean ± SEM Mean ± SEM p-value for M2-10B4 vs Raji

NT – unstained 1.000 0.017 0.792 0.029 >0.9999 NT – CD20 192.192 8.658 111.723 7.988 <0.0001 Ibrutinib – unstained 0.772 0.030 0.695 0.036 >0.9999 Ibrutinib – CD20 69.230 6.300 46.900 1.884 0.0117 p-value for Ibrutinib (CD20) <0.0001 <0.0001 vs NT (CD20)

Figure 6.10. Pre-treatment of Raji cells with ibrutinib leads to a reduction in the surface level of CD20. Raji cells were pre-treated with the BTK inhibitor ibrutinib at 10 μM for 1 hour, then seeded onto the stromal layer for 24 hours. After 24 hours, cells were collected and either left unlabelled, or incubated with isotype control (mouse IgG2b,κ-APC), or anti-CD20-APC antibody (0.24 μg/ml). Percentage of positivity to the antibodies was measured as % of PKH67- cells which were APC+. The MFI (geometric mean) measured for isotype controls was then subtracted by the MFI of stained samples. Data are the mean ± SEM of the MFI (shown as fold change over unstained control on plastic) of 2 replicates in 2 independent experiments. * p<0.05, **** p<0.0001, measured using a two-way Anova test.

197

D a u d i, C D 2 0 e x p re s s io n

* * * * 1 0 0

D a u d i s

l 8 0 + M 2 1 0 B 4

e * * * * o

v 6 0

r n

4 0

s M

n 2 0 u

0 N T Ib r u tin ib

DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value for M2-10B4 vs Daudi

NT – unstained 1.000 0.011 0.813 0.040 >0.9999 NT – CD20 86.356 3.047 46.203 3.397 <0.0001 Ibrutinib – unstained 0.696 0.059 0.665 0.052 >0.9999 Ibrutinib – CD20 44.626 3.596 20.892 1.803 <0.0001 p-value for Ibrutinib (CD20) <0.0001 0.0001 vs NT (CD20)

Figure 6.11. Pre-treatment of Daudi cells with ibrutinib leads to a reduction in the surface level of CD20. Daudi cells were pre-treated with the BTK inhibitor ibrutinib at 10 μM for 1 hour, then seeded onto the stromal layer for 24 hours. After 24 hours, cells were collected and either left unlabelled, or incubated with isotype control (mouse IgG2b,κ-APC), or anti-CD20-APC antibody (0.24 μg/ml). Percentage of positivity to the antibodies was measured as % of PKH67- cells which were APC+. The MFI (geometric mean) measured for isotype controls was then subtracted by the MFI of stained samples. Data are the mean ± SEM of the MFI (shown as fold change over unstained control on plastic) of 2 replicates in 2 independent experiments. **** p<0.0001, measured using a two-way Anova test.

198 6.3.3. Effect of co-culture with stroma on expression level of surface CD20 molecule

Previous studies had highlighted that co-culture between B-CLL cells and mesenchymal stromal cells leads to a reduction in CD20 surface expression [167]. This, subsequently, provokes a reduction in the ability of anti-CD20 monoclonal antibodies to bind and kill B cells, ultimately leading to a pro-survival effect. Such an effect was also observed in the current experimental settings – in figure 6.10 and 6.11, both tumour cell lines showed a reduced expression of CD20 in the presence of stromal cells in both untreated and ibrutinib-treated conditions. To investigate whether a similar reduction could be replicated with different stromal lines, M2-10B4 or HS-5 cells were co-cultured with Raji (figure 6.12) and Daudi (figure 6.13) cells for 24 hours. After co-culture, cells were collected, stained for surface CD20 and analysed by flow cytometry. Strikingly, surface levels of CD20 were significantly decreased after culture with stromal cells, compared to culture on plastic (MFI (geometric mean fluorescence intensity) relative to unstained controls: 62.9 on M2-10B4, 64.8 on HS-5 vs 94.4 on plastic for Raji, p<0.0001 for both stromal lines; 37.1 on M2-10B4, 44.6 on HS-5 vs 68.9 on plastic for Daudi, p=0.0005 for M2-10B4, p=0.0083 for HS-5). Detailed values are shown in figures 6.12 and 6.13 for Raji and Daudi respectively.

199 R a ji, C D 2 0 e x p re s s io n

* * * *

1 2 0 * * * *

R a ji s

l 1 0 0

+ M 2 1 0 B 4

e 8 0 + H S -5

a d

l 6 0

i I

a 4 0

M n

u 2 0

An ti-C D 2 0

RAJI + M2-10B4 + HS-5

Mean ± SEM Mean ± SEM p-value vs Raji Mean ± SEM p-value vs Raji

Unstained 1.000 0.025 0.845 0.048 >0.9999 0.882 0.053 >0.9999 Anti-CD20 94.396 6.281 62.951 5.483 <0.0001 64.793 4.270 <0.0001

Figure 6.12. Co-culture of Raji cells with stromal cells significantly decreases surface levels of the CD20 molecule. Raji cells were cultured for 24 hours on either plastic (green bars) or M2-10B4 (red bars) or HS-5 (blue bars). After 24 hours, cells were collected and either left unlabelled, or incubated with isotype control (mouse IgG2b-APC), or anti-CD20-APC antibody (0.24 μg/ml). Percentage of positivity to the antibodies was measured as % of PKH67- cells which were APC+. The MFI (geometric mean fluorescence intensity) measured for isotype controls was then subtracted by the MFI of stained samples. Data are the mean ± SEM of MFI (shown as MFI relative to unstained control on plastic) of 2 replicates in 3 independent experiments. **** p<0.0001, measured using a two-way Anova test.

200 D a u d i, C D 2 0 e x p re s s io n

* * 1 0 0 * * *

D a u d i s

l 8 0 + M 2 1 0 B 4

e + H S -5 o

v 6 0

r n

4 0

s M

n 2 0 u

An ti-C D 2 0

DAUDI + M2-10B4 + HS-5

Mean ± SEM Mean ± SEM p-value vs Daudi Mean ± SEM p-value vs Daudi

Unstained 1.000 0.020 0.844 0.023 >0.9999 0.890 0.015 >0.9999 Anti-CD20 68.948 11.137 37.075 3.756 0.0005 44.611 5.384 0.0083

Figure 6.13. Co-culture of Daudi cells with stromal cells significantly decreases surface levels of the CD20 molecule. Daudi cells were cultured for 24 hours on either plastic (green bars) or M2-10B4 (red bars) or HS-5 (blue bars). After 24 hours, cells were collected and either left unlabelled, or incubated with isotype control (mouse IgG2b-APC), or anti-CD20-APC antibody (0.24 μg/ml). Percentage of positivity to the antibodies was measured as % of PKH67- cells which were APC+. The MFI (geometric mean fluorescence intensity) measured for isotype controls was then subtracted by the MFI of stained samples. Data are the mean ± SEM of MFI (shown as MFI relative to unstained control on plastic) of 2 replicates in 3 independent experiments. ** p<0.01, *** p<0.001, measured using a two-way Anova test.

201 6.3.4. Role of cadherin-mediated cell-cell adhesion

The calcium (Ca2+)-dependent cell adhesion molecules (cadherins) are the main components of adherent junction complexes in epithelia and play a fundamental role in maintaining tissue morphology by regulating both homotypic and heterotypic cell-cell interactions [208]. Several reports have recently highlighted altered patterns of expression of cadherin molecules both in B-cell malignancies and other tumour types. The protocadherin family of cadherins, normally involved in the development of the nervous system, was shown to be up-regulated in both nodal (NFL) and duodenal (DFL) follicular lymphoma [209]. In B-CLL cells, functional E-cadherin RNA was found to be depressed compared to that of normal B cells via a loss-of-function mutation, and this event was responsible for the up-regulation of the Wnt/β-catenin pathway, which plays pro-survival roles in B-CLL [210]. Intriguingly, similar decreased levels of functional E-cadherin were also found to occur in Raji cells [210].

Micro-environmental cues are able to influence cadherin expression and activity in tumour cells. In mantle cell lymphoma, the adhesion of tumour cells to the human bone marrow cell line HS-5 induced an increase in E-cadherin protein content, and such an effect was only observed when direct contact between stromal and tumour cells was allowed [211]. The expression of N-cadherin on T-cell lymphoma cells was found to mediate both homotypic adhesion between tumour cells and heterotypic adhesion between T cells and fibroblasts, suggesting a putative role for N-cadherin in interacting with and enabling migration into the stromal layer [212]. Similarly, N-cadherin-mediated interactions between malignant CML cells and mesenchymal stromal cells led to an enhancement in the activity of the Wnt/β-catenin pathway, thus increasing the stroma-mediated protection from small molecule inhibitors [213].

To understand whether the enrichment observed for the cadherin-mediated cell-cell adhesion pathway in proteomic analyses matched an altered expression of cadherin molecules in the current in vitro settings, Raji and Daudi cells were cultured either on plastic or on PKH67-labelled stromal cells for 24 hours. Cells were then separated on the basis of their positivity to the PKH67 dye through the use of a FACS sorter. Tumour cells, negative to PKH67, were collected and lysed as described in 2.9. A western blot was then performed to visualise differences in total cadherin protein content, using an anti-pan cadherin antibody (used at 1:1000). As shown in figure 6.14, while no bands were observed in both Raji and Daudi cells cultured on plastic, proteins were bound by the antibody in cells cultured on stroma. As expected, the antibody bound approximately

202 around 130 kDa. This suggested that the contact with stroma leads to an increase in the amount of total cadherin molecule expressed by tumour cells (i.e. both membrane-bound and intracellular).

203

A Pan-cadherin

R RM D DM

179 kDa Pan-cadherin

124 kDa

40 kDa GAPDH

B P a n -c a d h e rin e x p re s s io n

5 0 * * P la s tic

y 4 0

t + M 2 1 0 B 4 i

s *

n e

t 3 0

e v

i 2 0

t a

l e

R 1 0

0 R a ji D a u d i

Plastic + M2-10B4

Mean ± SEM Mean ± SEM p-value

Raji 1.000 0.000 21.498 5.666 0.0300 Daudi 1.533 0.563 35.450 7.490 0.0019

Figure 6.14. Culture of tumour cells on stromal cells increases the expression of total cadherin. A) Raji and Daudi cells were cultured either on plastic (“R”, “D” respectively) or on stromal cells M2-10B4 (“RM, “DM” respectively) for 24 hours. Cells were then harvested and lysed. 20 µg of each lysate were loaded onto a SDS-PAGE, transferred to a PVDF membrane and incubated O/N with a rabbit polyclonal anti-pan cadherin antibody (used at 1:1000, bands at approx. 130 kDa). A rabbit monoclonal anti-GAPDH antibody (1:1000) was used as loading control (bands at approx. 37 kDa). An anti-rabbit IgG (H+L) HRP-conjugated secondary antibody (used at 1:2500) was then employed to visualise the bands by electrochemiluminescence. The western blot shown is representative of N=3 independent experiments. B) The graph shows the relative intensity of each band compared to the loading control (GAPDH), calculated using ImageJ to measure each band’s areas, normalised to Raji on plastic. Data are the mean ± SEM of 3 independent experiments. * p<0.05, ** p<0.01, measured using a two-way Anova test.

204 To determine whether such an increase in total amount of cadherin molecules could be responsible for the stroma-mediated protection, and not just a direct consequence of cell adhesion on a substrate, the same assay has been performed after culturing tumour cells on the ECM component fibronectin. Wells were coated with 5 µg/cm2 human fibronectin as described in 2.6, and Raji and Daudi tumour cells were added on the wells for 24 hours. Cells were then lysed as described in 2.9 and protein content was calculated. A western blot was then performed to visualise differences in total cadherin protein content, using an anti-pan cadherin antibody (1:1000). Figure 6.15 shows that culture of tumour cells on fibronectin does not lead to up-regulation of total cadherin molecules. In fact, no bands were seen approximately around 130 kDa in either tumour cells alone (“R”, “D”) or tumour cells on fibronectin (“RF”, “DF”). Of note, the presence of clear bands corresponding to the loading control (GAPDH, ̴ 37 kDa) showed that proteins from lysates were correctly loaded onto the gel (figure 6.15).

To further confirm the finding that the amount of total cadherin in proteins from tumour cells increases after co-culture with stromal cells, but not after culture on fibronectin- coated wells, the experiment was repeated by comparing the two different conditions at the same time. Raji and Daudi tumour cells were either cultured on plastic, or on stroma or on fibronectin. Cells were then separated through FACS sorting and tumour cells only were collected. The expression of total cadherin was then measured by performing a western blot. Figure 6.16 shows that, while the anti-pan cadherin antibody clearly bound to cadherin molecules in the bands containing lysates from tumour cells cultured on stroma (“RM” and “RD” for Raji and Daudi, respectively), the same outcome could not be observed in bands containing lysates from tumour cells cultured on fibronectin (“RF” and “DF” for Raji and Daudi, respectively). Again, the presence of bands corresponding to the loading control GAPDH demonstrates that approximately the same amount of proteins was loaded in each lane, ruling out the possibility that the absence of cadherin molecules from the bands corresponding to tumour cells cultured on fibronectin is just an artefact (figure 6.16). Of note, when measuring the intensity of each band relative to the loading control GAPDH, a significant difference in pixel intensity between cells on plastic or fibronectin and cells on stroma was not observed but as before there was a trend to increased cadherins in tumour cells cultured on stroma.

205

A Pan-cadherin

Pan-cadherin

GAPDH

B P a n -c a d h e rin e x p re s s io n 3 0

P la s tic y

t + F N

i s

n 2 0

e v

i n s

t 1 0

a n s

l e R

0 R a ji D a u d i

Plastic + FN

Mean ± SEM Mean ± SEM p-value

Raji 1.000 0.000 0.987 0.498 >0.9999 Daudi 2.801 1.470 3.312 1.651 0.9452

Figure 6.15. Culture of tumour cells on the ECM component fibronectin does not lead to an increase in the amount of total cadherin content. A) Raji and Daudi cells were cultured either on plastic (“R”, “D” respectively) or on 5 µg/cm2 fibronectin (“RF”, “DF” respectively) for 24 hours. Cells were then harvested and lysed. 20 µg of each lysate were loaded onto a SDS-PAGE, transferred to a PVDF membrane and incubated O/N with a rabbit polyclonal anti-pan cadherin antibody (used at 1:1000). An anti-rabbit IgG (H+L) HRP-conjugated secondary antibody (used at 1:2500) was then employed to visualise the bands by electrochemiluminescence. The western blot shown is representative of N=2 independent experiments. B) The graph shows the relative intensity of each band compared to the loading control (GAPDH), calculated using ImageJ to measure each band’s areas, normalised to Raji on plastic. Data are the mean ± SEM of 2 independent experiments. ns = non-significant, measured using a two-way Anova test.

206

A Pan-cadherin

Pan-cadherin

GAPDH

P a n -c a d h e rin e x p re s s io n B n s

H n s P la s tic

D 1 2 0 P + M 2 1 0 B 4

A n s G

9 0 + F N o

t n s

e v

i 6 0

e r

3 0 y

t 5

s n

e 0

t n

I R a ji D a u d i

Plastic + M2-10B4 + FN

Mean ± SEM Mean ± SEM p-value Mean ± SEM p-value vs Plastic vs Plastic Raji 1.000 0.000 70.421 38.422 0.0701 0.692 0.203 >0.9999

Daudi 1.809 0.477 41.903 11.251 0.3495 1.131 0.530 >0.9999

Figure 6.16. Culture of tumour cells on stromal cells, but not on the ECM component fibronectin, leads to an increase in the amount of total cadherin content. A) Raji and Daudi cells were cultured either on plastic (“R”, “D” respectively) or on stromal cells M2-10B4 (“RM, “DM” respectively) on 5 µg/cm2 fibronectin (“RF”, “DF” respectively) for 24 hours. Cells were then harvested and lysed. 20 µg of each lysate were loaded onto a SDS-PAGE, transferred to a PVDF membrane and incubated O/N with a rabbit polyclonal anti-pan cadherin antibody (used at 1:1000). An anti-rabbit IgG (H+L) HRP-conjugated secondary antibody (used at 1:2500) was then employed to visualise the bands by electrochemiluminescence. The western blot shown is representative of N=2 independent experiments. B) The graph shows the relative intensity of each band compared to the loading control (GAPDH), calculated using ImageJ to measure each band’s areas, normalised to Raji on plastic. Data are the mean ± SEM of 2 independent experiments. ns = non-significant, measured using a two-way Anova test.

207 The western immuno-blots confirmed an up-regulated expression of cadherins in the presence of stromal cells, but did not shed light into which of the numerous members of the cadherin family could be responsible for the increased amount of protein bound by the antibody. E-cadherin (CDH-1, CD324) and N-cadherin (CDH-2, CD325) are the most common cadherin molecules in human tissues. Given the cited literature, it seemed reasonable to focus the attention on E- and N-cadherin. Thus, a flow cytometric assay was performed. Tumour cells were cultured either on plastic or on stromal cells for 24 hours and then harvested and labelled with the appropriate antibody. The expression of E- cadherin was first measured, and interestingly, the analysis revealed a statistically significant decrease in the amount of E-cadherin expressed on the surface of Raji cells after culture on stromal cells (figure 6.17 A). The geometric mean fluorescence intensity relative to unstained control for Raji cells cultured on plastic was 2.771, but only 1.668 for cells cultured on stroma (p<0.0001). This result was not observed in Daudi cells (figure 6.17 B). Detailed geometric means and p-values are shown in figure 6.17.

Next, the expression of N-cadherin on the surface of tumour cells was measured. As observed for E-cadherin, the expression of transmembrane N-cadherin on the surface of Raji cells was decreased after culture with stromal cells (geometric mean fluorescence intensity relative to unstained control: 2.428) compared to culture on plastic (2.992, p=0.0003). However, such a statistically significant difference was not observed in Daudi cells (geometric mean fluorescence intensity relative to unstained control: 2.008 on stroma vs 2.375 on plastic, p=0.6907) (figure 6.18).

