The Effect of the Tumor Microenvironment on Oncolytic Virus Therapy for Glioblastoma

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Amy M. Haseley

Graduate Program in Neuroscience

The Ohio State University

2012

Dissertation Committee:

Dr. Balveen Kaur, Advisor

Dr. E. Antonio Chiocca

Dr. Deborah Parris

Dr. Mariano Viapiano

Amy M. Haseley

2012

Abstract

Glioblastoma multiforme is one of the most devastating diseases of the central nervous system, leaving patients with a median survival of 12-15 months following standard of care treatment. Oncolytic herpes simplex viruses (OV) are genetically modified to selectively infect and kill cancer cells by lytic destruction, and have been increasingly recognized as effective therapies against gliomas, reducing tumor burden and enhancing animal survival in pre-clinical studies. The efficacy of this therapy, however, is limited by the ever-changing tumor microenvironment which helps confer resistance to subsequent virus infection. In this thesis document we take a closer look at some of the changes which occur within the tumor microenvironment following OV therapy, and use the insight gained to create more sophisticated oncolytic viruses to combat glioblastoma.

To reduce the increase in angiogenesis reported following OV therapy, we first describe the construction and testing of a novel oncolytic HSV-1 derived virus: 34.5ENVE (viral

ICP34.5 is Expressed by Nestin promotor and Vstat120 Expressing). This virus showed significant glioma specific killing and anti-angiogenic effects in vitro and in vivo.

Treatment of mice bearing subcutaneous and intracranial glioma with 34.5ENVE resulted in a significant increase in animal survival, with 100% (subcutaneous) and 75%

(intracranial) of mice showing a complete response. Histology and dynamic contrast

ii enhanced magnetic resonance imaging (DCE MRI) revealed reduced microvessel density and increased tumoral necrosis in tumors treated with 34.5ENVE compared to tumors treated with a control virus. Collectively, these results describe the enhanced therapeutic efficacy of a transcriptionally driven OV by way of exploiting its impact on the tumor microenvironment.

Next, we describe the role of Cysteine rich 61 (CCN1) in the tumor microenvironment on

OV efficacy. CCN1 is a secreted extracellular matrix (ECM) protein elevated in cancer cells that modulates their adhesion and migration by binding cell surface receptors. We examined a hypothesized role for CCN1 in limiting the efficacy of oncolytic viral therapy for glioma, based on evidence of CCN1 induction that occurs in this setting. Expression is up-regulated in a variety of cancers, including glioma, resulting in a worse prognosis for these patients. As a significant induction of secreted CCN1 shortly following oncolytic viral therapy of glioma cells has been shown, we evaluated its role in the cellular response to viral infection. We found that exogenous CCN1 in glioma ECM orchestrates a cellular antiviral response that reduces viral replication and limits oncolytic virus efficacy. Gene expression profiling and real time PCR analysis revealed a significant induction of type-I interferon responsive genes in response to CCN1. Using function blocking antibodies we discovered this effect was mediated by CCN1 binding the α6β1 integrin receptor, resulting in the rapid secretion of IFNα which was essential for this innate antiviral effect. Collectively, these results describe the novel role of a CCN1-

iii integrin interaction in the activation of the type-I antiviral interferon response and ultimate inhibition of OV therapy.

Lastly, we investigate the relationship between CCN1 and macrophage-mediated oncolytic virus clearance in glioma. Using function-neutralizing antibodies, we show that inhibition of endogenously up-regulated CCN1 following OV therapy reduces OV mediated macrophage migration. Interestingly, CCN1‟s coordinated increase in macrophage migration was found due both to its direct interaction with macrophages, as evidenced by an enhancement in macrophage migration with purified CCN1 protein, as well as to its direct interaction with glioma cells, which results in an increased production of chemokines. CCN1 enhanced the pro-inflammatory activation of macrophages following OV infection, which led to an increase in macrophage-mediated viral clearance in vitro. Though knock-down of the integrin α6β1 on glioma cells did reduce the type I

IFN response presented above, it had no effect on CCN1‟s relationship with macrophages. Examination of the cell surface integrin αMβ2, known to mediate the

CCN1-macrophage interaction, revealed it to be the main effector for CCN1‟s effect. In vivo, use of an anti-CCN1 antibody in mice bearing subcutaneous gliomas treated with oncolytic rHSVQ-IE4/5-Luc revealed enhanced luciferase activity along with reduced macrophage infiltration. Taken together, our findings indicate CCN1 not only has a role in the immediate activation of the type I IFN response, thereby inhibiting virus infection, but also in increasing macrophage infiltration and activation resulting in a macrophage mediated reduction in viral oncolysis. Further, this study warrants investigation of

iv therapeutic strategies to reduce CCN1 following OV therapy, with the intention of creating a more suitable tumor microenvironment for virus therapy.