208 A R a ji, C D 3 2 4 (E -c a d h e rin ) e x p re s s io n

R a ji s

l 4 + M 2 1 0 B 4 o

o * * * *

e o

v 3

r n

s M

n 1 u

0 U n s ta in e d A n ti-C D 3 2 4

B D a u d i, C D 3 2 4 (E -c a d h e rin ) e x p re s s io n

D a u d i s

l 4 + M 2 1 0 B 4

r t

t n s

e o

v 3

r n

s M

n 1 u

0 U n s ta in e d A n ti-C D 3 2 4

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

Unstained 1.000 0.010 0.745 0.024 0.2212 1.000 0.013 0.869 0.019 0.8006 Anti-CD324 2.771 0.207 1.668 0.129 <0.0001 2.020 0.236 1.954 0.195 0.9442

Figure 6.17. E-cadherin expression on the surface of Raji, but not Daudi, tumour cells decreases after co-culture with stromal cells. Raji (A) and Daudi (B) tumour cells were cultured either on plastic (green bars) or on a confluent layer of M2-10B4 stromal cells (red bars) for 24 hours. Cells were then harvested and either left unstained, or stained with anti-human CD324 (E-cadherin)-PE antibody, or with a mouse IgG1-κ- PE isotype control (both used at 1 µg/ml). Percentage of positivity to the antibodies was measured as % of PKH67- cells which were PE+. The MFI (geometric mean fluorescence intensity) measured for isotype controls was then subtracted by the MFI of stained samples. Data are the mean ± SEM of the MFI (shown as geometric mean fluorescence intensity relative to unstained control on plastic) of 2 replicates in 3 independent experiments. ns = non-significant, **** p<0.0001, measured using a two-way Anova test.

209 A

R a ji, C D 3 2 5 (N -c a d h e rin ) e x p re s s io n

* * * R a ji

s l

o + M 2 1 0 B 4

r t

t 3

a d

l 2 e

e *

t s

M 1

n u

0 U n s ta in e d A n ti-C D 3 2 5

D a u d i, C D 3 2 5 (N -c a d h e rin ) e x p re s s io n

n s D a u d i

s l

o + M 2 1 0 B 4

r t

t 3

a d

l 2

t s

M 1

n u

0 U n s ta in e d A n ti-C D 3 2 5

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± SEM p-value Mean ± SEM Mean ± SEM p-value

Unstained 1.000 0.031 0.700 0.022 0.0250 1.000 0.010 0.765 0.039 0.8573 Anti-CD325 2.992 0.111 2.428 0.085 0.0003 2.375 0.464 2.008 0.474 0.6907

Figure 6.18. N-cadherin expression on the surface of Raji, but not Daudi, tumour cells decreases after co-culture with stromal cells. Raji (A) and Daudi (B) tumour cells were cultured either on plastic (green bars) or on a confluent layer of M2-10B4 stromal cells (red bars) for 24 hours. Cells were then harvested and either left unstained, or stained with anti-human CD325 (N-cadherin)-PE antibody, or with a mouse IgG1-κ- PE isotype control (both used at 1 µg/ml). Percentage of positivity to the antibodies was measured as % of PKH67- cells which were PE+. The MFI (geometric mean fluorescence intensity) measured for isotype controls was then subtracted by the MFI of stained samples. Data are the mean ± SEM of the MFI (shown as geometric mean fluorescence intensity relative to unstained control) of 2 replicates in 2 (A) or 3 (B) independent experiments. ns = non-significant, * p<0.05, *** p<0.001, measured using a two-way Anova test.

210 The finding that both E- and N-cadherin expression was decreased on Raji cell surface after contact with stroma seemed to contrast with the increase in total cadherin protein content observed by western blot. However, it is fundamental to keep into consideration that the expression of cadherin measured by flow cytometry only reflects the amount of molecule expressed on the cell’s surface. The amount of protein detected by western blot, on the other hand, is a measure of the total cadherin (so any members of the cadherin family) contained in the cells – this means membrane-bound, but also intracellular.

To further understand the role that the cadherin family of molecules plays in the tumour- stroma co-culture system, the extracellular expression of VE-cadherin (CD144) (figure 6.19) and P-cadherin (figure 6.20) was analysed. However, none of these molecules appeared to be expressed on the surface of either Raji or Daudi cells. Therefore, this data suggests that none of these molecules could be, at least in terms of extracellular expression, responsible for the increased levels of cadherins detected by western blot.

211

A R a ji, C D 1 4 4 (V E -c a d h e rin ) e x p re s s io n

n s 8 0 0 R a ji

s n s

l + M 2 1 0 B 4 o

o 6 0 0

o v

a d

l 4 0 0

t s

M 2 0 0 n

0 U n s ta in e d Is o ty p e A n ti-C D 1 4 4

B D a u d i, C D 1 4 4 (V E -c a d h e rin ) e x p re s s io n

n s 8 0 0 D a u d i

n s s

l + M 2 1 0 B 4 o

o 6 0 0

a d

l 4 0 0

e r

t s

M 2 0 0 n

0 U n s ta in e d Is o ty p e A n ti-C D 1 4 4

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± Mean ± p-value Mean ± Mean ± p-value SEM SEM SEM SEM Unstained 182.425 51.774 165.925 48.503 >0.9999 212.350 65.331 197.250 66.003 >0.9999 Isotype 508.750 65.483 359.500 56.117 0.4535 532.000 52.991 442.750 80.125 0.9960 Anti-CD144 513.750 19.817 393.750 26.348 0.7614 573.500 20.480 450.500 58.672 0.9336 p-value for isotype vs >0.9999 >0.9999 >0.9999 >0.9999 anti-CD144

Figure 6.19. VE-cadherin is not expressed on the surface of Raji and Daudi cells. Raji (A) and Daudi (B) tumour cells were cultured either on plastic (green bars) or on a confluent layer of M2-10B4 stromal cells (red bars) for 24 hours. Cells were then harvested and either left unstained, or stained with anti-human CD144 (VE-cadherin)-PE-Cy7 antibody, or with a mouse IgG1-PE-Cy7 isotype control (both used at 1 µg/ml). Percentage of positivity to the antibodies was measured as % of PKH67- cells which were PE-Cy7+. Data are the mean ± SEM of the MFI (shown as geometric mean fluorescence intensity relative to unstained control) of 2 replicates in 2 independent experiments. ns = non-significant, measured using a two-way Anova test.

212

A R a ji, P -c a d h e rin e x p re s s io n * 2 0 0 0

R a ji s

n s + M 2 1 0 B 4

o o

r 1 5 0 0

o v

a d

l 1 0 0 0

i I

t s

M 5 0 0 n u 0 U n s ta in e d Is o ty p e A n ti-P -c a d h

B D a u d i, P -c a d h e r in e x p re s s io n n s 2 0 0 0 n s D a u d i

l + M 2 1 0 B 4

o o

r 1 5 0 0

e o

a d

l 1 0 0 0

t s

M 5 0 0 n

0 U n s ta in e d Is o ty p e A n ti-P -c a d h

RAJI + M2-10B4 DAUDI + M2-10B4

Mean ± SEM Mean ± p-value Mean ± Mean ± SEM p-value SEM SEM Unstained 164.750 6.074 143.250 5.991 >0.9999 189.750 5.588 171.750 1.652 >0.9999 Isotype 1619.00 267.609 1048.50 96.755 0.0676 1418.50 89.385 1270.75 160.140 0.9965 Anti-Pcadh 964.500 82.104 766.000 76.607 0.9921 1044.75 51.737 921.250 157.736 0.9995 p-value for isotype vs 0.0241 0.8702 0.2326 0.3152 anti-P-cadh

Figure 6.20. P-cadherin is not expressed on the surface of Raji and Daudi cells. Raji (A) and Daudi (B) tumour cells were cultured either on plastic (green bars) or on a confluent layer of M2-10B4 stromal cells (red bars) for 24 hours. Cells were then harvested and either left unstained, or stained with anti-human P- cadherin-APC antibody, or with a mouse IgG2α-APC isotype control (both used at 1 µg/ml). Percentage of positivity to the antibodies was measured as % of PKH67- cells which were APC+. Data are the mean ± SEM of the MFI (shown as geometric mean fluorescence intensity relative to unstained control) of 2 replicates in 2 independent experiments. ns = non-significant, * p<0.05, measured using a two-way Anova test.

213 6.4. Discussion

The large-scale mass spectrometry-based proteomic study described in this chapter highlighted a number of pathways or functional clusters which are altered in tumour cells after co-culture with stroma, and therefore could be potentially responsible for the observed stroma-mediated protection from GA101-induced killing.

Based on both the in silico analysis and the published literature, two main clusters, one related to the BCR signalling axis and one related to (cadherin-mediated) cell-cell adhesion and epithelial adherens junctions remodelling have been reasoned to be likely involved in the phenotypic changes observed in tumour cells following tumour-stroma interactions.

The BCR signalling pathway has been investigated by specifically looking at three of the main kinases that initiate the downstream signalling cascades and for which well- characterised inhibitory molecules have been developed: SYK, PI3Kδ and BTK. However, the addition of the kinases’ specific inhibitors, namely R406, idelalisib and ibrutinib respectively, to GA101 treatment did not abrogate the stroma-mediated protective effect; on the contrary, it appeared to diminish GA101 efficacy – at least at inducing PCD. This could be dependent on the ability of these inhibitors to decrease the surface level of CD20 molecule on tumour B cells and, in turn, to “hide” the target cells from the antibody. Such an off-target effect had been previously observed [214,215], but no explanations as to why this occurred were provided. Interactions between the CD20 molecule and the BCR pathway have been previously described in several different B-cell NHL cell lines and patient samples [216], suggesting that there might be some shared effector molecules in the CD20-initiated signalling which require activation of the BCR pathway, or vice versa. Many questions regarding the reported overlap between CD20 and BCR signalling axes remain unanswered, delineating an interesting field of future research and at the same time posing crucial questions in regards to whether the use of small molecules inhibiting the BCR pathway in combination with anti-CD20 monoclonal antibodies can be clinically relevant. All the same, combination between GA101 and inhibitors of the BCR pathway did not manage to reduce survival of tumour cells when in co-culture with stromal cells. This demonstrated that the BCR signalling axis is not responsible for the microenvironment-mediated protective effect.

The second main cluster identified by proteomic analysis, which relates to cell-cell adhesion, epithelial adherens junctions remodelling and cadherin binding, was also investigated in vitro. Despite a less compelling literature supporting the involvement of

214 cadherins in the interactions between stroma and malignant B-cells, the findings that the stroma-mediated protection is dependent on contact and that the actin cytoskeleton undergoes drastic remodelling in the presence of stroma suggested that cadherin-mediated cell-cell adhesion could be playing an important role in the system. Therefore, a western blot was performed to visualise potential differences in the amount of cadherins expressed in tumour cells before and after contact with stroma. When on plastic, no bands at the expected size (̴ 130 kDa) were observed. However, a strong signal was measured after a 24-hour co-culture with stromal cells. Such an effect could not be recapitulated when tumour cells were cultured on fibronectin.

This result, however, needs to be carefully interpreted: in fact, whole lysates were used in this experiment, meaning that the observed differences could be due to the combination of both cell membrane-bound and intracellular cadherin molecules. Therefore, the increase in cadherin expression does not necessarily indicate an increased interaction with the stroma, as only surface-bound cadherins would be able to mediate the contact. For this reason, the extracellular expression of different members of the cadherin family was singularly analysed by flow cytometry. When looking at the most commonly expressed cadherins, i.e. E-cadherin and N-cadherin, no increase in the presence of stroma was observed – on the contrary, the expression appeared decreased in Raji cells. For E-cadherin, such a decrease was previously observed in both Raji cell line and B-CLL samples, and was found to lead to the up-regulation of the Wnt/β-catenin pathway [210]. Functional E-cadherin, in fact, plays an important role in the regulation and blockade of this pathway through its binding to β-catenin [217]. Its down-regulation and the subsequent activation of the signalling cascade downstream of Wnt/β-catenin, therefore, provide tumour cells with survival advantages [218]. On the other hand, N-cadherin was previously shown to mediate interactions between CML cells and stromal cells, and such interactions subsequently led to the activation of the pro-survival Wnt/β-catenin pathway. This observation seems controversial; however, different cadherin molecules could play different roles in different cell types. In the current system, surface expression of N-cadherin appeared reduced in Raji cells when cultured on stroma -but not on Daudi-, therefore excluding the possibility, at least for Raji cells, that a protective effect involving the N-cadherin-mediated interactions between tumour and stromal cells could take place. The fact that no changes in both E- and N-cadherin were observed in Daudi, moreover, suggests that the expression of these molecules is not linked to the stroma-mediated protection. Other cadherins, namely VE-cadherin and P-cadherin, were found to be important in mediating cell adhesion in the

215 literature [219,220], and their surface expression was therefore analysed; however, none of these seemed to be expressed on the surface of Raji or Daudi.

A more in-depth analysis of the role of each member of the cadherin family in the experimental settings is required to understand whether cadherin-mediated cell-cell adhesion plays a key role in mediating the contact with stroma and, thus, the protective effect from GA101-induced killing. For instance, a western blot of lysates containing membrane-bound proteins only could be performed, in order to understand whether the same increase in cadherin expression after co-culture seen in whole lysates can be observed in surface cadherins only. Intracellular flow cytometry could also help to define which cadherins are mostly responsible for the increased levels observed with whole-lysate western. Finally, measuring the activity of the Wnt/β-catenin pathway in tumour cells after co-culture could help to understand whether there is correlation between this signalling axis and cadherin expression and whether this changes in the presence of stromal cells. In addition to cadherins, cell-cell adhesion can be mediated by many different subsets of adherent molecules. Antibody arrays of adhesion molecules are commercially available, and could be performed to highlight single adhesion molecules that undergo expression changes after contact with stroma and that could thus explain the in silico findings obtained from the proteomic analysis.

In addition to BCR signalling and adherent junctions, the proteomic analysis performed highlighted other differentially expressed clusters of pathways. For instance, changes in the expression of cell cycle-related molecules were identified. Since several studies reported the ability of stromal cells to provoke arrest of cell cycle and subsequent increase in drug resistance in B-NHL and MM [211,221], a deeper analysis of the effects of co-culture on tumour cell proliferation in the current system is needed.

Importantly, an actin cytoskeleton-related cluster, including several signalling axes involved in actin dynamics and remodelling, was also identified. Given that actin reorganisation to the cell-cell junction points is a fundamental step leading to GA101- induced PCD [87] and given the observed actin remodelling that occurred in the presence of stromal cells (see chapter 3), it would be interesting to unveil the role that each of these actin-related signalling axes, such as Rac, RhoA or Cdc42 signalling, could be playing in the context of contact-mediated protection. One of the pathways identified by IPA, the NRF2-mediated oxidative stress response, appeared in silico to be up-regulated after co- culture. Time prevented the in vitro validation of the role played by such a pathway; however, the importance that ROS generation has in the execution of PCD suggests that

216 such a signalling axis, if in fact activated in the presence of stroma, could be involved in mediating the reduced efficacy of GA101-induced PCD.

Finally, a cluster involving eIF2 signalling axis and, in general, translation-related pathways was found highly enriched in both IPA and DAVID. Whilst, on the one hand, such an enrichment could merely reflect the numerous changes that occur upon co-culture and that require synthesis of new proteins, on the other hand, a direct role of the translational machinery in stroma-mediated protection cannot be excluded. In a recent study, Burwick et al. showed that blockade of the heme-regulated eIF2α (HRI) kinase could provoke apoptosis of MM cells, with a high degree of cell death observed even in the presence of the stromal microenvironment [222]. Intriguingly, the eIF2 signalling axis appears to regulate several stress-related molecules that lie downstream of eIF2 [223]. Such an observation could therefore warrant the need for a more in-depth analysis on this pathway.

In summary, the proteomic analysis performed has led to the identification of a number of pathways that are altered in tumour cells after co-culture with stroma. The question as to whether these pathways are indeed responsible for the protective effect mediated by stromal cells, however, remains unanswered. Therefore, additional studies and in vitro validation experiments are required to further determine the role of each of the signalling axes highlighted in the in silico analyses.

217 7. In vivo effect of immune cell mobilisation on mAb efficacy

The induction of direct cell death appears to be a potentially important mechanism of action through which GA101 induces tumour cell death in vitro [86,87]. In contrast, the in vivo efficacy of type-II anti-CD20 mAbs is mainly mediated by engagement of immune effector cells and the subsequent execution of ADCC and ADCP [53,114,224]. By virtue of its glycoengineered Fc region, GA101 is superior to rituximab and other type-I antibodies at inducing both NK cell-dependent ADCC and monocyte- and macrophage- mediated ADCP in vitro [51]. Therefore, targeting the microenvironment in an effort to further increase GA101 ability to recruit immune effector cells and induce ADCC and ADCP could improve tumour clearance in vivo and ultimately enhance therapeutic outcomes.

7.1. Effect of CXCR-4 blockade in combination with GA101 in vivo

Despite the fact that the blockade of CXCR-4/CXCL-12 signalling axis through the use of inhibitors or antibodies (i.e. plerixafor and anti-CXCL-12 antibody) did not overcome the protective effect mediated by stroma in vitro (see chapter 3), several published observations pointed out that blocking these receptors could still increase anti-CD20 mAb efficacy in an in vivo setting. Specifically, the use of the CXCR-4 inhibitor plerixafor (PXF) was shown to provoke mobilisation of CD34+ cells from the bone marrow into the circulation, leading to the egression of neutrophils and subsequently stronger induction of ADCC/ADCP upon rituximab treatment [20,225]. Therefore, it was hypothesised that mobilisation of CD34+ cells could increase GA101 therapeutic efficacy in syngeneic murine models of B- and T-cell lymphoma.

First, it was questioned whether treatment with PXF was able to mobilise CD34+ cells into the circulation. To address this question, female C57BL/6 mice were inoculated with murine lymphoma cells gene-modified to express human CD20 (EL4-huCD20) and treated with either GA101, or PXF, or the combination GA101+PXF. 3 hours after each PXF injection on day 1, day 7 or day 14, mice were bled from the tail vein and immune cells in

218 the blood were counted via a Sysmex analyser. A schematic treatment schedule is shown in figure 7.1.

219

Figure 7.1. Treatment schedule used to analyse in vivo efficacy of combination between PXF and GA101. A syngeneic murine model of T-cell lymphoma was established by injecting female C57BL/6 mice with 5 x 105 EL4 huCD20 cells (day 0). PXF was injected i.p. three times a week from day 1 until day 21, GA101 was injected i.p. twice a week starting at day 7 until day 21. In the combination arm, GA101 was injected 2 hours after PXF. Mice were bled from the tail vein on day 1, 7 and 14 approx. 3 hours after the PXF injections and blood was analysed using a Sysmex counter to observe changes in the immune cell subsets.

220 Blood cell counts confirmed that treatment with PXF significantly increased number of neutrophils, monocytes, lymphocytes and eosinophils in the circulation. At day 1, the number of all analysed immune cells subsets in the PXF-treated group doubled compared to the saline group (figure 7.2). Lymphocyte count increased from a median of 56.4 x 108 in saline group to 102 x 108 in PXF-treated group (p<0.0001). An even higher increase was observed in neutrophils (from 66 x 107 to 439 x 107, respectively, p<0.0001). Monocyte and eosinophil counts also significantly increased after treatment (p<0.0001 for both). A similar outcome was obtained at day 7 (figure 7.3), with the PXF-treated groups (PXF and PXF+GA101) showing significant increases in the counts of each immune cell subset, compared to saline (524 x 107 for PXF, 416 x 107 for PXF+GA101 vs 81 x 107 in saline group, p<0.0001 for both PXF and PXF+GA101 in neutrophils; 176.9 x 108 for PXF, 150.4 x 108 for PXF+GA101 vs 54.4 x 108 in saline group, p<0.0001 for both PXF and PXF+GA101 in lymphocytes; similar results in monocytes and in eosinophils, detailed values are shown in figure 7.3.). Similarly, at day 14 (figure 7.4) there was a visible increase in immune cells for lymphocytes, monocytes and eosinophils for PXF and PXF+GA101, despite the differences between saline and PXF-treated groups being lessened (detailed values are shown in figure 7.4).

221 D A Y 1

4  1 0 1 0 * * * *

2  1 0 1 0 * * * *

u n

o 1 0

3  1 0 u 1 0

c o

1 .5  1 0

l i y 1 0 1 0 2  1 0 h

c 1  1 0

t p

1 0 u 9 m 1  1 0 5 1 0

e 

N L

0 0 S a lin e P X F S a lin e P X F

9 * * * *

6  1 0 2 .5  1 0 9 * * * *

t n

n 9 u

u 2  1 0

o o

9 c

c 4  1 0

s s 9

l 1 .5  1 0

y p

c 9 o

o 9 1  1 0

2  1 0 n

s o

o 8

M 5  1 0 E

0 0 S a lin e P X F S a lin e P X F

Saline PXF

Median N = Median N = p-value

Lymphocytes 564 x 107 26 1020 x 107 26 <0.0001 Neutrophils 66 x 107 26 439 x 107 26 <0.0001 Monocytes 102 x 107 26 202 x 107 26 <0.0001 Eosinophils 13 x 107 26 93 x 107 26 <0.0001

Figure 7.2. Sysmex count of immune cell subsets at day 1 demonstrates that plerixafor is able to mobilise CD34+ cells. Mice were injected with PXF (i.p.) and bled from the tail vein after 3 hours at day 1. A Sysmex analyser was used to count the number of immune cells present in the circulation after PXF injections. The numbers of lymphocytes, monocytes, neutrophils and eosinophils are shown for each mouse analysed (single dots), with or without PXF. Of note, at day 1 only saline and PXF groups are shown, as GA101 treatment was started at day 7. Data are the median ± SEM pooled from 2 independent experiments, 7 mice per group in each experiment (less might be shown, when some were culled before the time-points analysed). **** p<0.0001, data analysed by using a Mann-Whitney U test.

222 D A Y 7

* * * * * * * * * * * * * * * * 1 0

1  1 0 1 0 3  1 0

t t

0 9 n n

8  1 0 u

o o c 1 0

2  1 0

6 1 0 0 9 s

s 

h c

p 0 9 o

o 4  1 0 h

r 1 0

t 1  1 0

u m e 0 9

2  1 0 y

N L

0 0 S a lin e G A 1 0 1 P X F G A 1 0 1 + P X F S a lin e G A 1 0 1 P X F G A 1 0 1 + P X F

* * * * * * * *

0 9 0 9 1 .5  1 0

4  1 0 * * * * * * * *

t n

n 0 9 u

u 3  1 0

o 0 9 o

c 1 .0  1 0

l i

e 0 9 t

2  1 0 h

c o

o 0 8

n i n 5 .0  1 0

0 9 s

o 1  1 0

M E

0 0 S a lin e G A 1 0 1 P X F G A 1 0 1 + P X F S a lin e G A 1 0 1 P X F G A 1 0 1 + P X F

Saline GA101 PXF GA101+PXF

Median N = Median N = Median N = p-value vs Median N = p-value vs saline saline Neutrophils 81 x 107 14 125 x 107 14 524 x 107 12 <0.0001 416 x 107 13 <0.0001 Lymphocytes 544 x 107 14 656 x 107 14 1769 x 107 12 <0.0001 1504 x 107 13 <0.0001 Monocytes 85 x 107 14 77 x 107 14 282 x 107 12 <0.0001 190 x 107 13 <0.0001 Eosinophils 11 x 107 13 10 x 107 14 58 x 107 12 <0.0001 44 x 107 13 <0.0001

Figure 7.3. Sysmex count of immune cell subsets at day 7 demonstrates that plerixafor is able to mobilise CD34+ cells. Mice were injected with PXF (i.p.) and bled from the tail vein after 3 hours at day 7. A Sysmex analyser was used to count the number of immune cells present in the circulation after PXF injections. The numbers of lymphocytes, monocytes, neutrophils and eosinophils are shown for each mouse analysed (single dots), with or without PXF. Data are the median ± SEM pooled from 2 independent experiments, 7 mice per group in each experiment (less might be shown, when some were culled before the time-points analysed). **** p<0.0001, data analysed by using a Mann-Whitney U test.

223 D A Y 1 4

* * * *

* * * 2 .5  1 0 1 0 * * *

1  1 0 1 0 t

n 2 .0 1 0 1 0

u  t

0 9 o n

8  1 0 c

s 1 0

o 1 .5  1 0

c t

0 9

6  1 0 y

l c

i 1 0 o

h 1 .0  1 0 h

p 0 9 p

o 4  1 0

r m t 5 .0 1 0 0 9

y 

u L

e 2  1 0 0 9 N 0 0 S a lin e G A 1 0 1 P X F G A 1 0 1 + P X F S a lin e G A 1 0 1 P X F G A 1 0 1 + P X F

* * * * 0 9 4  1 0 * * 0 9

1 .5  1 0 * * * *

t n 3 1 0 0 9 n

u 

u o

o 0 9 c

c 1 .0  1 0

l e 0 9 i

t 2  1 0

c o

o 0 8

n n i 5 .0  1 0

0 9 s

o 1  1 0

M E

0 0 S a lin e G A 1 0 1 P X F G A 1 0 1 + P X F S a lin e G A 1 0 1 P X F G A 1 0 1 + P X F

Saline GA101 PXF GA101+PXF

Median N = Median N = Median N = p-value vs Median N = p-value vs saline saline Neutrophils 92 x 107 9 76 x 107 14 510 x 107 10 0.0003 439 x 107 10 0.0005 Lymphocytes 834 x 107 9 406 x 107 14 1623 x 107 10 0.0004 1278 x 107 10 0.0220 Monocytes 120 x 107 9 64 x 107 14 247 x 107 10 0.0021 205 x 107 10 0.0021 Eosinophils 18 x 107 9 11 x 107 14 60 x 107 10 <0.0001 44 x 107 10 0.0030

Figure 7.4. Sysmex count of immune cell subsets at day 14 demonstrates that plerixafor is able to mobilise CD34+ cells. Mice were injected with PXF (i.p.) and bled from the tail vein after 3 hours at day 14. A Sysmex analyser was used to count the number of immune cells present in the circulation after PXF injections. The numbers of lymphocytes, monocytes, neutrophils and eosinophils are shown for each mouse analysed (single dots), with or without PXF. Data are the median ± SEM pooled from 2 independent experiments, 7 mice per group in each experiment (less might be shown, when some were culled before the time-points analysed). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, data analysed by using a Mann- Whitney U test.

224 Despite the promising increase in the number of immune effector cells in the circulation, however, addition of PXF to GA101 did not increase survival compared to GA101 alone (p=0.98). Instead, the therapeutic efficacy of GA101 in combination with PXF was slightly lower than GA101 as a single treatment (survival proportions at day 100: 28.6% for GA101 vs 21.4% for GA101+PXF). Both the treatment groups had significantly increased survival compared to saline group (p=0.045 for GA101 vs saline; p=0.035 for GA101+PXF vs saline) (figure 7.5).

In a Eµmyc-huCD20 syngeneic model of B-cell lymphoma, despite an overall increase in the count of lymphocytes, neutrophils, monocytes and eosinophils (data not shown), no survival advantages were observed for GA101- and GA101+PXF-treated groups compared to saline (p=0.4 and p=0.39, respectively) (figure 7.6).

Since the treatment schedule employed in the previous studies did not lead to any increases in survival, an earlier treatment was tested to determine whether this might improve the therapeutic outcome in a syngeneic model of T-cell lymphoma. EL4-huCD20 cells were injected into female C57BL/6 mice as previously shown, and both PXF and GA101 treatments were started at day 1, three and two times per week respectively. Despite the much higher degree of survival achieved with GA101 as a single treatment (85.7% vs 14.3% in saline group, p=0.005), there were no significant improvements when mice were treated with the combination of GA101+PXF (42.8% vs 14.3% in saline group, p=0.06). The GA101-treated group showed a (non-statistically significant but) better response compared to the combination arm (85.7% vs 42.8%, respectively, p=0.09) (figure 7.7).

225 E L 4 -h u C D 2 0

1 0 0 S a lin e

G A 1 0 1

8 0 l

a P X F

i v

r 6 0 G A 1 0 1 + P X F

n 4 0

c r

e n s

P 2 0 *

0 0 2 0 4 0 6 0 8 0 1 0 0 D a y s

p-value vs p-value vs p-value vs saline GA101 PXF

Saline - 0.0451 0.9398 GA101 0.0451 - 0.0445 PXF 0.9398 0.0445 - GA101+PXF 0.0348 0.9846 0.0140

Figure 7.5. Combination of GA101 and PXF did not lead to significant survival advantages in a syngeneic model of T-cell lymphoma. Female C57BL/6 mice were injected i.v. with 5x105 EL4-huCD20 cells, then treated with PXF (5 mg/kg, i.p.), started at day 1, GA101 (50 μg, i.p.), started at day 7, or a combination of the two. The saline group was injected with PBS (i.p.). Endpoints were weight loss 20-25%, or development of moribund signs. Kaplan-Meier survival curve is shown. Data is pooled from 2 independent experiments, 7 mice per group per experiment. Statistical analysis of survival curve was performed by using a Mantel-Cox test. * p<0.05, ns=non-significant.

226

E u m y c -h u C D 2 0

1 0 0 S a lin e

l 8 0 G A 1 0 1

a v

i P X F v

r 6 0 u

s G A 1 0 1 + P X F

n 4 0

r e

P 2 0 n s

0 0 2 0 4 0 6 0 8 0 D a y s

p-value p-value vs p-value vs vs GA101 PXF saline Saline - 0.4017 0.8737 GA101 0.4017 - PXF 0.8737 0.0625 - GA101+PXF 0.3935 0.8854 0.0620

Figure 7.6. Treatment with GA101 and combination of GA101 and PXF did not lead to significant survival advantages in a syngeneic model of B-cell lymphoma. Female C57BL/6 mice were injected i.v. with 1x106 Eµmyc-huCD20 cells, then treated with PXF (5 mg/kg, i.p.), started at day 1, GA101 (50 μg, i.p.), started at day 7, or a combination of the two. The saline group was injected with PBS (i.p.). Endpoints were weight loss 20-25%, or development of moribund signs (7 mice per group). Kaplan-Meier survival curve is shown. Statistical analysis of survival curve was performed by using a Mantel-Cox test. ns=non-significant.

227 E L 4 -h u C D 2 0

1 0 0 S a lin e * * v s s a lin e l 8 0 G A 1 0 1 d 1

a n s

v i

v v s G A 1 0 1 + P X F P X F r 6 0

u G A 1 0 1 d 1

t n s + P X F n 4 0

e v s s a lin e

r e

P 2 0

0 0 2 0 4 0 6 0 8 0 1 0 0 D a y s

p-value p-value vs p-value vs vs GA101 d1 PXF saline Saline - 0.0050 0.9484 GA101 d1 0.0050 - 0.0029 PXF 0.9484 0.0029 - GA101 d1+PXF 0.0664 0.0933 0.0658

Figure 7.7. Earlier treatment with GA101 and PXF did not lead to significant survival advantages in a syngeneic model of CD20-expressing EL4-huCD20. Female C57BL/6 mice were injected i.v. with 5x105 EL4-huCD20 cells, then treated with PXF (5 mg/kg, i.p.), GA101 (50 μg, i.p.) (both started at day 1) or a combination of the two. The saline group was injected with PBS (i.p.). Endpoints were weight loss 20-25%, or development of moribund signs. Kaplan-Meier survival curve is shown (7 mice per group). Statistical analysis of survival curve was performed by using a Mantel-Cox test. ** p<0.01, ns=non-significant.

228 7.2.Effect of TGF-β blockade in combination with GA101 in vivo

Previously published studies suggested that the blockade of transforming growth factor (TGF)-β receptor II could improve survival in breast and colorectal cancer-bearing mice through increased activity of NK cells and CTLs and increased number of tumour- infiltrating T cells [226]. When given in combination with chemotherapy, anti-TGF-β receptor II mAb was effective against both primary tumour and metastasis. Specifically, NK cell activity was highly increased, as shown by a greater release of IFN-γ.

It was hypothesised that this approach could therefore induce a higher degree of ADCC in combination with anti-CD20 mAbs as previously demonstrated with TLR7 agonists [54]. Hence, a syngeneic model of B-cell lymphoma was used to test whether combining the type-II anti-CD20 antibody tositumumab with an anti-TGF-β receptor antibody, namely 1D11, could lead to an increased overall survival. Eµmyc-huCD20 cells were injected into female C57BL/6 mice on day 0, and treatment with 1D11 was started at day 4, three times a week for a week. Tositumumab (Tos) was injected starting from day 7, for a total of four times. A schematic treatment schedule is shown in figure 7.8.

Combining Tos with 1D11, however, did not seem to increase the therapeutic efficacy of anti-CD20 mAbs in the murine model used (figure 7.9). Although Tos as a single treatment caused a significant survival advantage (p=0.0089 vs saline group), the addition of 1D11 did not improve survival further (p=0.01 for combination vs saline). There were no significant differences between Tos as a single treatment and Tos in combination with 1D11 (p=0.94).

229

Figure 7.8. Treatment schedule used to analyse in vivo efficacy of combination between 1D11 and tositumumab. A syngeneic murine model of B-cell lymphoma was established by injecting female C57BL/6 mice with 1 x 106 Eµmyc huCD20 cells (day 0). 1D11 was injected i.p. three times a week for one week from day 4, Tos was injected i.p. four times starting at day 7 until day 18. In the combination arm, Tos was injected 4 hours after 1D11.

230 E u m y c -h u C D 2 0

1 0 0 S a lin e

l 8 0 T o situ m u m a b

v i

v 1 D 1 1 r 6 0 u to s vs s a lin e

s * * T o s + 1 D 1 1

n 4 0 * to s + 1 D 1 1 vs s a lin e

e c

r n s to s + 1 D 1 1 vs to s e

P 2 0

0 0 2 0 4 0 6 0 8 0 1 0 0 D a y s

p-value vs p-value vs p-value vs saline Tositumumab 1D11

Saline - 0.0089 0.8926 Tositumumab 0.0089 - 0.0157 1D11 0.8926 0.0157 - Tos+1D11 0.0101 0.9362 0.0154

Figure 7.9. Combination of Tositumumab and 1D11 did not lead to significant survival advantages in a syngeneic model of B-cell lymphoma. Female C57BL/6 mice were injected i.v. with 1x106 Eµmyc-huCD20 cells, then treated with saline (i.p.), 1D11 (10 mg/kg, i.p.), Tos (50 μg, i.p.) or a combination of the two, 7 mice per group. Endpoints were weight loss 20-25%, or development of moribund signs. Kaplan-Meier survival curve is shown. Statistical analysis of survival curve was performed by using a Mantel-Cox test. * p<0.05, ** p<0.01, ns=non-significant.

231 7.3. Migration of tumour cells to the bone marrow in vivo

The lack of improvements in the efficacy of GA101 when in combination with plerixafor cannot be attributed to a lack of egression of immune cells from the bone marrow, as the data obtained on immune cell count show that plerixafor is efficiently increasing the number of neutrophils, lymphocytes, monocytes and eosinophils in the circulation (see figures 7.2, 7.3, 7.4). These results therefore suggest that, despite the increase in number, bone marrow-derived immune effector cells are either not recruited by GA101, or not involved in ADCC/ADCP, or unable to perform effective ADCC/ADCP. Alternatively, tumour cells could escape systemic targeting by GA101, for example by migrating out of the circulation and into the bone marrow.

The possibility that the bulk of tumour cells could have migrated into the bone marrow and thus could be protected by TME-mediated signals was therefore interrogated by inoculating mice with luciferase-iRes-GFP-expressing EL4-huCD20 and Raji cell lines, in order to be able to visualise by bioluminescence the metastatic spread of tumour cells over 14 days. Since luciferase-iRes-GFP can be an immunogenic complex and thus tends to be rejected by immuno-competent mice [227], for this experiment the immuno-deficient NOD-SCID-γ chain-deficient (NSG) mice were used. 5 x 105 cells of each cell line were inoculated intravenously in NSG female mice and both bioluminescence images and X-ray images were taken dorsally on days 1, 7 and 14. A schematic treatment schedule and survival are shown in figure 7.10.

The images acquired showed that, while no tumour cells were visualised on day 1 (figure 7.11), a bulk of tumour cells could be observed on day 7. Both Raji-luc-iGFP and EL4- huCD20-luc-iGFP cells were mainly concentrated in an area which overlapped with the femurs, spine and shoulder (mouse 2.1) visualised by X-ray. However, given the close proximity of the lymph nodes (e.g. inguinal or mesothelial) to these bones it was not possible to say with certainty where the tumour was located. Interestingly, mouse 2.2 exhibited a tumour near the eye, suggesting migration to cranium. Mouse 2.3 presented, in addition, a bulk of tumour near the site of injection (tail vein) (figure 7.12).

Interestingly, at day 14, two mice out of the three inoculated with Raji-luc-iGFP cells did not display the presence of tumour bulk anywhere in the body. This could be due to the movement of the tumour bulk towards the ventral side of the mouse – which would not be visible by dorsal imaging. Mouse 1.1 presented tumour burden in areas corresponding to femurs and mesothelial lymph nodes. In mice inoculated with EL4-huCD20-luc-iGFP, on

232 the other hand, tumour bulks could still be observed, with a strong bioluminescence signal overlapping with the femur, brain and hips (mouse 2.2) or site of injection and spleen (2.3) (figure 7.13).