Collectively, the results presented in this doctoral thesis reveal very real limitations to sustainable OV therapy, brought on by the response of the tumor microenvironment to virus infection. We show these limitations may be reduced by harnessing the insight gained from a broader understanding of the tumor microenvironment to create more sophisticated OVs. Future studies will focus on the clinical relevance of 34.5ENVE for the treatment of patients suffering from glioblastoma multiforme. In addition we will continue to investigate the role for CCN1 and its inhibition in the improvement of OV therapy.

I dedicate this document to my Mom and Dad: thank you for your unconditional love and support and your constant encouragement in all facets of my life. And to my husband Chris: thank you for always encouraging me to “think outside the box”.

Acknowledgments

First and foremost I would like to acknowledge my PhD advisor, Dr. Balveen Kaur. Thank you for your unyielding support and utmost enthusiasm for my scientific endeavors. You have truly enabled my growth throughout graduate school on both an academic level, as well as a personal level. I would not be the scientist I am today without your encouragement and, at times, quite necessary criticism. Nothing was ever sugar-coated, but only straight-forward and honest. Thank you also for your patience and guidance; it has been an honor to be a member of your laboratory.

I would also like to thank my committee members, Dr. Nino Chiocca, Dr. Debbie Parris, and Dr. Mariano Viapiano. I have had the great privilege of learning from some of the finest scientists with this collection of mentors and have learned a great deal from their expertise. Thank you for your support and sincere interest in my research and my career.

I would like to thank the Neuroscience Graduate Studies Program, and in particular Dr. John Oberdick and Dr. Georgia Bishop. It has been a real pleasure to work with you throughout my time in graduate school; you have each been a great mentor to me from the beginning. And I would like to thank Keri Bantz whose effort has made NGSP the truly fantastic program it is today.

In the Dardinger Laboratory for Neurosciences, I would like to thank Dr. Nina Dmitrieva, Dr. M. Oskar Nowicki, Dr. Hiroshi Nakashima, Dr. Kazue Kasai, Dr. Kazuhiko Kurozumi, Dr. Jakub Godlewski, and Lisa Denning for their support and collaborations. I would also like to acknowledge the current and past members of the Kaur Lab: Dr. Ji Young Yoo, Jeffrey Wojton, Hans Meisen, Dr. Jayson Hardcastle, Dr. Christopher Alvarez-Breckenridge, Dr. Kazuo Okemoto, Dr. Jun-Ge Yu, Sean Boone, Hector Cordero-Nieves, and Chelsea Bolyard. In your own ways, each and every one of you has helped to shape me into the scientist that I am today.

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Vita

2002...... North Olmsted High School 2007...... B.S. Biology, The Ohio State University 2007...... B.S. Psychology, The Ohio State University 2007 to present ...... Graduate Research Associate, Department of Neurological Surgery, The Ohio State University

Publications

Haseley A, Alvarez-Breckenridge C, Chaudary AR, Kaur B. Advances in oncolytic viral therapy for glioma. Recent Patents on CNS Drug Discovery, 2009, 4, 1-13.

Yoo J+, Haseley A+, Bratasz A, Chiocca E, Zhang J, Cain D, Powell K, Kaur B (2011). Anti-tumor efficacy of 34.5ENVE: a transcriptionally retargeted and “anti-angiogenic armed” oncolytic virus. Molecular Therapy, 2012, 20(2):287-97 + these authors contributed equally

Haseley A, Boone S, Wojton J, Yu L, Yoo JY, Yu J, Kurozumi K, Glorioso JC, Caligiuri MA, Kaur B. Extracellular matrix protein CCN1 limits oncolytic efficacy in glioma. Cancer Research, 2012, 72(6):1353-62.

Yoo JY, Pradarelli J, Haseley A, Wojton J, Kaka A, Bratasz A, Alvarez-Breckenridge A, Yu J-G, Powell K, Mazar A, Teknos TN, Chiocca EA, Glorioso JC, Old M, Kaur B. Copper chelation enhances anti-tumor efficacy and systemic delivery of oncolytic HSV. Clinical Cancer Research, 2012, Epub ahead of print

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Fields of Study

Major Field: Neuroscience Graduate Studies Program

Table of Contents

Abstract ...... ii

Acknowledgments...... vii

Vita ...... viii

Publications ...... viii

Table of Contents ...... x

List of Tables ...... xiv

List of Figures ...... xv

Chapter 1: Introduction ...... 1

Section 1: Glioblastoma Overview ...... 1

Section 2: Current Approaches to GBM ...... 2

Section 3: Oncolytic Virus Therapy ...... 6

Section 4: Herpes-Simplex Derived Oncolytic Viruses ...... 7

Section 5: Future developments in oncolytic viral therapy ...... 13

Section 5.1: Anti-angiogenic developments in oncolytic virus therapy ...... 14

Section 5.2: Host immunity to oncolytic virus infection ...... 18

Section 5.3: Enhancing viral spread throughout a solid tumor ...... 21 x

Section 6: Cysteine-rich 61 (CCN1) ...... 25

Section 7: Hypothesis and Overview ...... 27

Section 8: Figures and Tables ...... 29

Chapter 2: Anti-tumor efficacy of 34.5ENVE: a transcriptionally retargeted and

“Vstat120” expressing oncolytic virus...... 32

Introduction ...... 32

Results ...... 34

Discussion ...... 40

Materials and methods ...... 42

Figures and Tables ...... 49

Chapter 3: Cloning and characterization of Cy-1 tetracycline-inducible cells ...... 62

Introduction ...... 62

Results ...... 62

Cy-1 cells express CCN1 following dox treatment ...... 62

CCN1 inhibits glioma cell proliferation ...... 63

CCN1 enhances glioma cell migration and invasion ...... 64

Discussion ...... 65

Materials and Methods ...... 67

Figures and Tables ...... 70

Chapter 4: Extracellular matrix protein CCN1 limits oncolytic efficacy in glioma ...... 77

Introduction ...... 77

Results ...... 79

CCN1 gene expression is upregulated by virus but not by chemotherapy or radiation

treatment ...... 79

Extracellular CCN1 expression inhibits viral transgene expression, replication, and

oncolysis ...... 79

CCN1 mediated OV inhibition is dependent on α6β1 integrin receptor-mediated IFNα

secretion ...... 83

Discussion ...... 86

Materials and Methods ...... 88

Figures and Tables ...... 92

Chapter 5: Inhibition of CCN1 enhances oncolytic virus therapy by reducing macrophage mediated viral clearance ...... 109

Introduction ...... 109

Results ...... 111

CCN1 increases macrophage infiltration toward oncolytic HSV-1 infected tumors in vivo

...... 111

CCN1 increases macrophage migration toward OV infected glioma cells ...... 111

CCN1 induces MCP-1 and MCP-3 gene expression by infected glioma cells...... 112 xii

CCN1 directly effects macrophage migration by binding integrin αMβ2 ...... 113

The above results show that CCN1 can induce glioma cells to increase secretion of MCP-

1 and MCP-3 to increase macrophage chemotaxis towards infected cells. CCN1 is a secreted ECM molecule that has been shown to increase adhesion of murine macrophages and monocytes [119, 203]. To determine if CCN1 directly increased migration of monocytes and macrophages in the absence of infected glioma cells, we measured the migration of RAW264.7 murine macrophages and human monocytic THP-1 cells through transwells coated with either purified CCN1 protein or BSA. Quantification of migrated cells revealed a significant increase in migration of both macrophage and monocytic cells

(Figures 34a, b)...... 113

Enhanced activation of macrophages by CCN1 leads to increased viral clearance ...... 114

Discussion ...... 116

Materials and Methods ...... 119

Figures and Tables ...... 125

Conclusions and Future Directions ...... 134

References ...... 138

xiii

List of Tables

Table 1: List of completed and on-going oncolytic virus trials for patients with GBM ... 31

Table 2: CCN1 fold induction...... 106

Table 3: CCN1 induces expression of type-I interferon response gene in the presence and absence of OV ...... 107