233

Figure 7.10. Schematic treatment schedule and survival of NSG mice after tumour inoculation. Female NSG nude mice were inoculated intravenously with 5 x 105 Raji-luc-iGFP (group 1, mice 1.1, 1.2, 1.3) or 5 x 105 EL4-huCD20-luc-iGFP (group 2, mice 2.1, 2.2, 2.3) at day 0. At day 1, 7 and 14, mice were imaged by X-ray and bioluminescence using a Bruker system. Mice were culled on the days circled in blue, when presenting moribund signs.

234

A 1.1 1.2 1.3 B 2.1 2.2 2.3

Figure 7.11. Images showing localisation of tumour burden in NSG mice at day 1. NSG mice inoculated with either Raji-luc-iGFP (A) or EL4-huCD20-luc-iGFP (B) were imaged using a Bruker system. The Bruker software was used to merge X-ray and bioluminescence images. Colour represents tumour burden – no signal was observed for bioluminescence. This experiment was performed once.

235

A 1.1 1.2 1.3 B 2.1 2.2 2.3

Figure 7.12. Images showing localisation of tumour burden in NSG mice at day 7. NSG mice inoculated with either Raji-luc-iGFP (A) or EL4-huCD20-luc-iGFP (B) were imaged using a Bruker system. The Bruker software was used to merge X-ray and bioluminescence images. Colour represents tumour burden, a scale measuring photon/sec/mm2 is reported at the bottom. This experiment was performed once.

236

1.1 1.2 1.3 2.1 2.2 2.3 A B

Figure 7.13. Images showing localisation of tumour burden in NSG mice at day 14. NSG mice inoculated with either Raji-luc-iGFP (A) or EL4-huCD20-luc-iGFP (B) were imaged using a Bruker system. The Bruker software was used to merge X-ray and bioluminescence images. Colour represents tumour burden, a scale measuring photon/sec/mm2 is reported at the bottom. This experiment was performed once.

237 7.4. Discussion

By blocking the interactions of CXCR-4 with the chemokine CXCL-12, the CXCR-4 inhibitor plerixafor has been shown to be able to mobilise CD34+ cells from the bone marrow into the circulation [228]. This therapeutic intervention also results in an increase in the number of immune cells mobilised to the peripheral blood which could therefore facilitate the recruitment of immune effector cells by monoclonal antibodies and potentially increase therapeutic efficacy [20]. In a syngeneic model of CD20-expressing EL4-huCD20 lymphoma, as expected, treatment with plerixafor led to a sustained increase in blood counts of neutrophils, lymphocytes, monocytes and eosinophils. However, such an increase in these peripheral immune effector cells did not lead to an enhancement in GA101-mediated clearance of tumour cells. To understand whether this result was due to the migration of tumour cells into the bone marrow and the following interactions with the protective microenvironment, Raji-luc-iGFP and EL4-huCD20-luc-iGFP cells were inoculated into NSG mice and their migration was monitored by bioluminescence imaging.

While no tumour burden was observed at day 1, probably because the injected cells had not replicated enough to create a bulk that could be visualised, a strong signal was observed at both day 7 and day 14. At day 7, cells seemed to mainly accumulate in areas that overlapped femurs and spine, suggesting an involvement of either the bone marrow, or lymph nodes that are present in those areas. At day 14, additional areas appeared involved, such as brain, hips, spleen or lymph nodes. Interestingly, two out of three mice inoculated with Raji-luc-iGFP, which presented tumour bulk in areas corresponding to femurs at day 7, did not seem to exhibit any measurable tumour burden by day 14. As these mice displayed moribund signs and were culled at day 19 (see figure 7.10), the absence of signal is unlikely be due to tumour regression. Instead, cells could have been spreading again in the whole body, thus reducing the signal in a few specific areas. It is also possible that tumour cells could have moved towards the opposite side of the body – imaging the opposite side of each mouse could help in understanding whether this is the case. Occasionally, i.p. injections of luciferin can also fail and so reimaging may be successful. Since this experiment was only performed once, it is difficult to make any final conclusions. To further extrapolate information, when repeating the experiment bone marrow and lymph nodes could be dissected after luciferin injection and individual tissues could be imaged. This would help to confirm whether cell migration involved lymph nodes, or bones, or both. However, it is possible that the migration of EL4-huCD20-luc- iGFP cells into the bone marrow of NSG mice after as little as 7 days from inoculation

238 could explain the lack of efficacy observed when combining GA101 with the CXCR-4 inhibitor plerixafor. In fact, as shown in figure 7.1, while treatment with plerixafor was started at day 1, GA101 injections were only performed from day 7. If EL4-huCD20 migrated into the bone marrow of mice by day 7, the therapeutic efficacy of GA101 would therefore be hampered, as tumour cells would not be present in the circulation and therefore would not be targeted. Instead, protective cues from the microenvironment could mediate increased survival and ultimately determine therapeutic failure. Indeed, when treatment started at day 1 (figure 7.7), a higher therapeutic efficacy could be observed – however, combination treatment still did not lead to survival advantages.

Another consideration that is worth making is the limited sensitivity of the Bruker imager, where only extended tumour burden can be properly visualised. A dissection of bone marrow followed by flow cytometry in order to measure the amount of luc-iGFP- expressing cells contained could therefore be a more accurate way to determine whether migration towards stroma actually occurs.

The mouse models used in the migration experiments have some major limitations and do not recapitulate well the models used to determine survival after GA101 treatment or the clinical situation. In fact, because of the immunogenicity of the luc-iGFP construct used to visualise and monitor tumour cells, a syngeneic model could not be used – thus the need to employ immuno-deficient mice. Whilst EL4-huCD20 cells more closely replicate the in vivo survival experiments performed, where the murine line was used, the use of Raji cells was justified by an effort to reproduce the in vitro experimental system, where Burkitt’s lymphoma cell lines were studied – these two models, therefore, cannot demonstrate that tumour cells would definitely migrate into the bone marrow of immunocompetent mice in a syngeneic model of CD20-expressing EL4-huCD20 lymphoma. These experiments do not therefore exclude the possibility that these therapeutic interventions could potentially increase the efficacy of GA101 in other preclinical models or indeed in the clinic. Therefore further efforts should be put into developing additional models and testing these strategies in the clinic. For the preclinical experiments, this would involve less immunogenic constructs that allow long-term visualisation of tumour cells, in order to be able to use immuno-competent mice with no risks of tumour rejection.

239 8. Conclusions and future directions

The introduction of immunotherapy in the treatment of B-cell malignancies has led to important improvements in patients’ survival. Rituximab, first approved by the FDA in 1997, marked the beginning of a new era in the management of B-NHL. Combining the standard chemotherapeutic regimens with monoclonal antibodies, in fact, could improve therapeutic outcomes without inducing additional remarkable side effects. Since rituximab approval, a great amount of effort has been focused on developing even more efficient antibodies, with the aim of further increasing survival probabilities by augmenting B cell clearance. Recent advances have led to the development of third generation, glycoengineered antibodies with higher ability to recruit immune effector cells and activate Fc-dependent mechanisms of action [48]. One such antibody, obinutuzumab (GA101), has been recently approved by the FDA for use in FL and B-CLL, and phase-III clinical trials using GA101 in other B-NHL are on-going [229].

Despite the great success obtained with anti-CD20 monoclonal antibodies, there are still a percentage of patients that will not respond to therapy, or who relapse. One of the putative causes of such a failure in treatment could lie in the existence of a protective microenvironment. Tumour B cells could extravasate from the circulation and migrate into the bone marrow following chemotactic signals. In the bone marrow, interactions with the microenvironment components, specifically stromal cells, ECM and soluble factors, could mediate resistance to treatments and lead to therapeutic failure.

Against this background, the project carried out during this PhD aimed to investigate the role of the tumour microenvironment, and in particular the stromal component, in protecting malignant B cells from the type-II anti-CD20 monoclonal antibody GA101.

In this chapter, the main findings of the thesis will be discussed, followed by a discussion of the future work that is needed, in order to address those questions that remain unanswered. Finally, the research undertaken and its impact will be analysed in the light of the current knowledge and novel discoveries in the field.

240 8.1. Summary of main findings

Since GA101 is able to induce death of B cell tumours through three main mechanisms of action, namely PCD, ADCP and ADCC, the first step has been to understand whether interactions between tumour and stromal microenvironment affected each of these mechanisms. The stromal microenvironment has been mimicked by culturing bone marrow stromal cells of either murine or human origin (M2-10B4 and HS-5, respectively) with two human Burkitt’s lymphoma cell lines, Raji and Daudi. Both stromal lines were able to mediate tumour cell protection from GA101-induced PCD, with a stronger protective effect observed for the murine line M2-10B4 (figures 3.2 and 3.3). Compelling evidence suggested that the CXCR-4/CXCL-12 signalling axis played a key role in mediating stroma-mediated resistance of B-cell lymphoma and B-CLL cell lines [19,20,166]; however, the inhibition of this pathway did not abrogate the protective effect in the studied system (figures 3.9 and 3.10). Treatment of tumour cells with GA101 was then performed in the presence of either stroma-conditioned media, or tumour/stroma-conditioned media, or in transwell plates containing a membrane that separated the two cell populations (stromal and tumour cells). This revealed that the microenvironment-mediated protective effect from GA101-induced PCD is entirely dependent on direct contact between tumour and stromal cells (figures 3.11, 3.12 and 3.13). GA101-induced PCD has been recently characterised [86,87]. Given its dependence on homotypic adhesion and actin cytoskeleton reorganisation towards the cell-cell junction points, the effect of co-culture with stromal cells on the ability of GA101 to induce homotypic adhesion was studied. M2-10B4 stromal cells were able to remodel the actin cytoskeleton and lead to the detachment of cells that had previously undergone homotypic adhesion from each other (figures 3.16 to 3.19). Since the stroma-mediated protective effect was found to occur even after removal of contact between stromal and tumour cells and lasted for at least 20 hours post co-culture, (figure 3.22), the ability of GA101 to induce homotypic adhesion in Raji tumour cells previously co-cultured with stroma was analysed. Stroma-conditioned tumour cells were unable to reorganise their actin cytoskeleton upon treatment with GA101 (figure 3.23), suggesting that stromal cells protect from GA101-induced PCD by interfering with the actin cytoskeleton reorganisation and homotypic adhesion of B cells. In addition to this, culture of tumour cells on the ECM component fibronectin did not protect from GA101- induced PCD and was not able to interfere with the actin reorganisation and homotypic adhesion induced upon GA101 treatment (figures 3.24 to 3.28).

241 To understand whether the presence of a stromal microenvironment could affect the efficacy of GA101 at inducing ADCP, human monocytes were isolated and added to the co-culture system, before treating the wells with GA101. In the presence of stromal cells, ADCP induced by human monocytes was strongly reduced (figure 4.2), and such a reduction was observed after 2, 4, 8 or 24 hours of treatment (figures 4.3 and 4.4). When treatment was started immediately after addition of tumour cells to the stromal layer, no reduction in phagocytosis was observed; a co-culture time between stromal and tumour cells of at least 2 hours was in fact required for a protective effect to be achieved (figure 4.6 and 4.7). As observed with PCD, the culture of tumour cells with stroma- and tumour/stroma-conditioned media did not lead to a decreased efficacy of GA101 at inducing ADCP (figures 4.8 and 4.9). Similarly, culture on the ECM component fibronectin did not affect ADCP (figure 4.10). Since stromal cells expressed CD172a (SIRP-α) (figure 4.11), a receptor that ligates to the don’t-eat-me signal CD47, it was reasoned that activation of CD47 by stroma could lead to reduced GA101-induced ADCP. However, the blockade of CD172a did not abrogate the protective effect observed in the presence of stromal cells (figure 4.12). Finally, the ability of different phagocytes at mediating GA101-induced ADCP was assessed. Whilst treatment in the presence of macrophages of both murine and human origins recapitulated the protective effect observed with human monocytes (figures 4.17 and 4.18), when tumour cells were treated in the presence of neutrophils, the stroma-mediated protection from ADCP did not appear biologically relevant (figure 4.19).

The ability of GA101 to activate NK cells and, therefore, induce NK cell-mediated ADCC was assessed by measuring production of IFN-γ by NK cells. Again, the presence of stromal cells affected the ability of GA101 to activate NK cells (figure 5.2). This effect was abrogated by culturing tumour cells in stroma-conditioned media, revealing that protection from ADCC is dependent on direct contact between tumour and stroma (figure 5.3). Interestingly, stromal cells were able to protect from NK cell activation even when treatment was started immediately after the beginning of co-culture between tumour and stroma, suggesting that stroma could potentially affect the activity of NK cell population (figures 5.4 and 5.5). As observed for PCD and ADCP, culture of tumour cells with fibronectin did not affect NK cell activation (figure 5.6).

From the experiments performed, direct interactions between stromal and tumour cells appeared to initiate certain signalling events that were ultimately responsible for the observed protection from GA101-mediated killing. To investigate which specific pathways

242 could be altered in tumour cells upon culture with the stromal microenvironment, a mass spectrometry-based proteomic analysis was performed. Tumour cells were cultured on the stromal layer for 24 hours and differentially expressed proteins were identified. With the aid of two analysis software, clusters of pathways enriched in the differentially expressed proteins were selected. Previously published literature was consulted to determine those pathways that could be involved in the system and that could be further analysed in vitro, in order to ascertain the signalling axis (or axes) responsible for the stroma-mediated increased survival. The analysis led to the identification of several main clusters that could have a role in the studied system (figures 6.1 to 6.4, 6.7); among those, the BCR signalling pathway and the cadherin-mediated adherens junctions were validated in vitro. The blockade of different effector molecules of the BCR signalling axis, such as PI3Kδ, Syk and BTK, did not lead to the abrogation of stroma-mediated protective effect; instead, the overall efficacy of GA101 appeared reduced when in combination with PI3Kδ, Syk and BTK inhibitors (figures 6.8 and 6.9). The BTK inhibitor was indeed able to reduce the surface expression of CD20 molecule on tumour cells, suggesting that this effect could be influencing GA101 efficacy (figure 6.10 and 6.11). A similar reduction in CD20 expression could be observed, in tumour cells, after co-culture with stroma, revealing another putative mechanism that could potentially be responsible for the decreased killing mediated by GA101 (figure 6.12 and 6.13). The expression of the cadherin family of molecules before and after co-culture was assessed by western blot, revealing an increase in total expression of cadherins after culture with stroma, compared to culture on plastic or on fibronectin (figure 6.17). However, when looking at the extracellular expression of single cadherins, the same effect was not recapitulated: in fact, surface expression of E- cadherin and N-cadherin decreased (Raji) or showed no differences (Daudi) on stroma (figures 6.18 and 6.19).

Given that the blockade of CXCR-4 has previously been shown to lead to the egression of CD34+ cells from the bone marrow into the circulation [20], the CXCR-4 inhibitor plerixafor has been used in combination with GA101 in in vivo syngeneic models of CD20+ lymphomas. Treatment with plerixafor did indeed increase the number of immune effector cells available systemically (figures 7.2 to 7.4); however, such an increase did not correspond to a better therapeutic efficacy of GA101 (figure 7.5 and 7.6). Such a lack of improvement in GA101 efficacy could be due to the migration of tumour cells to the bone marrow, and therefore to an impaired possibility of targeting tumour cells because of both their physical location and molecular protective cues derived from the microenvironment. To investigate this possibility, luciferase-iRes-GFP-expressing tumour cells were

243 inoculated into immune-deficient mice and monitored through bioluminescence imaging. This experiment suggested that, at 7 and 14 days from inoculation, tumour cells do indeed migrate towards femurs, spine, brain, spleen and hips of NSG mice (figures 7.12 and 7.13). However, this preliminary experiment cannot on its own confirm that tumour cells do actually migrate into the bone marrow of the bones – therefore, further studies are warranted to confirm the exact location of migrated tumour cells.

8.2. Discussion of methodology and future work

The tumour cells employed in the experiments, Raji and Daudi cells, are human Burkitt’s lymphoma cell lines which have been routinely used in the past to study B-NHL. Whilst the use of cell lines has several advantages over primary samples from patients, such as the constant availability and the large number of cells that can be used for each experiment, limitations of this approach should also be considered. In this context, two main aspects need to be taken into account: 1) cell lines such as Raji and Daudi are transformed, and therefore contain the EBV herpesvirus [230,231]. This could therefore modify their behaviour compared to the original primary tumour from which they originate; 2) compared to other B-NHL tumours, the Burkitt’s lymphoma’s tumour microenvironment has peculiar characteristics: in fact, tumour-associated macrophages appear the main component of the Burkitt’s lymphoma’s microenvironment [232]. Therefore, interactions with stromal components might not be as relevant as in other B-cell malignancies. Further studies with cell types from different B-cell malignancies are therefore warranted to confirm the reproducibility of these findings.

The M2-10B4 and HS-5 stromal cell lines were selected and used in the tumour/stroma co- culture system because they were routinely used and demonstrated to protect from spontaneous apoptosis and drug-induced killing in previously published literature [21,150,177,233]. Both M2-10B4 and HS-5 were shown to mediate protection of Raji and Daudi tumour cells from GA101-induced PCD (figures 3.2 and 3.3). This suggested that the ability of stroma to protect from GA101-induced killing was not affected by the use of a human/human vs murine/human system, even though the degree of protection appeared to be cell line-dependent. Although it is well-known that different stromal lines can mediate protection through different mechanisms [123], it was reasoned that the comparison of two different systems (i.e. human/human vs murine/human) would not have been possible, given the limited time. Therefore, in an attempt to generate the best possible

244 model to mimic the microenvironment-mediated resistance to anti-CD20 antibody therapy, i.e. which mediated the greatest level of protection, the murine line M2-10B4 has been selected and used in the following experiments.

Whilst a strong and reproducible protective effect is observed in Raji and Daudi cells cultured on M2-10B4, such a protection should be validated by using primary samples from patients with B-cell malignancies, in terms of both tumour cells and bone marrow stromal cells. In fact, such a system would more closely represent the body’s real conditions, ensuring that the actual interactions that occur between tumour and stroma are studied. Primary samples, in fact, tend to be more heterogeneous than cell lines, and could display differences in behaviour that can be both tissue- and patient-dependent. Therefore, the ability to reproduce the observed findings in these settings would further increase the relevance of this study.