Table 4: Primer Sequences ...... 108

Table 5: Primer Sequences ...... 133

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List of Figures

Figure 1: Classification of Astrocytomas ...... 29

Figure 2: A modular representation of the human CCN1 protein ...... 30

Figure 3: Structure and Characterization of 34.5ENVE ...... 49

Figure 4: Increased virus replication and cytopathic effect of 34.5ENVE in high nestin expressing cells...... 51

Figure 5: Real Time-PCR analysis ...... 52

Figure 6: Anti-angiogenic effect of 34.5ENVE in vitro ...... 53

Figure 7: Reduced angiogenesis in tumors treated with 34.5ENVE ...... 55

Figure 8: Anti-tumor effects of 34.5ENVE in vivo ...... 56

Figure 9: OV treatment induced tumor regression ...... 57

Figure 10: Contrast enhancement of PBS and OV treated tumors ...... 58

trans Figure 11: K and Ve for rQnestin34.5 and 34.5ENVE treated tumors...... 59

Figure 12: Effect of 34.5ENVE on tumor necrosis ...... 60

Figure 13: Schematic showing tetracycline-inducible tet-on system ...... 70

Figure 14: Induction of CCN1 in vitro and in vivo ...... 71

Figure 15: CCN1 inhibits proliferation in vitro and in vivo...... 72

Figure 16: CCN1 blocks glioma cell mitosis at G1-S phase ...... 74

Figure 17: CCN1 increases glioma cell migration and invasion ...... 76

Figure 18: CCN1 gene expression is upregulated by virus but not by chemotherapy or radiation therapy...... 92

Figure 19: Extracellular CCN1 expression inhibits viral transgene expression, replication, and cell killing...... 93

Figure 20: CCN1 inhibition of viral transgene expression & CCN1 expression ...... 94

Figure 21: Doxycycline dose response & effect on LN229 glioma cells...... 95

Figure 22: Quantification of virus expressed luciferase ...... 96

Figure 23: CCN1 in the ECM limits OV replication and cytotoxicity ...... 97

Figure 24: Survival data of athymic nude mice implanted subcutaneously with U251T3

1.5x107 glioma cells following ENVE virus therapy...... 98

Figure 25: Transcript profiling of Cy-1 cells induced to express CCN1: ...... 99

Figure 26: Functional activation of a type-I IFN response by CCN1 mediates OV inhibition...... 100

Figure 27: CCN1 mediated OV inhibition is dependent on its interaction with cell surface

α6β1 integrin independently of its ability to bind to αvβ3 and αvβ5...... 101

Figure 28: ITGA6 expression, IFNα expression, & CCN1 effect on U87∆EGFR glioma cells ...... 103

Figure 29: Confocal fluorescent and bright field images of GFP positive infected LN229 glioma cells and JiEGFR HSV-resistant cells infected with rHsvQ1 MOI=5...... 104

Figure 30: Model for CCN1 mediated OV inhibition...... 105

Figure 31: CCN1 inhibition decreases macrophage infiltration toward oncolytic HSV-1 infected tumors, increasing OV activity, in vivo...... 125

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Figure 32: CCN1 increases RAW cell migration towards infected glioma cells ...... 127

Figure 33: CCN1 induces chemokine expression in glioma cells ...... 128

Figure 34: CCN1 protein increases migration of macrophages and monocytes by directly binding integrin αMβ2 ...... 130

Figure 35: CCN1 increases macrophage pro-inflammatory gene expression ...... 131

Figure 36: CCN1 increases macrophage mediated viral clearance by binding integrin

αMβ2 ...... 132

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Chapter 1: Introduction

Section 1: Glioblastoma Overview

Astrocytomas are broadly classified as either diffusely infiltrating or localized

(circumscribed). Localized astrocytomas are of lower grades, as classified by the World

Health Organization (WHO), and have a potential of cure following surgical resection.

These include pilocytic astrocytoma (WHO grade I), subependymal giant cell astrocytoma (WHO grade I) and pleomorphic xanthoastrocytoma (WHO grade I). The higher grade infiltrating astrocytomas (grades II, III and IV) are more biologically aggressive and resistant to therapy. Diffusely infiltrating astrocytomas are intrinsically invasive tumors with unfavorable prognosis and include diffuse astrocytoma (WHO grade II), characterized by the presence of cytologic atypia, anaplastic astrocytoma