The use of the 7-AAD/Annexin V staining to determine survival percentages within the tumour cell population represents a reliable method to assess cell viability. However, the presence of a stromal component could complicate the analysis: in fact, dead tumour cells could be taken up by stromal cells, and if the stain was lost in the process such possibility could not be accounted for. A way to demonstrate that such an event does not occur and that survival percentages are an actual representation of stroma-mediated protection would consist in enumerating tumour cells through the use of counting beads. This could confirm that the same number of tumour cells, but a lower percentage of dead cells, is measured in the presence of stroma, ensuring that a protective effect from GA101-mediated PCD is actually observed.

As previously described (see chapter 3), the protective effect mediated by M2-10B4 stromal cells on GA101-induced PCD was not due to soluble factors, but instead appeared entirely dependent on direct contact between stromal and tumour cells. This seems to contrast with the previously published literature, where a role for soluble factors in bringing about protection of tumour cells, even though partial, is often acknowledged [18,234]. The different mechanisms of protection observed could be explained by 1) differences in the tumour and stromal cell lines studied; 2) differences in the co-culture times and ratios; 3) differences in the mechanisms of protection that could result from the use of each cytotoxic drug or antibody studied. Since no other studies have looked at the interactions between M2-10B4 stromal cells and Raji/Daudi B-cell lymphoma cells in the context of GA101-induced killing, it is not possible to compare the results obtained in this study with findings from other laboratories.

245 Another important consideration that needs to be made is the apparent lack of a role for the component of the ECM fibronectin in the microenvironment-mediated protection from GA101. As shown, culture of Raji and Daudi tumour cells on fibronectin did not affect the efficacy of GA101 at inducing PCD, ADCP and ADCC, suggesting that the interactions initiated upon contact with stromal cells that are at the basis of the protective effect are not recapitulated in the presence of fibronectin. Several studies reported a role for the ECM in mediating drug resistance in B-cell malignancies: for instance, De La Fuente et al. cultured CLL samples on fibronectin and found that adhesion to the substrate increased the ratio of anti-apoptotic to pro-apoptotic molecules in CLL cells, leading to increased tumour cell survival compared to culture on plastics. This was mediated by the interaction of the integrin α4β1 (VLA-4) with fibronectin [235]. A following study from the same group demonstrated that interactions between VLA-4 in CLL cells and fibronectin were also responsible for CLL resistance to the chemotherapeutic drug fluradabine [156]. However, further studies analysing the role of culture on fibronectin in the Burkitt’s lymphoma cell line Daudi demonstrated that, differently from what was observed with CLL cells, adhesion to fibronectin induced only a weak protection of tumour B cells from doxorubicin – despite the fact that a strong adhesion of Daudi cells to the substrate was observed [236]. Thus, the fibronectin-mediated protection appears to be cell line- and possibly drug- dependent. In fact, to date there are no reports that look at the ability of adhesion on fibronectin to mediate protection from anti-CD20 antibody therapy and it is therefore difficult to infer conclusions on whether the use of a given therapeutic strategy can affect the substrate’s capacity to protect tumour cells. In addition to this, the blockade of VLA-4 in the presence of stroma did not lead to any increase in GA101-mediated killing, thus suggesting that the stroma-mediated protection was not via the same pathway as fibronectin-mediated protection of CLL cells from chemotherapy. Importantly, adhesion to different substrates is likely to have different effects on viability and survival of tumour cells; therefore, other substrates, such as laminin, collagen or matrix-associated proteoglycans [237], should be employed to confirm that the ECM does not affect GA101- induced cell death.

A limitation of this project that needs to be taken into account is the lack of characterisation of the stromal line M2-10B4 before and after co-culture with B-cell lymphoma cells. In fact, it is well-known that normal, healthy fibroblasts can be activated by cancer cells to become cancer-associated fibroblasts (CAFs) [238]. Such cells present surface markers that are typical of a cancer-mediated activation, such as α-smooth muscle actin (SMA) [239] or fibroblast activation protein (FAP) [240] and are actively involved in

246 driving tumour progression and resistance to cytotoxic drugs in haematological malignancies and many other cancers (reviewed in [128]). The M2-10B4 line does protect tumour cells from GA101; however, whether it is the contact with tumour cells in the first place that activates stromal cells and alters their behaviour in order to drive the observed protective effect was not investigated. As several studies recently highlighted the important role played by reciprocal interactions between stromal and tumour cells in mediating tumour cell survival (see 1.6.3), it is fundamental that future research addresses this point. A way to understand whether stromal cells are able to mediate protection of tumour cells only after being activated would involve co-culturing stromal and tumour cells, then separating the two population and comparing the ability of naïve stromal cells vs tumour- conditioned stromal cells to protect GA101-pre-treated tumour cells from killing. However, such an experiment would still require a contact, although short, between tumour and stromal cells. A better option, although laborious, would consist of performing a proteomic analysis of naïve stromal cells vs tumour-conditioned stromal cells. This could identify pathways that are altered in the stromal component upon contact with tumour cells, highlighting putative changes that could be responsible for the stromal activation and ability to protect malignant cells from therapies. As previous studies have highlighted the existence of a contact-dependent up-regulation of PKC-βII in stroma following co-culture with B-CLL cells [131], which in turn led to the activation of NF-κB and subsequent B- CLL drug resistance, it would be interesting to understand whether a similar mechanism occurs in the studied system.

One interesting finding was that the stroma-mediated protection of tumour cells from PCD lasted for several hours after removal of contact from stromal cells. In fact, survival of tumour cells post treatment with GA101 following co-culture with stroma and subsequent separation through FACS sorting was significantly greater in stroma-conditioned tumour cells than in naïve tumour cells at 0 and 4 hours post-sort. This protection gradually decreased overtime and disappeared after 20 hours. Such a transient but long-term protection indicates the existence of pro-survival signalling pathways which initiate upon contact between tumour and stromal cells and are sustained over time. Importantly, it was not determined whether the protective effect lasts after removal of contact in ADCP and ADCC. If similar kinetics were observed in post-contact protection from PCD, ADCP and ADCC, this would suggest that similar, if not the same, pathways might be altered following contact with M2-10B4 stromal cells in order to bring about the survival advantages observed. Whilst it has not been possible to determine this within the time frame of the project, the many similarities observed in stroma-mediated protection between

247 the different modes of action (namely PCD, ADCP and ADCC) might suggest that this could be the case.

The idea that the protective effect could be independent of the features of each specific mechanism of action of GA101, however, seems to contrast with the observation that the tumour actin cytoskeleton undergoes drastic remodelling when in contact with stromal cells, possibly interfering with, or counterbalancing, the ability of GA101 to induce homotypic adhesion and, subsequently, PCD. In fact, if protection from PCD did indeed derive from the stroma-mediated reorganisation of the actin cytoskeleton, it could not be explained why such mechanism also protected from ADCP or ADCC. Whilst remodelling of the actin cytoskeleton in phagocytes is essential for phagocytosis [241], actin dynamics in target cells are not known to influence ADCP. However, remodelling of the actin cytoskeleton could still modify expression of surface receptors and therefore indirectly affect the ability of phagocytes to bind and engulf tumour cells. At the same time, the actin remodelling could also be a “side effect” of the M2-10B4/B cell co-culture: in fact, a functional role for this process in mediating protection from PCD has not been shown. Investigating this point has, however, proven difficult: in fact, the blockade of actin dynamics through the use of actin polymerisation or depolymerisation inhibitors, such as the Rho kinase (ROCK) inhibitor H1152 [242], even though at low concentrations has led to the death of stromal cells, likely because of a reduction of their adhesive capacity to plastic (data not shown). In addition, by interfering with actin dynamics, these inhibitors could potentially block the activity of GA101. Therefore, it is tricky to determine whether remodelling of the actin cytoskeleton is indeed affecting PCD.

The proteomic analysis performed identified several enriched pathways which were actin- related, such as Rho signalling, Rac signalling and Cdc42 signalling. This finding supports the hypothesis that remodelling of the actin cytoskeleton upon co-culture does indeed play a role in the protective effect mediated by stroma. Interestingly, published literature suggests that these molecules might be involved in mediating adhesion of tumour cells to stromal cells, and their blockade could potentially abrogate such an adhesion [243,244]. With protection from GA101-induced killing (PCD, ADCP and ADCC) being dependent on contact between tumour and stroma, the possibility of impairing the adhesive capacities of tumour cells to stroma constitutes an interesting strategy to abrogate the protective effect. Analysing each of these signalling axes in the context of GA101-induced killing could help to understand 1) whether protection can be abrogated by the inhibition of any effector molecules that are part of these axes; 2) whether mechanisms of action other than

248 PCD, such as ADCP or ADCC, could be affected by the stroma-mediated remodelling of the actin cytoskeleton.

Similarly, an important role in stroma-mediated protection might be played by the NRF2- mediated oxidative stress response, which in the in silico analysis appeared to be up- regulated upon co-culture. NRF2 is a transcription factor that mediates cellular response to oxidative stress by inducing the expression of stress response genes [245]. In fact, it is known that GA101-induced PCD is initiated by homotypic adhesion of B cells, followed by a cascade of events leading to the release of ROS and subsequent cell death [87]. Therefore, the ability of stromal cells to activate a pathway involved in the response to oxidative stress could decrease the efficacy of GA101 at inducing PCD. In terms of ADCP, it is harder to imagine how oxidative stress levels could affect phagocytosis: in fact, the engulfment of target cells by phagocytes should be dependent on surface receptors that guide and initiate phagocytosis (discussed in 4.4). However, the hypothesis that tumour cells could modify their surface receptors upon activation of oxidative stress response pathways, leading to a reduced ability to be recognised and engulfed by phagocytes, cannot be excluded. In regards to ADCC, on the other hand, the possibility that an activation of stress response-related pathways could affect the ability of NK cells to bind to target cells and influence ADCC (and in particular the NK cell-mediated IFN-γ release) has been previously studied. In fact, Soriani et al. observed that, in MM cell lines, the drug-induced production of ROS led to the E2F1-dependent up-regulation of NK cell-activating ligand, thus increasing the ability of these immune effector cells to target tumour cells [246]. Therefore, the stroma-dependent activation of the NRF2-mediated oxidative stress response could interfere with this mechanism by reducing cellular ROS levels and subsequently impeding the up-regulation of NK cell-activating ligand. Given these observations, the role of oxidative stress response pathways in decreasing ADCC efficacy requires further investigation.

Intriguingly, studies looking at the NRF2-mediated oxidative stress response pathway highlighted the role of an actin-binding molecule, namely Keap1, in regulating degradation and activation of NRF2 [247]. Therefore, these two pathways appear to be tightly linked, and modifications in one could bring about changes in the other, leading to subsequent cellular responses. In the context of the current experimental system, one could speculate that the actin reorganisation initiated upon contact with stromal cells might, among other effects, modify the activation status of NRF2 through Keap1, leading to an increased

249 ability of tumour cells to respond to oxidative stress and ultimately augmenting cell survival.

Another important aspect that is worth considering is the fact that stromal cells are able to decrease the surface levels of CD20 molecule. This finding had been previously shown [167]; however, whether such an effect could be responsible for the decreased activity of GA101 has not been determined. Future steps in this regards could include assessing the degree of CD20 molecule on tumour cells’ surface after culture in stroma-conditioned media or culture with stromal cells in the presence of transwell plates. If a decrease in CD20 level is observed in these conditions, one could infer that stroma-mediated protection from GA101-induced death, because dependent on direct contact, is not linked to CD20 levels, and different stroma-mediated mechanisms cause the increased resistance to GA101 and the reduction in CD20. However, it is possible that neither conditioned media nor culture in transwell plates are able to recapitulate the decrease in CD20 observed in conditions of direct contact with stroma. In this scenario, counteracting the down- regulation in CD20 expression in the presence of stroma would be required to confirm the role that this decrease in CD20 has in the stroma-mediated increased survival of tumour cells. Importantly, there exist commercially available compounds, such as farnesyltransferase inhibitors (FTIs), which can mediate an increase in the expression of CD20 [248]. Therefore, it would be interesting to investigate the effect that such an increase can have on GA101-induced modes of killing. If these compounds are able to reverse the decrease in CD20 brought about by contact with stroma, it would be important to determine whether such a modification in CD20 expression can abrogate, at least partly, the protective effect. If this was the case, further in vivo research looking at combining GA101 treatment with FTIs would be needed to confirm the efficacy of such a treatment strategy.

Importantly, while both monocyte- and macrophage-mediated phagocytosis was affected by the presence of stromal cells, a strong stroma-mediated protective effect was not observed with neutrophils. This suggests that stromal cells influence pathways in tumour cells that in turn modulate different receptors. Whilst the ones that mediate interactions between tumour and monocytes could be affected by the stromal microenvironment, the same might not occur for those that are responsible for the interactions between tumour cells and neutrophils. Performing a proteomic analysis of membrane receptors in monocytes and neutrophils and comparing the two could therefore be useful to investigate

250 which differentially expressed molecules might be mediating the different interactions with tumour cells, and in turn how the stromal microenvironment can affect them.

Moreover, future research should be aimed at further analysing different models of in vitro co-culture systems, with the objective of characterising stromal cells on the basis of their ability to protect from type-II antibody-induced killing. If a lack of tumour cell’s increased survival is observed, a comparison between the protecting and non-protecting stromal lines could be made, in terms of 1) surface markers (i.e. activated vs non-activated); 2) whether the different GA101-induced mechanisms of actions lead to differences in the protective ability; 3) protein expression changes in tumour cells induced by co-culture with a protecting line vs a non-protecting line. Such a study, although laborious, could help to delineate those features that are signature of a B-cell lymphoma-associated stromal microenvironment and also narrow down the putative pathways that might be responsible for the protective effect observed. In fact, clusters of pathways that are found to be present in tumour cells cultured with both protecting and non-protecting stromal cells would not be involved in the increased survival experimentally observed and could therefore be excluded from further functional analyses.

Despite the latest developments in the field, mass spectrometry-based proteomic analyses present a number of limitations that should be kept in mind when interpreting the in silico results. Whilst the development of novel methodologies and instruments has generally improved detection and identification of proteins [249] and the use of the SILAC technique has made possible the relative quantification of analytes in samples [198], there still remain issues with both technical and analytical aspects. For instance, while on the one hand SILAC increases the user’s confidence in protein identification (i.e. each protein will be independently identified in both the medium-labelled and heavy-labelled sample), on the other hand, those peptides that do not display labelled amino acids in their sequences will not be taken into account, because it will not possible to determine which cell population they belong to. However, this issue is partly overcome by the use of the protease trypsin, which specifically cleaves C-terminal to lysine and arginine residues, ensuring the presence of one or the other labelled amino acid in each cleaved fragment. Another important limitation lies in the fact that no analyses have been performed to measure the post-translational modifications (PTM) in each identified protein. In fact, each protein can play a different role in a system based on whether specific phosphorylation, acetylation, cleavage or ubiquitination events (to mention a few) have taken place [250]. Whilst software such as IPA can help predict the activation status of enriched pathways based on

251 the expression fold change of each protein, only the accurate detection of PTM events can ensure to confidently determine whether a given protein is performing a certain activity in the cellular system. To increase the ability to accurately interpret results from a proteomic analysis, therefore, the study could be repeated by performing additional steps with the aim of enriching a protein mixture in phospho-peptides, for instance, before proceeding to the mass spectrometry analysis.

Animal models of CD20-expressing tumours were established in vivo to understand whether blocking the CXCR-4/CXCL-12 signalling axis could lead to an egression of CD34+ cells from the bone marrow into the circulation. Whilst an increase in the numbers of neutrophils, monocytes, lymphocytes and eosinophils was observed in the circulation of both EL4-huCD20- and Eµmyc-huCD20-bearing mice, this did not lead to a stronger induction of B cell tumour clearance upon GA101 treatment. Whether this effect was due to the migration of tumour cells into the bone marrow and the subsequent survival cues delivered by the protective microenvironment could not be determined, although preliminary imaging data suggested the presence of tumour burden in bones by day 7. Additional experiments involving dissection of bones and analysis of the cellular components through flow cytometry could help determine whether tumour cells are actually present in the marrow compartment. To confirm that migration into the bone marrow does affect the efficacy of GA101, however, further experiments are needed. For instance, in the case tumour cells do migrate in the bone marrow of mice, these cells could be collected, treated with GA101 ex vivo and compared to naïve tumour B cells, in order to observe whether the in vivo microenvironment does confer resistance to antibody therapy. Given that the project performed is microenvironment-focused, it is fundamental to be able to recapitulate in in vivo settings the observations made in vitro. Therefore, more work should be performed on this aspect.

A limiting factor in these experiments is the fact that, because of their immunogenicity, luciferase-iRes-GFP-expressing cells can only be inoculated without risk of rejection in immuno-compromised mice. However, since antibody therapy does rely on the immune system to induce tumour cell clearance, this technique cannot accurately recapitulate the actual activity of GA101 in mice, and in particular cannot take into account the ability of GA101 to kill through recruitment of NK cells and subsequent ADCC (of note, NSG mice do not develop functional T, B and NK cells). This is an important aspect that needs to be considered when designing in vivo experiments. The development of less immunogenic cell lines that enable visualisation of cells by bioluminescence are therefore warranted, in

252 order to be able to use immune-competent mice and syngeneic models in the study of immunotherapy.

Finally, since GA101 has been recently approved for use in not only B-NHL (and in particular, follicular lymphoma [251,252]) but also in B-CLL [253], confirming that the observations made in terms of stromal protection are also valid in B-CLL cell lines and/or B-CLL samples would further augment the relevance of this study.

8.3. Impact of research in the field

The project carried out aimed to investigate the stroma-mediated resistance of tumour cells to GA101 therapy, in order to ascertain the mechanisms that are at the basis of the protection from GA101-mediated tumour cell killing in the presence of stroma. Overall, the performed experiments determined that the stromal microenvironment is able to protect tumour cells from GA101-mediated PCD, ADCP and ADCC. The bases of such a stroma- mediated protection have been analysed and a number of pathways interrogated, with the aim of characterising the interactions between stromal and tumour cells and, possibly, delineating a strategy to manipulate such interactions. The anticipated final goal, therefore, consisted of blocking those pathways considered responsible for the stroma-mediated protection and ultimately restoring the efficacy of GA101 treatment in in vitro and in vivo models of B-cell malignancies.

The role of the microenvironment in counteracting tumour cell killing is well documented in haematopoietic malignancies [254]. With regards to B-cell malignancies, B-ALL, B- CLL and many B-NHL subtypes were shown to be affected by the presence of microenvironment components in in vitro and in vivo studies [255] - reiterating the importance of the surrounding environment in driving tumour development, progression and survival to therapeutic strategies. Therefore, when designing treatment options, interactions between tumour B cells and their surrounding environment need to be taken into account in order to obtain tangible improvements in the management of patients with B-cell malignancies.

With regards to anti-CD20 monoclonal antibodies, the role of the microenvironment, in particular the stromal component, in impairing antibody therapy has recently been elucidated. Whilst a great effort has been made in understanding the biological mechanisms of microenvironment-mediated protection of malignant cells from the type-I

253 antibody rituximab [19-21], the activity of type-II antibodies in the context of TME has not been extensively studied. Therefore, whilst on the one hand enormous progresses have been made in the development of novel antibodies, on the other hand greater efforts should be put into understanding how the microenvironment can affect the efficacy of such antibodies. The work presented has been carried out with the aim of increasing our knowledge on how stromal components impact on GA101 modes of action and what specific interactions between tumour and TME are at the basis of the decreased killing efficacy, in order to inform clinical strategies when managing patients with CD20+ B-cell malignancies. As described, direct interactions with stromal components do impact upon the efficacy of GA101. Several aspects of such a protective effect were elucidated; however, a mechanism responsible for the decreased ability of GA101 to induce tumour cell death was not identified. More work is therefore warranted in order to further explore the modalities by which stromal components mediate resistance of tumour cells to type-II antibody therapy.

Whilst it is more and more evident that the stromal microenvironment does play a key role in conferring tumour cells’ resistance to both conventional and monoclonal antibody therapy, other interactions occur between a tumour cell and the surrounding environment in the body. In fact, B-cell malignancies, arisen either in bone marrow or in lymphoid organs, would develop, proliferate and then enter the circulation, before eventually migrating into secondary organs. In this process, tumour cells encounter and possibly interact with other different tissues and cell types, namely endothelial cells, mesenchymal stromal cells, cancer-associated fibroblasts and immune cells, such as tumour-associated macrophages. Each of these cell types can affect the behaviour of tumour B cells, regulating their growth, resistance to therapies and migratory capabilities. Therefore, while the analysis of tumour/stroma interactions can help delineate the impact that the stromal component has on tumour cell survival and identify new putative targets, it is important to remember that many of the elements that similarly affect tumour cell survival are not taken into account in the study.

Whilst the development of a strategy that ensures the inhibition of critical interactions between tumour and stromal cells could mean the successful abrogation of the in vitro stroma-mediated protection from GA101, the feasibility of such an approach in an in vivo and, eventually, clinical context should be carefully evaluated. In fact, although the crosstalk between tumour and stromal component has been shown to mediate tumour resistance to therapies, it is important to bear in mind that the stromal environment also

254 plays essential roles in maintaining normal tissue homeostasis, structure and functions. The targeting of the tumour microenvironment, thus, could potentially lead to detrimental effects for mice – or, eventually, patients. Therefore, the development of strategies that could target interactions between stromal and tumour cells without affecting the crosstalk of stroma and other healthy cells should be pursued.

In summary, this research sheds light onto characteristics of the stroma-mediated protection of tumour cells from anti-CD20 monoclonal antibodies and identified potential candidate pathways that could be responsible for the increased resistance to therapeutic strategies. More work needs to be performed in order to elucidate the effect that the targeting of these pathways might have on stroma-mediated protection – and also to increase our understanding of whether the same mechanistic data can be observed with different stromal cell lines, different components of the microenvironment and different tumour cell lines or patient samples. A more in-depth knowledge of the biology of the CD20 molecule could also help to understand how to further exploit its targeting in B-cell malignancies, and is therefore warranted. The hope is that this work, and any subsequent analyses performed in this field, can eventually be translated into clinical practice, improve the management of these diseases and ultimately increase patients’ survival.

255 9. Bibliography

1. 2015 Teenagers’ and young adults’ cancers mortality statistics. . 14/04/18 2. 2018 HMRN - QuickStats. . 14/04/18 3. Molina A. A decade of rituximab: Improving survival outcomes in non-Hodgkin's lymphoma. Annual Review of Medicine. Vol 59, Annual Review of Medicine. Palo Alto: Annual Reviews; 2008. p 237-250. 4. Stashenko P, Nadler LM, Hardy R, Schlossman SF. Characterization of a human lymphocyte-B-specific antigen. Journal of Immunology 1980;125:1678-1685. 5. Rosenthal P, Rimm IJ, Umiel T, et al. Ontogeny of human hematopoietic-cells - Analysis utilizing monoclonal-antibodies. Journal of Immunology 1983;131:232-237. 6. Griffiths R, Mikhael J, Gleeson M, Danese M, Dreyling M. Addition of rituximab to chemotherapy alone as first-line therapy improves overall survival in elderly patients with mantle cell lymphoma. Blood 2011;118:4808-4816. 7. Coiffier B, Lepage E, Briere J, et al. . CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. New England Journal of Medicine 2002;346:235-242. 8. Woyach JA, Ruppert AS, Heerema NA, et al. . Treatment with Fludarabine and Rituximab Produces Extended Overall Survival (OS) and Progression-Free Survival (PFS) in Chronic Lymphocytic Leukemia (CLL) without Increased Risk of Second Malignancy: Long-Term Follow up of CALGB Study 9712. Blood 2009;114:226-226. 9. Hallek M, Fischer K, Fingerle-Rowson G, et al. . Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open- label, phase 3 trial. Lancet 2010;376:1164-1174. 10. Smith MR. Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance. Oncogene 2003;22:7359-7368. 11. Goede V, Fischer K, Busch R, et al. . Obinutuzumab plus Chlorambucil in Patients with CLL and Coexisting Conditions. New England Journal of Medicine 2014;370:1101-1110. 12. Mossner E, Brunker P, Moser S, et al. . Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity. Blood 2010;115:4393-4402. 13. Morschhauser FA, Cartron G, Thieblemont C, et al. . Obinutuzumab (GA101) Monotherapy in Relapsed/Refractory Diffuse Large B-Cell Lymphoma or Mantle-Cell Lymphoma: Results From the Phase II GAUGUIN Study. Journal of Clinical Oncology 2013;31:2912-9. 14. Trentin L, Cabrelle A, Facco M, et al. . Homeostatic chemokines drive migration of malignant B cells in patients with non-Hodgkin lymphomas. Blood 2004;104:502-508. 15. Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood 1999;94:3658-3667. 16. Lwin T, Hazlehurst LA, Li Z, et al. . Bone marrow stromal cells prevent apoptosis of lymphoma cells by upregulation of anti-apoptotic proteins associated with activation of NF-kappa B (RelB/p52) in non-Hodgkin's lymphoma cells. Leukemia 2007;21:1521-1531. 17. Taylor ST, Hickman JA, Dive C. Survival signals within the tumour microenvironment suppress drug-induced apoptosis: lessons learned from B lymphomas. Endocrine-Related Cancer 1999;6:21-23. 18. Kay NE, Shanafelt TD, Strege AK, Lee YK, Bone ND, Raza A. Bone biopsy derived marrow stromal elements rescue chronic lymphocytic leukemia B-cells from spontaneous and drug induced cell death and facilitates an "angiogenic switch". Leukemia Research 2007;31:899-906.

256 19. Beider K, Ribakovsky E, Abraham M, et al. . Targeting the CD20 and CXCR4 Pathways in Non-Hodgkin Lymphoma with Rituximab and High-Affinity CXCR4 Antagonist BKT140. Clinical Cancer Research 2013;19:3495-3507. 20. Hu YP, Gale M, Shields J, et al. . Enhancement of the anti-tumor activity of therapeutic monoclonal antibodies by CXCR4 antagonists. Leukemia & Lymphoma 2012;53:130-138. 21. Mraz M, Zent CS, Church AK, et al. . Bone marrow stromal cells protect lymphoma B-cells from rituximab-induced apoptosis and targeting integrin alpha-4-beta-1 (VLA-4) with natalizumab can overcome this resistance. British Journal of Haematology 2011;155:53- 64. 22. Vonfreedenjeffry U, Vieira P, Lucian LA, McNeil T, Burdach SEG, Murray R. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. Journal of Experimental Medicine 1995;181:1519-1526. 23. Carsetti R. The development of B cells in the bone marrow is controlled by the balance between cell-autonomous mechanisms and signals from the microenvironment. Journal of Experimental Medicine 2000;191:5-8. 24. Harris NL, Stein H, Coupland SE, et al. . New approaches to lymphoma diagnosis. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program 2001:194-220. 25. Chen JZ, Alt FW. Gene rearrangement and B-cell development. Current Opinion in Immunology 1993;5:194-200. 26. Dal Porto JM, Haberman AM, Shlomchik MJ, Kelsoe G. Antigen drives very low affinity B cells to become plasmacytes and enter germinal centers. Journal of Immunology 1998;161:5373-5381. 27. Zhang Y, Garcia-Ibanez L, Toellner KM. Regulation of germinal center B-cell differentiation (vol 270, pg 8, 2016). Immunological Reviews 2016;272:202-202. 28. Shaffer AL, Young RM, Staudt LM. Pathogenesis of Human B Cell Lymphomas. In: Paul WE, editor. Annual Review of Immunology, Vol 30. Vol 30, Annual Review of Immunology. Palo Alto: Annual Reviews; 2012. p 565-610. 29. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V-H genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94:1848-1854. 30. Kikushige Y, Ishikawa F, Miyamoto T, et al. . Self-Renewing Hematopoietic Stem Cell Is the Primary Target in Pathogenesis of Human Chronic Lymphocytic Leukemia. Cancer Cell 2011;20:246-259. 31. Blombery PA, Wall M, Seymour JF. The molecular pathogenesis of B-cell non-Hodgkin lymphoma. European Journal of Haematology 2015;95:280-293. 32. Cobaleda C, Sanchez-Garcia I. B-cell acute lymphoblastic leukaemia: towards understanding its cellular origin. Bioessays 2009;31:600-609. 33. Tedder TE, Engel P. CD20 - A regulator of cell-cycle progression of B-lymphocytes. Immunology Today 1994;15:450-454. 34. Stamenkovic I, Seed B. Analysis of 2 cDNA clones encoding the lymphocyte-B antigen- CD20 (B1, BP35), a type-III integral membrane-protein. Journal of Experimental Medicine 1988;167:1975-1980. 35. Einfeld DA, Brown JP, Valentine MA, Clark EA, Ledbetter JA. Molecular-cloning of the human B-cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. Embo Journal 1988;7:711-717. 36. Tedder TF, Forsgren A, Boyd AW, Nadler LM, Schlossman SF. Antibodies reactive with the B1 molecule inhibit cell-cycle progression but not activation of human lymphocytes-B. European Journal of Immunology 1986;16:881-887. 37. Bubien JK, Zhou LJ, Bell PD, Frizzell RA, Tedder TF. Transfection of the CD20 cell-surface molecule into ectopic cell-types generates a Ca2+ conductance found constitutively in B- lymphocytes. Journal of Cell Biology 1993;121:1121-1132. 38. O'Keefe TL, Williams GT, Davies SL, Neuberger MS. Mice carrying a CD20 gene disruption. Immunogenetics 1998;48:125-132.

257 39. Ishibashi K, Suzuki M, Sasaki S, Imai M. Identification of a new multigene four- transmembrane family (MS4A) related to CD20, HTm4 and beta subunit of the high- affinity IgE receptor. Gene 2001;264:87-93. 40. Kuijpers TW, Bende RJ, Baars PA, et al. . CD20 deficiency in humans results in impaired T cell-independent antibody responses. Journal of Clinical Investigation 2010;120:214-222. 41. Poljak RJ, Amzel LM, Avey HP, Chen BL, Phizackerley RP, Saul F. 3-dimensional structure of Fab' fragment of a human immunoglobulin at 2.8-A resolution. Proceedings of the National Academy of Sciences of the United States of America 1973;70:3305-3310. 42. Schroeder HW, Cavacini L. Structure and function of immunoglobulins. Journal of Allergy and Clinical Immunology 2010;125:S41-S52. 43. 2018 Antibody effector functions. . 24/07/18 44. Bournazos S, Ravetch JV. Fc gamma receptor pathways during active and passive immunization. Immunological Reviews 2015;268:88-103. 45. Hamaguchi Y, Xiu Y, Komura K, Nimmerjahn F, Tedder TF. Antibody isotype-specific engagement of Fc gamma receptors regulates B lymphocyte depletion during CD20 immunotherapy. Journal of Experimental Medicine 2006;203:743-753. 46. Teeling JL, French RR, Cragg MS, et al. . Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood 2004;104:1793-1800. 47. Stein R, Qu ZX, Chen S, et al. . Characterization of a new humanized anti-CD20 monoclonal antibody, IMMU-106, and its use in combination with the humanized anti-CD22 antibody, epratuzumab, for the therapy of non-Hodgkin's lymphoma. Clinical Cancer Research 2004;10:2868-2878. 48. Robak T, Robak E. New Anti-CD20 Monoclonal Antibodies for the Treatment of B-Cell Lymphoid Malignancies. Biodrugs 2011;25:13-25. 49. Umana P, Moessner E, Bruenker P, et al. . Novel 3(rd) generation humanized type IICD20 antibody with glycoengineered fc and modified elbow hinge for enhanced ADCC and superior apoptosis induction. Blood 2006;108:72A-72A. 50. Forero-Torres A, de Vos S, Pohlman BL, et al. . Results of a Phase 1 Study of AME-133v (LY2469298), an Fc-Engineered Humanized Monoclonal Anti-CD20 Antibody, in Fc gamma RIIIa-Genotyped Patients with Previously Treated Follicular Lymphoma. Clinical Cancer Research 2012;18:1395-1403. 51. Herter S, Birk MC, Klein C, Gerdes C, Umana P, Bacac M. Glycoengineering of Therapeutic Antibodies Enhances Monocyte/Macrophage-Mediated Phagocytosis and Cytotoxicity. Journal of Immunology 2014;192:2252-2260. 52. Friess T, Gerdes C, Nopora A, et al. . GA101, a novel humanized type IICD20 antibody with glycoengineered Fe and enhanced cell death induction, mediates superior efficacy in a variety of NHL xenograft models in comparison to rituximab. Blood 2007;110:691A-692A. 53. Abes R, Gelize E, Fridman WH, Teillaud JL. Long-lasting antitumor protection by anti-CD20 antibody through cellular immune response. Blood 2010;116:926-934. 54. Cheadle EJ, Lipowska-Bhalla G, Dovedi SJ, et al. . A TLR7 agonist enhances the antitumor efficacy of obinutuzumab in murine lymphoma models via NK cells and CD4 T cells. Leukemia 2017;31:1611-1621. 55. Hilchey SP, Hyrien O, Mosmann TR, et al. . Rituximab immunotherapy results in the induction of a lymphoma idiotype-specific T-cell response in patients with follicular lymphoma: support for a "vaccinal effect" of rituximab. Blood 2009;113:3809-3812. 56. Cartron G, Dacheux L, Salles G, et al. . Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor Fc gamma RIIIa gene. Blood 2002;99:754-758. 57. Flieger D, Renoth S, Beier I, Sauerbruch T, Schmidt-Wolf I. Mechanism of cytotoxicity induced by chimeric mouse human monoclonal antibody IDEC-C2B8 in CD20-expressing lymphoma cell lines. Cellular Immunology 2000;204:55-63.

258 58. Cragg MS, Morgan SM, Chan HTC, et al. . Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts. Blood 2003;101:1045-1052. 59. Cragg MS, Glennie MJ. Antibody specificity controls in vivo effector mechanisms of anti- CD20 reagents. Blood 2004;103:2738-2743. 60. Teeling JL, Mackus WJM, Wiegman L, et al. . The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20. Journal of Immunology 2006;177:362-371. 61. Chan HTC, Hughes D, French RR, et al. . CD20-induced lymphoma cell death is independent of both caspases and its redistribution into Triton X-100 insoluble membrane rafts. Cancer Research 2003;63:5480-5489. 62. Klein C, Lammens A, Schafer W, et al. . Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties. Mabs 2013;5:22-33. 63. Niederfellner G, Lammens A, Mundigl O, et al. . Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies. Blood 2011;118:358-367. 64. Tobinai K, Klein C, Oya N, Fingerle-Rowson G. A Review of Obinutuzumab (GA101), a Novel Type II Anti-CD20 Monoclonal Antibody, for the Treatment of Patients with B-Cell Malignancies. Advances in Therapy 2017;34:324-356. 65. Golay J, Zaffaroni L, Vaccari T, et al. . Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 2000;95:3900-3908. 66. Clynes R, Maizes JS, Guinamard R, Ono M, Takai T, Ravetch JV. Modulation of immune complex-induced inflammation in vivo by the coordinate expression of activation and inhibitory Fc receptors. Journal of Experimental Medicine 1999;189:179-185. 67. Smyth MJ, Cretney E, Kelly JM, et al. . Activation of NK cell cytotoxicity. Molecular Immunology 2005;42:501-510. 68. Srivastava RM, Lee SC, Andrade PA, et al. . Cetuximab-Activated Natural Killer and Dendritic Cells Collaborate to Trigger Tumor Antigen-Specific T-cell Immunity in Head and Neck Cancer Patients. Clinical Cancer Research 2013;19:1858-1872. 69. Nimmerjahn F, Ravetch JV. Divergent immunoglobulin G subclass activity through selective Fc receptor binding. Science 2005;310:1510-1512. 70. Dhodapkar KM, Kaufman JL, Ehlers M, et al. . Selective blockade of inhibitory Fc gamma receptor enables human dendritic cell maturation with IL-12p70 production and immunity to antibody-coated tumor cells. Proceedings of the National Academy of Sciences of the United States of America 2005;102:2910-2915. 71. Boruchov AM, Heller G, Veri MC, Bonvini E, Ravetch JV, Young JW. Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions. Journal of Clinical Investigation 2005;115:2914-2923. 72. Ferrara C, Stuart F, Sondermann P, Brukner P, Umana P. The carbohydrate at Fc gamma RIIIa Asn-162 - an element required for high affinity binding to non-fucosylated IgG glycoforms. Journal of Biological Chemistry 2006;281:5032-5036. 73. Kaneko Y, Nimmerjahn F, Ravetch EV. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 2006;313:670-673. 74. Lefebvre M-L, Krause SW, Salcedo M, Nardin A. Ex vivo-activated human macrophages kill chronic lymphocytic leukemia cells in the presence of rituximab: mechanism of antibody- dependent cellular cytotoxicity and impact of human serum. Journal of immunotherapy 2006;29:388-397. 75. Uchida JJ, Hamaguchi Y, Oliver JA, et al. . The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy. Journal of Experimental Medicine 2004;199:1659-1669. 76. Church AK, VanDerMeid KR, Baig NA, et al. . Anti-CD20 monoclonal antibody dependent phagocytosis of chronic lymphocytic leukemia cells by autologous macrophages. Clinical and experimental immunology 2016;183:90-101.