(WHO grade III), which have cytologic atypia and the presence of mitotic activity, and glioblastoma multiforme (GBM) (WHO grade IV), which show presence of tumor cell necrosis and/or micro vascular proliferation (angiogenesis), in addition to cytologic atypia and mitotic activity (Figure 1). Apart from tumor grade, other prognostic factors affecting patient survival include age at diagnosis, tumor location, and extent of tumor resection. However, regardless of these variables, overall prognosis of astrocytomas remains dismal. While patients diagnosed with grade II tumors usually survive more than

5 years, those with grade III tumors do not survive beyond 2-3 years. And the outlook for

1 patients suffering from GBM is the worst with patients having a median survival of les than 15 months following an aggressive course of radiation and chemotherapy [1]. GBM can develop as a primary tumor (de novo) or by progression from a lower grade astrocytoma (secondary GBM). It is now known that these two broad subtypes of GBM are genetically unique and distinct disease processes make them different in their genesis.

While the primary GBM is remarkable for both loss of heterozygosity (LOH) on chromosome arm 10q (70%) and EGFR amplification (36%), the secondary GBM most frequently demonstrates mutation of the TP53 tumor suppressor gene, which is already present in 60% of the precursor lower grade astrocytomas [2]. Given the poor prognosis of this disease, there is a desperate need for novel methods of intervention.

Section 2: Current Approaches to GBM

In addition to being the most dismal of the astrocytomas, glioblastoma is also the most common intrinsic brain tumor; it is estimated there will be 9,000 new cases diagnosed in the United States each year (CBTRUS). Prior to the 1940s surgical resection of brain tumors was the only form of treatment. Later, recognition of external beam radiotherapy enabled its use as a beneficial adjuvant therapy [3]. In 1952, the perfusion of nitrogen mustard derivatives into the brains of patients led the way in chemotherapeutics, but it wasn‟t until the 1970s that treatment of malignant brain tumors became evidence-based, with large studies devoted to comparing radiotherapy and surgical resection, in addition to nitrosourea compounds [4]. Some of the best studied agents have tended to be drugs which are nonionized and highly lipid soluble in order to penetrate the CNS most

2 efficiently. These agents include alkylating agents including the aforementioned nitrogen mustards and nitrosoureas in addition to procarbozine and temozolomide. Temozolomide became of interest after the discovery that it‟s conversion to the active metabolite, MTIC, is not dependent on hepatic metabolism. Temozolomide instead is able to cross into the

CNS and then spontaneously hydrolyze to its active metabolite at physiologic pH [5].

MTIC then converts spontaneously to the reactive methyldiazonium cation which then goes on to methylate the N7 and O6 position of guanine and the N3 position of adenine, mediating the cytotoxic effects of temozolomide [6]. In cell lines where there is an active

O6-methylguanine-DNA-methyltransferase (MGMT), which repairs methylation at the O6 position of guanine, there is resistance to temozolomide, which can be reduced by depletion of MGMT [7]. The current standard of care protocol for the treatment of glioblastoma consists of 6 weeks of 60-gy fractionated radiation therapy, given concurrently with 42 days of daily temozolomide, given at 75 mg/m2. This regimen is followed by 6 to 12 cycles of 150-200mg/m2 temozolomide given every 5 days for a 38 day cycle [1]. This current standard of care affords patients an approximate 3 month increase in median survival which, though significant, is a far cry from cure with a 2 year survival rate of 26.5%.

Most malignant gliomas recur within 2 cm of the original tumor focus, and it has been postulated that local delivery of therapeutic agents may offer a promising solution. Two such techniques are the use of Gliadel Wafers and Convection-enhanced delivery. The use of Gliadel wafers placed along the surface of the resection cavity that release

3 carmustine (BCNU) over a 3 week period was found to only modestly improve survival

[8]. Convection enhanced delivery (CED) has been used for the infusion of drugs into the brain via bulk fluid flow. A phase III clinical trial comparing CED of IL13-PE38QQR (a chimeric cytotoxin composed of IL13 fused to pseudomonas aeruginosa exotoxin A) to

Gliadel wafers was recently completed. Final analysis between these two modalities indicated no significant difference in survival benefit, however this was thought to be due to several factors including poor drug specificity, suboptimal catheter placement, and absence of drug specific imaging [9]. The use of nanoparticles which encapsulate large amounts of drug have also been developed to address the challenges met by lack of sustainable release and penetration within the brain. Indeed, preclinical studies using both liposomal and polymeric nanoparticles have shown feasibility and efficacy of this strategy [10, 11].