259 77. Dyall R, Vasovic LV, Clynes RA, Nikolic-Zugic J. Cellular requirements for the monoclonal antibody-mediated eradication of an established solid tumor. European Journal of Immunology 1999;29:30-37. 78. Kono K, Takahashi A, Ichihara F, Sugai H, Fujii H, Matsumoto Y. Impaired antibody- dependent cellular cytotoxicity mediated by Herceptin in patients with gastric cancer. Cancer Research 2002;62:5813-5817. 79. Bowles JA, Wang SY, Link BK, et al. . Anti-CD20 monoclonal antibody with enhanced affinity for CD16 activates NK cells at lower concentrations and more effectively than rituximab. Blood 2006;108:2648-2654. 80. Deligne C, Metidji A, Fridman WH, Teillaud JL. Anti-CD20 therapy induces a memory Th1 response through the IFN-γ/IL-12 axis and prevents protumor regulatory T-cell expansion in mice. Leukemia 2015;29:947-957. 81. Glennie MJ, French RR, Cragg MS, Taylor RP. Mechanisms of killing by anti-CD20 monoclonal antibodies. Molecular Immunology 2007;44:3823-3837. 82. Shan DM, Ledbetter JA, Press OW. Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunology Immunotherapy 2000;48:673- 683. 83. Hofmeister JK, Cooney D, Coggeshall KM. Clustered CD20 induced apoptosis: Src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3- dependent apoptosis. Blood Cells Molecules and Diseases 2000;26:133-143. 84. Ivanov A, Beers SA, Walshe CA, et al. . Monoclonal antibodies directed to CD20 and HLA- DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells. Journal of Clinical Investigation 2009;119:2143-2159. 85. Kroemer G, Jaattela M. Lysosomes and autophagy in cell death control. Nature Reviews Cancer 2005;5:886-897. 86. Alduaij W, Ivanov A, Honeychurch J, et al. . Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies. Blood 2011;117:4519-4529. 87. Honeychurch J, Alduaij W, Azizyan M, et al. . Antibody-induced nonapoptotic cell death in human lymphoma and leukemia cells is mediated through a novel reactive oxygen species-dependent pathway. Blood 2012;119:3523-3533. 88. Reff ME, Carner K, Chambers KS, et al. Depletion of B-cells in-vivo by a chimeric mouse- human monoclonal-antibody to CD20. Blood 1994;83:435-445. 89. McLaughlin P, Grillo-Lopez AJ, Link BK, et al. . Rituximab chimeric Anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: Half of patients respond to a four-dose treatment program. Journal of Clinical Oncology 1998;16:2825-2833. 90. Davis TA, Grillo-Lopez AJ, White CA, et al. . Rituximab anti-CD20 monoclonal antibody therapy in non-Hodgkin's lymphoma: Safety and efficacy of re-treatment. Journal of Clinical Oncology 2000;18:3135-3143. 91. Olejniczak SH, Stewart CC, Donohue K, Czuczman MS. A quantitative exploration of surface antigen expression in common B-cell malignancies using flow cytometry. Immunological Investigations 2006;35:93-114. 92. Johnson NA, Boyle M, Bashashati A, et al. . Diffuse large B-cell lymphoma: reduced CD20 expression is associated with an inferior survival. Blood 2009;113:3773-3780. 93. Prevodnik VK, Lavrencak J, Horvat M, Novakovic BJ. The predictive significance of CD20 expression in B-cell lymphomas. Diagnostic Pathology 2011;6:6. 94. Davis TA, Czerwinski DK, Levy R. Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression. Clinical Cancer Research 1999;5:611- 615. 95. Foran JM, Norton AJ, Micallef INM, et al. . Loss of CD20 expression following treatment with rituximab (chimaeric monoclonal anti-CD20): a retrospective cohort analysis. British Journal of Haematology 2001;114:881-883. 96. Beum PV, Kennedy AD, Williams ME, Lindorfer MA, Taylor RP. The shaving reaction: Rituximab/CD20 complexes are removed from mantle cell lymphoma and chronic

260 lymphocytic leukemia cells by THP-1 monocytes. Journal of Immunology 2006;176:2600- 2609. 97. Hudrisier D, Aucher A, Puaux AL, Bordier C, Joly E. Capture of target cell membrane components via trogocytosis is triggered by a selected set of surface molecules on T or B cells. Journal of Immunology 2007;178:3637-3647. 98. Boross P, Jansen JHM, Pastula A, van der Poel CE, Leusen JHW. Both activating and inhibitory Fc gamma receptors mediate rituximab-induced trogocytosis of CD20 in mice. Immunology Letters 2012;143:44-52. 99. Beers SA, French RR, Chan HTC, et al. . Antigenic modulation limits the efficacy of anti- CD20 antibodies: implications for antibody selection. Blood 2010;115:5191-5201. 100. Wu JM, Edberg JC, Redecha PB, et al. . A novel polymorphism of Fc gamma RIIIa (CD16) alters receptor function and predisposes to autoimmune disease. Journal of Clinical Investigation 1997;100:1059-1070. 101. Weng WK, Levy R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. Journal of Clinical Oncology 2003;21:3940-3947. 102. Dornan D, Spleiss O, Yeh RF, et al. . Effect of FCGR2A and FCGR3A variants on CLL outcome. Blood 2010;116:4212-4222. 103. Ghesquieres H, Cartron G, Seymour JF, et al. . Clinical outcome of patients with follicular lymphoma receiving chemoimmunotherapy in the PRIMA study is not affected by FCGR3A and FCGR2A polymorphisms. Blood 2012;120:2650-2657. 104. Kenkre VP, Hong FX, Cerhan JR, et al. . Fc Gamma Receptor 3A and 2A Polymorphisms Do Not Predict Response to Rituximab in Follicular Lymphoma. Clinical Cancer Research 2016;22:821-826. 105. Patz M, Isaeva P, Forcob N, et al. . Comparison of the in vitro effects of the anti-CD20 antibodies rituximab and GA101 on chronic lymphocytic leukaemia cells. British Journal of Haematology 2011;152:295-306. 106. Dalle S, Reslan L, de Horts TB, et al. . Preclinical Studies on the Mechanism of Action and the Anti-Lymphoma Activity of the Novel Anti-CD20 Antibody GA101. Molecular Cancer Therapeutics 2011;10:178-185. 107. Cang SD, Mukhi N, Wang KM, Liu DL. Novel CD20 monoclonal antibodies for lymphoma therapy. Journal of Hematology & Oncology 2012;5:64. 108. Cheson BD. Ofatumumab, a Novel Anti-CD20 Monoclonal Antibody for the Treatment of B-Cell Malignancies. Journal of Clinical Oncology 2010;28:3525-3530. 109. Rafiq S, Butchar JP, Cheney C, et al. . Comparative Assessment of Clinically Utilized CD20- Directed Antibodies in Chronic Lymphocytic Leukemia Cells Reveals Divergent NK Cell, Monocyte, and Macrophage Properties. Journal of Immunology 2013;190:2702-2711. 110. Golay J, Da Roit F, Bologna L, et al. . Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab. Blood 2013;122:3482-3491. 111. Wang SY, Racila E, Taylor RP, Weiner GJ. NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement. Blood 2008;111:1456-1463. 112. Kern DJ, James BR, Blackwell S, Gassner C, Klein C, Weiner GJ. GA101 induces NK-cell activation and antibody-dependent cellular cytotoxicity more effectively than rituximab when complement is present. Leukemia & Lymphoma 2013;54:2500-2505. 113. Herter S, Herting F, Mundigl O, et al. . Preclinical Activity of the Type II CD20 Antibody GA101 (Obinutuzumab) Compared with Rituximab and Ofatumumab In Vitro and in Xenograft Models. Molecular Cancer Therapeutics 2013;12:2031-2042. 114. Herter S, Herting F, Muth G, et al. . GA101 P329GLALA, a variant of obinutuzumab with abolished ADCC, ADCP and CDC function but retained cell death induction, is as efficient as rituximab in B-cell depletion and antitumor activity. Haematologica 2018;103:E78-E81. 115. Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011;144:646- 674.

261 116. Nicholas NS, Apollonio B, Ramsay AG. Tumor microenvironment (TME)-driven immune suppression in B cell malignancy. Biochimica Et Biophysica Acta-Molecular Cell Research 2016;1863:471-482. 117. Panayiotidis P, Jones D, Ganeshaguru K, Foroni L, Hoffbrand AV. Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro. British Journal of Haematology 1996;92:97-103. 118. Lagneaux L, Delforge A, Bron D, De Bruyn C, Stryckmans P. Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood 1998;91:2387-2396. 119. Medina DJ, Goodell L, Glod J, Gelinas C, Rabson AB, Strair RK. Mesenchymal stromal cells protect mantle cell lymphoma cells from spontaneous and drug-induced apoptosis through secretion of B-cell activating factor and activation of the canonical and non- canonical nuclear factor kappa B pathways. Haematologica-the Hematology Journal 2012;97:1255-1263. 120. Damiano JS, Cress AE, Hazlehurst LA, Shtil AA, Dalton WS. Cell adhesion mediated drug resistance (CAM-DR): Role of integrins and resistance to apoptosis in human myeloma cell lines. Blood 1999;93:1658-1667. 121. Mohle R, Failenschmid C, Bautz F, Kanz L. Overexpression of the chemokine receptor CXCR4 in B cell chronic lymphocytic leukemia is associated with increased functional response to stromal cell-derived factor-1 (SDF-1). Leukemia 1999;13:1954-1959. 122. Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell'Aquila M, Kipps TJ. Blood-derived nurse- like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood 2000;96:2655-2663. 123. Kurtova AV, Balakrishnan K, Chen R, et al. . Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance. Blood 2009;114:4441-4450. 124. Tabe Y, Jin LH, Tsutsumi-Ishii Y, et al. . Activation of integrin-linked kinase is a critical prosurvival pathway induced in leukemic cells by bone marrow-derived stromal cells. Cancer Research 2007;67:684-694. 125. Hoellenriegel J, Zboralski D, Maasch C, et al. . The Spiegelmer NOX-A12, a novel CXCL12 inhibitor, interferes with chronic lymphocytic leukemia cell motility and causes chemosensitization. Blood 2014;123:1032-1039. 126. Gehrke I, Gandhirajan RK, Poll-Wolbeck SJ, Hallek M, Kreuzer KA. Bone Marrow Stromal Cell-Derived Vascular Endothelial Growth Factor (VEGF) Rather Than Chronic Lymphocytic Leukemia (CLL) Cell-Derived VEGF Is Essential for the Apoptotic Resistance of Cultured CLL Cells. Molecular Medicine 2011;17:619-627. 127. Polyak K, Haviv I, Campbell IG. Co-evolution of tumor cells and their microenvironment. Trends in Genetics 2009;25:30-38. 128. Raffaghello L, Vacca A, Pistoia V, Ribatti D. Cancer associated fibroblasts in hematological malignancies. Oncotarget 2015;6:2589-2603. 129. Ding W, Nowakowski GS, Knox TR, et al. . Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression. British Journal of Haematology 2009;147:471-483. 130. Ding W, Knox TR, Tschumper RC, et al. . Platelet-derived growth factor (PDGF)-PDGF receptor interaction activates bone marrow-derived mesenchymal stromal cells derived from chronic lymphocytic leukemia: implications for an angiogenic switch. Blood 2010;116:2984-2993. 131. Lutzny G, Kocher T, Schmidt-Supprian M, et al. . Protein Kinase C-beta-Dependent Activation of NF-kappa B in Stromal Cells Is Indispensable for the Survival of Chronic Lymphocytic Leukemia B Cells In Vivo. Cancer Cell 2013;23:77-92. 132. Orimo A, Weinberg RA. Stromal fibroblasts in cancer - A novel tumor-promoting cell type. Cell Cycle 2006;5:1597-1601.

262 133. Durig J, Schmucker U, Duhrsen U. Differential expression of chemokine receptors in B cell malignancies. Leukemia 2001;15:752-756. 134. Ma Q, Jones D, Borghesani PR, et al. . Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 1998;95:9448-9453. 135. Burger JA, Kipps TJ. CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment. Blood 2006;107:1761-1767. 136. Arai J, Yasukawa M, Yakushijin Y, Miyazaki T, Fujita S. Stromal cells in lymph nodes attract B-lymphoma cells via production of stromal cell-derived factor-1. European Journal of Haematology 2000;64:323-332. 137. Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 Pathway in Cancer. Clinical Cancer Research 2010;16:2927-2931. 138. Burger M, Hartmann T, Krome M, et al. . Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells. Blood 2005;106:1824-1830. 139. Randhawa S, Cho BS, Ghosh D, et al. . Effects of pharmacological and genetic disruption of CXCR4 chemokine receptor function in B-cell acute lymphoblastic leukaemia. British Journal of Haematology 2016;174:425-436. 140. Burns JM, Summers BC, Wang Y, et al. . A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. Journal of Experimental Medicine 2006;203:2201-2213. 141. Sun XQ, Cheng GC, Hao MG, et al. . CXCL12/CXCR4/CXCR7 chemokine axis and cancer progression. Cancer and Metastasis Reviews 2010;29:709-722. 142. Puddinu V, Casella S, Radice E, et al. . ACKR3 expression on diffuse large B cell lymphoma is required for tumor spreading and tissue infiltration. Oncotarget 2017;8:85068-85084. 143. Burwick N, Roccaro AM, Moreau AS, et al. . CXCR7 Regulates SDF-1 Induced, Adhesion, Homing, and Signaling in Multiple Myeloma. Clinical Lymphoma & Myeloma 2009;9:S149- S149. 144. Melo RDC, Longhini AL, Bigarella CL, et al. CXCR7 Is Highly Expressed in Acute Lymphoblastic Leukemia and Potentiates CXCR4 Response to CXCL12. Plos One 2014;9:12. 145. Melo RDC, Ferro KPV, Duarte ADS, Saad STO. CXCR7 participates in CXCL12-mediated migration and homing of leukemic and normal hematopoietic cells. Stem Cell Research & Therapy 2018;9:5. 146. Wang JH, Shiozawa YS, Wang JC, et al. . The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. Journal of Biological Chemistry 2008;283:4283-4294. 147. Hattermann K, Held-Feindt J, Lucius R, et al. . The Chemokine Receptor CXCR7 Is Highly Expressed in Human Glioma Cells and Mediates Antiapoptotic Effects. Cancer Research 2010;70:3299-3308. 148. Li WJ, Ding QL, Ding YX, et al. . Oroxylin A Reverses the Drug Resistance of Chronic Myelogenous Leukemia Cells to Imatinib Through CXCL12/CXCR7 Axis in Bone Marrow Microenvironment. Molecular Carcinogenesis 2017;56:863-876. 149. Baldini L, Cro L, Calori R, Nobili L, Silvestris I, Maiolo AT. Differential expression of very late activation antigen-3 (VLA-3)/VLA-4 in B-cell non-Hodgkin lymphoma and B-cell chronic lymphocytic leukemia. Blood 1992;79:2688-2693. 150. Kurtova AV, Tamayo AT, Ford RJ, Burger JA. Mantle cell lymphoma cells express high levels of CXCR4, CXCR5, and VLA-4 (CD49d): importance for interactions with the stromal microenvironment and specific targeting. Blood 2009;113:4604-4613. 151. Koopman G, Keehnen RM, Lindhout E, et al. . Adhesion through the LFA-1 (CD11a/CD18)- ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells. The journal of immunology 1994;152:3760-3767. 152. Simmons PJ, Masinovsky B, Longenecker BM, Berenson R, Torok-Storb B, Gallatin WM. Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of hematopoietic progenitor cells. Blood 1992;80:388-395.

263 153. Terol MJ, López-Guillermo A, Bosch F, et al. . Expression of beta-integrin adhesion molecules in non-Hodgkin's lymphoma: correlation with clinical and evolutive features. Journal of clinical oncology 1999;17:1869-1875. 154. Shalapour S, Hof J, Kirschner-Schwabe R, et al. . High VLA-4 expression is associated with adverse outcome and distinct gene expression changes in childhood B-cell precursor acute lymphoblastic leukemia at first relapse. Haematologica 2011;96:1627-1635. 155. Taylor ST, Hickman JA, Dive C. Epigenetic determinants of resistance to etoposide regulation of Bcl-X(L) and Bax by tumor microenvironmental factors. Journal of the National Cancer Institute. 2000;92:18-23. 156. de la Fuente MT, Casanova B, Moyano JV, et al. Engagement of alpha4beta1 integrin by fibronectin induces in vitro resistance of B chronic lymphocytic leukemia cells to fludarabine. Journal of leukocyte biology 2002;71:495-502. 157. de la Fuente MT, Casanova B, Cantero E, et al. Involvement of p53 in alpha4beta1 integrin-mediated resistance of B-CLL cells to fludarabine. Biochemical and biophysical research communications 2003;311:708-712. 158. Danoch H, Kalechman Y, Albeck M, Longo DL, Sredni B. Sensitizing B- and T- cell Lymphoma Cells to Paclitaxel/Abraxane-Induced Death by AS101 via Inhibition of the VLA- 4-IL10-Survivin Axis. Molecular Cancer Research 2015;13:411-422. 159. Peled A, Kollet O, Ponomaryov T, et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 2000;95:3289-3296. 160. Hidalgo A, Sanz-Rodríguez F, Rodríguez-Fernández JL, et al. Chemokine stromal cell- derived factor-1alpha modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells. Experimental hematology 2001;29:345-355. 161. Ngo HT, Leleu X, Lee J, et al. . SDF-1/CXCR4 and VLA-4 interaction regulates homing in Waldenstrom macroglobulinemia. Blood 2008;112:150-158. 162. Jacamo R, Chen Y, Wang Z, et al. Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation of NF-κB mediates chemoresistance. Blood 2014;123:2691-2702. 163. Kurtova A, Tamayo A, Ford RJ, Burger JA. Migratory Activity and Stromal Cell Adhesion of Mantle Cell Lymphoma Cells Can Be Diminished by Blocking of VLA-4 and CXCR4 Receptors. Blood 2008;112:976-976. 164. Kim Y-R, Eom K-S. Simultaneous Inhibition of CXCR4 and VLA-4 Exhibits Combinatorial Effect in Overcoming Stroma-Mediated Chemotherapy Resistance in Mantle Cell Lymphoma Cells. Immune network 2014;14:296-306. 165. Sivina M, Kreitman RJ, Peled A, Ravandi F, Burger JA. Adhesion of Hairy Cells Leukemia (HCL) Cells to Stromal Cells Can Be Inhibited by Blocking VLA-4 Integrins and CXCR4 Chemokine Receptors. Blood 2011;118:768-768. 166. Buchner M, Brantner P, Stickel N, et al. . The microenvironment differentially impairs passive and active immunotherapy in chronic lymphocytic leukaemia-CXCR4 antagonists as potential adjuvants for monoclonal antibodies. British Journal of Haematology 2010;151:167-178. 167. Marquez ME, Hernández-Uzcátegui O, Cornejo A, Vargas P, Da Costa O. Bone marrow stromal mesenchymal cells induce down regulation of CD20 expression on B-CLL: implications for rituximab resistance in CLL. Br J Haematol 2015;169:211-218. 168. O'Callaghan K, Lee L, Nguyen N, et al. . Targeting CXCR4 with cell-penetrating pepducins in lymphoma and lymphocytic leukemia. Blood 2012;119:1717-1725. 169. Sauter B, Albert ML, Francisco L, Larsson M, Somersan S, Bhardwaj N. Consequences of cell death: Exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. Journal of Experimental Medicine 2000;191:423-433. 170. Selenko N, Majdic O, Draxler S, et al. . CD20 antibody (C2B8)-induced apoptosis of lymphoma cells promotes phagocytosis by dendritic cells and cross-priming of CD8(+) cytotoxic T cells. Leukemia 2001;15:1619-1626.

264 171. Wahlin BE, Sundstrom C, Holte H, et al. T Cells in Tumors and Blood Predict Outcome in Follicular Lymphoma Treated with Rituximab. Clinical Cancer Research 2011;17:4136- 4144. 172. Cheadle EJ, Gilham DE, Hawkins RE. The combination of cyclophosphamide and human T cells genetically engineered to target CD19 can eradicate established B-cell lymphoma. British Journal of Haematology 2008;142:65-68. 173. Zhang J, Xin L, Shan BZ, et al. PEAKS DB: De Novo Sequencing Assisted Database Search for Sensitive and Accurate Peptide Identification. Molecular & Cellular Proteomics 2012;11: M111.010587. 174. 2018. DAVID - Help. . 11/05/18. 175. Hosack DA, Dennis G, Sherman BT, Lane HC, Lempicki RA. Identifying biological themes within lists of genes with EASE. Genome Biology 2003;4:8. 176. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols 2009;4:44-57. 177. Simon-Gabriel CP, Foerster K, Saleem S, et al. . Microenvironmental stromal cells abrogate NF-kappa B inhibitor-induced apoptosis in chronic lymphocytic leukemia. Haematologica 2018;103:136-147. 178. Duda DG, Kozin SV, Kirkpatrick ND, Xu L, Fukumura D, Jain RK. CXCL12 (SDF1 alpha)- CXCR4/CXCR7 Pathway Inhibition: An Emerging Sensitizer for Anticancer Therapies? Clinical Cancer Research 2011;17:2074-2080. 179. Kalatskaya I, Berchiche YA, Gravel S, Limberg BJ, Rosenbaum JS, Heveker N. AMD3100 Is a CXCR7 Ligand with Allosteric Agonist Properties. Molecular Pharmacology 2009;75:1240- 1247. 180. Weekes CD, Kuszynski CA, Sharp JG. VLA-4 mediated adhesion to bone marrow stromal cells confers chemoresistance to adherent lymphoma cells. Leukemia & Lymphoma 2001;40:631-645. 181. Edelmann J, Klein-Hitpass L, Carpinteiro A, et al. . Bone marrow fibroblasts induce expression of PI3K/NF-kappa B pathway genes and a pro-angiogenic phenotype in CLL cells. Leukemia Research 2008;32:1565-1572. 182. Ramirez PA, Rettig MP, Holt M, DiPersio JF. M2-10B4 mesenchymal stromal cells confer an in vitro protective effect of murine mCGPR/+ acute promyelocytic leukemic cells against chemotherapy. Blood 2007;110:836A-837A. 183. Ohkouchi S, Block GJ, Katsha AM, et al. . Mesenchymal Stromal Cells Protect Cancer Cells From ROS-induced Apoptosis and Enhance the Warburg Effect by Secreting STC1. Molecular Therapy 2012;20:417-423. 184. Liu JZ, Masurekar A, Johnson S, et al. . Stromal cell-mediated mitochondrial redox adaptation regulates drug resistance in childhood acute lymphoblastic leukemia. Oncotarget 2015;6:43048-43064. 185. Barclay AN, van den Berg TK. The Interaction Between Signal Regulatory Protein Alpha (SIRP alpha) and CD47: Structure, Function, and Therapeutic Target. Annual Review of Immunology, Vol 32 2014;32:25-50. 186. Koskinen C, Persson E, Baldock P, et al. . Lack of CD47 Impairs Bone Cell Differentiation and Results in an Osteopenic Phenotype in Vivo due to Impaired Signal Regulatory Protein alpha (SIRP alpha) Signaling. Journal of Biological Chemistry 2013;288:29333-29344. 187. Wong KL, Tai JJY, Wong WC, et al. . Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets. Blood 2011;118:E15-E30. 188. Le Blanc K, Davies LC. Mesenchymal stromal cells and the innate immune response. Immunology Letters 2015;168:140-146. 189. Gordon S. Phagocytosis: An Immunobiologic Process. Immunity 2016;44:463-475. 190. Brown S, Heinisch I, Ross E, Shaw K, Buckley CD, Savill J. Apoptosis disables CD31- mediated cell detachment from phagocytes promoting binding and engulfment. Nature 2002;418:200-203.

265 191. Ziegler-Heitbrock L. Blood monocytes and their subsets: established features and open questions. Frontiers in Immunology 2015;6:423. 192. Wong KL, Yeap WH, Tai JJY, Ong SM, Dang TM, Wong SC. The three human monocyte subsets: implications for health and disease. Immunologic Research 2012;53:41-57. 193. Cros J, Cagnard N, Woollard K, et al. . Human CD14(dim) Monocytes Patrol and Sense Nucleic Acids and Viruses via TLR7 and TLR8 Receptors. Immunity 2010;33:375-386. 194. Zawada AM, Rogacev KS, Rotter B, et al. . SuperSAGE evidence for CD14(++)CD16(+) monocytes as a third monocyte subset. Blood 2011;118:E50-E61. 195. Bryceson YT, March ME, Ljunggren HG, Long EO. Synergy among receptors on resting NK cells for the activation of natural cytotoxicity and cytokine secretion. Blood 2006;107:159- 166. 196. Scharton TM, Scott P. Natural-killer-cells are a source of interferon-gamma that drives differentiation of CD4+ T-cell subsets and induces early resistance to Leishmania-major in mice. Journal of Experimental Medicine 1993;178:567-577. 197. Steen H, Mann M. The ABC's (and XYZ's) of peptide sequencing. Nature Reviews Molecular Cell Biology 2004;5:699-711. 198. Ong SE, Blagoev B, Kratchmarova I, et al. . Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Molecular & Cellular Proteomics 2002;1:376-386. 199. Ong SE, Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nature Protocols 2006;1:2650-2660. 200. Sonenberg N, Hinnebusch AG. Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets. Cell 2009;136:731-745. 201. Kramer A, Green J, Pollard J, Tugendreich S. Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics 2014;30:523-530. 202. Buchner M, Baer C, Prinz G, et al. . Spleen tyrosine kinase inhibition prevents chemokine- and integrin-mediated stromal protective effects in chronic lymphocytic leukemia. Blood 2010;115:4497-4506. 203. Meadows SA, Vega F, Kashishian A, et al. . PI3K delta inhibitor, GS-1101 (CAL-101), attenuates pathway signaling, induces apoptosis, and overcomes signals from the microenvironment in cellular models of Hodgkin lymphoma. Blood 2012;119:1897-1900. 204. Farooqui MZH, Valdez J, Martyr S, et al. . Ibrutinib for previously untreated and relapsed or refractory chronic lymphocytic leukaemia with TP53 aberrations: a phase 2, single-arm trial. Lancet Oncology 2015;16:169-176. 205. Herman SEM, Mustafa RZ, Jones J, Wong DH, Farooqui M, Wiestner A. Treatment with Ibrutinib Inhibits BTK- and VLA-4-Dependent Adhesion of Chronic Lymphocytic Leukemia Cells In Vivo. Clinical Cancer Research 2015;21:4642-4651. 206. Da Roit F, Engelberts PJ, Taylor RP, et al. . Ibrutinib interferes with the cell-mediated antitumor activities of therapeutic CD20 antibodies: implications for combination therapy. Haematologica 2015;100:77-86. 207. Bojarczuk K, Siernicka M, Dwojak M, et al. . B-cell receptor pathway inhibitors affect CD20 levels and impair antitumor activity of anti-CD20 monoclonal antibodies. Leukemia 2014;28:1163-1167. 208. Halbleib JM, Nelson WJ. Cadherins in development: cell adhesion, sorting, and tissue morphogenesis. Genes & Development 2006;20:3199-3214. 209. Takata K, Tanino M, Ennishi D, et al. Duodenal follicular lymphoma: Comprehensive gene expression analysis with insights into pathogenesis. Cancer Science 2014;105:608-615. 210. Sharma S, Lichtenstein A. Aberrant splicing of the E-cadherin transcript is a novel mechanism of gene silencing in chronic lymphocytic leukemia cells. Blood 2009;114:4179- 4185. 211. Lwin T, Hazlehurst LA, Dessureault S, et al. Cell adhesion induces p27Kip1-associated cell- cycle arrest through down-regulation of the SCFSkp2 ubiquitin ligase pathway in mantle- cell and other non-Hodgkin B-cell lymphomas. Blood 2007;110:1631-1638.

266 212. Kawamura-Kodama K, Tsutsui J, Suzuki ST, Kanzaki T, Ozawa M. N-cadherin expressed on malignant T cell lymphoma cells is functional, and promotes heterotypic adhesion between the lymphoma cells and mesenchymal cells expressing N-cadherin. Journal of Investigative Dermatology 1999;112:62-66. 213. Zhang B, Li M, McDonald T, et al. . Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling. Blood 2013;121:1824-1838. 214. Bojarczuk K, Siernicka M, Dwojak M, et al. . B-cell receptor pathway inhibitors affect CD20 levels and impair antitumor activity of anti-CD20 monoclonal antibodies. Leukemia 2014;28:1163-1167. 215. Skarzynski M, Niemann CU, Lee YS, et al. . Interactions between Ibrutinib and Anti-CD20 Antibodies: Competing Effects on the Outcome of Combination Therapy. Clinical Cancer Research 2016;22:86-95. 216. Franke A, Niederfellner GJ, Klein C, Burtscher H. Antibodies against CD20 or B-Cell Receptor Induce Similar Transcription Patterns in Human Lymphoma Cell Lines. Plos One 2011;6:11. 217. Orsulic S, Huber O, Aberle H, Arnold S, Kemler R. E-cadherin binding prevents beta- catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. Journal of Cell Science 1999;112:1237-1245. 218. Lu DS, Zhao YD, Tawatao R, et al. . Activation of the Wnt signaling pathway in chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America 2004;101:3118-3123. 219. Kudo S, Caaveiro JMM, Nagatoishi S, et al. Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin. Scientific Reports 2017;7:15. 220. Akers SM, O'Leary HA, Minnear FL, et al. . VE-cadherin and PECAM-1. enhance ALL migration across brain microvascular endothelial cell monolayers. Experimental Hematology 2010;38:733-743. 221. Hao M, Zhang L, An G, et al. . Bone marrow stromal cells protect myeloma cells from bortezomib induced apoptosis by suppressing microRNA-15a expression. Leukemia & Lymphoma 2011;52:1787-1794. 222. Burwick N, Zhang MY, de la Puente P, et al. . The eIF2-alpha kinase HRI is a novel therapeutic target in multiple myeloma. Leukemia Research 2017;55:23-32. 223. Harding HP, Novoa I, Zhang YH, et al. . Regulated translation initiation controls stress- induced gene expression in mammalian cells. Molecular Cell 2000;6:1099-1108. 224. Beers SA, Chan CHT, James S, et al. . Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation. Blood 2008;112:4170-4177. 225. Devi S, Wang YL, Chew WK, et al. . Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow. Journal of Experimental Medicine 2013;210:2321-2336. 226. Zhong ZJ, Carroll KD, Policarpio D, et al. . Anti-Transforming Growth Factor beta Receptor II Antibody Has Therapeutic Efficacy against Primary Tumor Growth and Metastasis through Multieffects on Cancer, Stroma, and Immune Cells. Clinical Cancer Research 2010;16:1191-1205. 227. Baklaushev VP, Kilpelainen A, Petkov S, et al. . Luciferase Expression Allows Bioluminescence Imaging But Imposes Limitations on the Orthotopic Mouse (4T1) Model of Breast Cancer. Scientific Reports 2017;7:7715. 228. Devine SM, Flomenberg N, Vesole DH, et al. Rapid mobilization of CD34+cells following administration of the CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin's lymphoma. Journal of Clinical Oncology 2004;22:1095-1102. 229. 2018 Clinical Trials Using Obinutuzumab - National Cancer Institute. . 27/04/18

267 230. Updated: 5/18/2018 Raji ATCC Â® CCL-86 Homo sapiens lymphoblast Burkitt's lymph. . 24/07/18. 231. 2018 Daudi ATCC Â® CCL-213 Homo sapiens peripheral blood Burkitt. . 24/07/18. 232. Gregory C, Forbes S, Petrova S, (MRC) MRC. 2012. 'Cellular microenvironment in Burkitt’s lymphoma: gene expression profiling of tumour-associated macrophages in situ'. PhD, University of Edinburgh, Scotland, UK. 233. Lwin T, Crespo LA, Wu A, et al. . Lymphoma cell adhesion-induced expression of B cell- activating factor of the TNF family in bone marrow stromal cells protects non-Hodgkin's B lymphoma cells from apoptosis. Leukemia 2009;23:170-177. 234. Cols M, Barra CM, He B, et al. . Stromal Endothelial Cells Establish a Bidirectional Crosstalk with Chronic Lymphocytic Leukemia Cells through the TNF-Related Factors BAFF, APRIL, and CD40L. Journal of Immunology 2012;188:6071-6083. 235. de la Fuente MT, Casanova B, Garcia-Gila M, Silva A, Garcia-Pardo A. Fibronectin interaction with alpha 4 beta 1 integrin prevents apoptosis in B cell chronic lymphocytic leukemia: correlation with Bcl-2 and Bax. Leukemia 1999;13:266-274. 236. Huang YJ, Xu XH, Ji LL, et al. . Expression of far upstream element binding protein 1 in B- cell non-Hodgkin lymphoma is correlated with tumor growth and cell-adhesion mediated drug resistance. Molecular Medicine Reports 2016;14:3759-3768. 237. Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. Journal of Cell Science 2010;123:4195-4200. 238. Shiga K, Hara M, Nagasaki T, Sato T, Takahashi H, Takeyama H. Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth. Cancers 2015;7:2443- 2458. 239. Sappino AP, Skalli O, Jackson B, Schurch W, Gabbiani G. Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues. International Journal of Cancer 1988;41:707-712. 240. Kraman M, Bambrough PJ, Arnold JN, et al. . Suppression of Antitumor Immunity by Stromal Cells Expressing Fibroblast Activation Protein-alpha. Science 2010;330:827-830. 241. May RC, Machesky LM. Phagocytosis and the actin cytoskeleton. Journal of Cell Science 2001;114:1061-1077. 242. Ikenoya M, Hidaka H, Hosoya T, Suzuki M, Yamamoto N, Sasaki Y. Inhibition of Rho-kinase- induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor. Journal of Neurochemistry 2002;81:9-16. 243. Azab AK, Azab F, Blotta S, et al. . RhoA and Rac1 GTPases play major and differential roles in stromal cell-derived factor-1-induced cell adhesion and chemotaxis in multiple myeloma. Blood 2009;114:619-629. 244. Kumar A, Bhattacharyya J, Jaganathan BG. Adhesion to stromal cells mediates imatinib resistance in chronic myeloid leukemia through ERK and BMP signaling pathways. Scientific Reports 2017;7:14. 245. Motohashi H, Yamamoto M. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends in Molecular Medicine 2004;10:549-557. 246. Soriani A, Iannitto ML, Ricci B, et al. . Reactive Oxygen Species- and DNA Damage Response-Dependent NK Cell Activating Ligand Upregulation Occurs at Transcriptional Levels and Requires the Transcriptional Factor E2F1. Journal of Immunology 2014;193:950-960. 247. Kensler TW, Wakabayash N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annual Review of Pharmacology and Toxicology. Vol 47, Annual Review of Pharmacology and Toxicology. Palo Alto: Annual Reviews; 2007. p 89- 116.

268 248. Winiarska M, Nowis D, Bil J, et al. . Prenyltransferases Regulate CD20 Protein Levels and Influence Anti-CD20 Monoclonal Antibody-mediated Activation of Complement- dependent Cytotoxicity. Journal of Biological Chemistry 2012;287:31983-31993. 249. Angel TE, Aryal UK, Hengel SM, et al. . Mass spectrometry-based proteomics: existing capabilities and future directions. Chemical Society Reviews 2012;41:3912-3928. 250. Duan GY, Walther D. The Roles of Post-translational Modifications in the Context of Protein Interaction Networks. Plos Computational Biology 2015;11:23. 251. Research CfDEa. 2018 Approved Drugs - FDA approves obinutuzumab for previously untreated follicular lymphoma. Center for Drug Evaluation and Research . 27/04/18. 252. Research CfDEa. 2018 Approved Drugs - Obinutuzumab. Center for Drug Evaluation and Research . 27/04/18. 253. 2018 FDA Approval for Obinutuzumab - National Cancer Institute. . 27/04/18. 254. Zhou JB, Mauerer K, Farina L, Gribben JG. The role of the tumor microenvironment in hematological malignancies and implication for therapy. Frontiers in Bioscience 2005;10:1581-1596. 255. Ahmadzadeh V, Tofigh R, Farajnia S, Pouladi N. The Central Role for Microenvironment in B-Cell Malignancies: Recent Insights into Synergistic Effects of its Therapeutic Targeting and Anti-CD20 Antibodies. International Reviews of Immunology 2016;35:136-155.

269