Another approach to combat glioblastoma is by targeted molecular therapy. However, efficacy has been met with challenges given the dense, interconnection of robust signaling pathways within this disease. Nevertheless, advances in the understanding of glioblastoma biology have come from failed trials with small molecule inhibitors and antibodies against many of the “key players” in the oncogenesis of this disease, and new treatment regimens have emerged with promising outlooks. For example, though monotherapy trials using mTOR inhibitors have demonstrated modest results due to the counterproductive activation of PI3K/AKT [12], production of second-generation

4 inhibitors which are able to inhibit both mTOR and PI3K have shown promise pre- clinically and clinical trials are underway [13].

Glioblastomas are highly vascularized tumors and are characterized by activation of multiple proangiogenic signaling pathways. The “angiogenic switch” occurs when growing tumors reach a size, typically 1-2mm in diameter, when the existing blood supply can no longer support the tumor and angiogenesis must occur in response to hypoxia. [14-16]. In GBM, the formation of new, aberrant and tortuous blood vessels is a key event in tumor formation and led to the advent of antiangiogenic therapies for the treatment of this disease. Initially, antiangiogenic drugs were thought to prevent new blood vessel formation, essentially choking the tumor [17]. However, the process of vascular normalization has been recently postulated by Jain, which describes the result of antiangiogenic agents as a revertion of abnormal tumor vasculature to a more normalized state, enhancing drug delivery and radiotherapy efficacy [18]. The most commonly used antiangiogenic agent for glioblastoma is Bevacizumab, a recombinant, humanized monoclonal antibody that inhibits VEGR-mediated cell signaling by sequestration of its ligand VEGF-A. The biggest set-backs for the use of antiangiogenic agents are the rare but serious complications, most notably hypertension [19], and the reduction in contrast enhancement on postcontrast T1-weighted MRI images [20]. Ultimately, the results from trials with antiangiogenic agents have been only mildly encouraging.

Collectively, standard approaches to combating glioblastoma have shown a modest improvement in survival. Currently, there is no cure, and almost all patients progress eventually. The use of novel therapies therefore needs to be investigated for their use alone and in conjunction with current FDA approved treatment modalities. Oncolytic viruses are one such novel approach to combating glioblastoma and will be discussed in the next section.

Section 3: Oncolytic Virus Therapy

Oncolytic virus (OV) therapy is based on the concept of using replication competent viruses to selectively infect and replicate in cancer cells, with minimal destruction of non- neoplastic tissue. While the concept of using live viruses to infect and destroy tumors dates back to nearly over a century, advances in molecular biology and virology have accelerated the development of OVs in the last two decades [21]. Conditionally replication competent viruses are genetically engineered or selected to be avirulent in normal cells but can exploit the aberrant molecular/genetic pathways in tumors resulting in their efficient replication within cancer cells and the lytic destruction of the infected malignant cell. Oncolytic virus therapy is a novel treatment option for GBM patients and several have been used in preclinical and clinical trials to evaluate safety and efficacy.

These viruses are natural or genetically engineered from a variety of viruses including

Herpes Simplex Virus-1 (HSV-1), Adenovirus (Ad), New Castle Disease Virus (NDV), and Reovirus (RV). In all of these trials intratumoral administration of infectious oncolytic viral particles was found to be safe but significant evidence of efficacy remains

6 to be established (Table 1). Several innovative strategies to enhance intratumoral viral spread and antitumor efficacy without compromising its safety are currently under investigation. The very innovative approaches being used to improve therapeutic efficacy include the following: design of viruses which can express cytokines to activate a systemic antitumor immune response, inclusion of angiostatic genes to combat the tumor vasculature, and also the inclusion of enzymes capable of digesting tumor extracellular matrix (ECM) in order to enhance viral spread through solid tumors. These studies have resulted in the development of several viruses which may contribute towards the advancement of future therapeutics. In this thesis we will focus on the advances that have been made and the barriers which exist within the tumor microenvironment with the use of oncolytic herpes simplex viruses.

Section 4: Herpes-Simplex Derived Oncolytic Viruses

Herpes Simplex Virus-1 (HSV-1) is an enveloped, double-stranded DNA virus containing a large, well characterized, fully sequenced genome of about 152kb of DNA that encodes more than 80 genes. While the large size makes genetic manipulations cumbersome, it also provides ample opportunities to remove genes that are not essential for replication (estimated to be about 30kb) [22]. Removal of these genes allows for the insertion of therapeutic transgenes within the viral backbone. Furthermore, the ability of

HSV-1 to remain as an episome avoids the possibility of any insertional mutagenesis of the infected cell, and the easy availability of antiherpetic drugs in order to keep viral replication in check makes it a very desirable vector for therapeutic applications. Taken 7 together, these qualities of HSV-1 have led to the development of several oncolytic viruses for the treatment of CNS tumors.

HSV-1 viruses mutated in the viral genes ICP6 and/or RL-1 have been tested in human patients (Table 1). ICP6 encodes for the viral counterpart of mammalian ribonucleotide reductase (RR) which is required for the de novo synthesis of deoxyribonucleotides, essential for viral DNA synthesis and replication. A deficiency in ICP6 would make the virus replication competent only in mitotic cells which express the mammalian counterpart of the enzyme. This enzyme is normally not expressed in non-replicating cells, and hence viruses deficient in ICP6 would be replication incompetent in normal non cycling cells. To test this hypothesis, a mutant virus, deficient for the large subunit of

RR (ICP6/RR), was created by the insertion of an Eschericia coli LacZ gene within the

UL39 gene locus [23]. Cytopathic and drug sensitivity assays using this attenuated virus

(hrR3) revealed not only efficient cancer cell killing in vitro and but also similarly effective tumor killing in animal models of tumorigenesis [24, 25]. More recent evidence also indicates that the RR mutant HSV-1 may be “molecularly targeted” to replicate more efficiently in cells with specific oncogenic mutations such as homozygous deletion of the p16 gene [26]. The HSV-1 ICP34.5 (RL-1) gene was also identified as neurovirulent as attenuation of both copies of this gene led to a reduction in HSV-1 replication in vitro and in vivo [27, 28]. Upon viral infection, PKR becomes rapidly activated, phosphorylating the α-subunit of eIF-2α, leading to a total shutoff of host protein synthesis. This response is overcome, however, by the HSV-1 gene RL-1/ 34.5, which encodes for the ICP34.5 8 protein. ICP34.5 leads to the dephosphorylation of the α-subunit of eIF-2α and disinhibition of protein synthesis. Deletion of the viral RL-1 gene therefore makes HSV-1 unable to replicate in normal cells. However, proliferating cancer cells often have an activated Ras/Mitogen activated protein kinase kinase (MEK) pathway rendering the cells deficient in the antiviral PKR response [29]. Hence an HSV-1 deficient in the viral 34.5 gene can selectively replicate in Ras/MEK activated proliferating cancer cells. More significantly this mutant virus retained the ability to replicate in actively dividing mouse embryo cells but not in confluent mouse embryo cultures [28].

Consistent with these findings, an RL-1 null HSV-1 mutant (1716), which fails to produce

ICP34.5 is able to efficiently replicate in and cause subsequent cell death of a majority of glioma cell lines and primary tumor derived cells [30]. Oncolytic HSV-1 1716, similarly attenuated, was found to have significant antitumor efficacy in vivo when administered to mice bearing experimental brain tumors [31, 32]. Pathological examination of mice injected intracranially with HSV1716, revealed a finite, self-limiting host response to

HSV1716, highlighting the potential of using it for therapeutic purposes.

Based on its safety profile in animals, the mutated HSV1716 was tested in the UK for use in human patients with malignant brain tumors [33]. This clinical study represented the first foray of inoculating mutated oncolytic HSV vectors into the brains of human patients with malignant brain tumors, and for concerns of safety, only a very moderate

9 dosage of the virus was permitted to be tested. In this study, nine patients with malignant glioma were treated with escalating doses of HSV1716 to a maximum of 105 p.f.u. by direct intra-tumoral injection. Patients were closely monitored for any signs of treatment induced toxicity. No adverse clinical events which could be attributed to the administration of HSV1716 were observed. No evidence of encephalitis, viral shedding, or reactivation of endogenous latent virus was seen in any patient [33]. Five of these nine patients underwent subsequent tumor progression and biopsy samples from these patients showed no signs of HSV1716 or wild type HSV by PCR analysis. In a subsequent report by the investigators, it was noted that two of these nine patients were still living and stable at four years and four months and three years and seven months, respectively, following treatment [34]. Two more clinical trials were initiated to test the therapeutic efficacy of treating patients with malignant glioma by HSV1716. In one of the clinical trials, evidence of viral replication in human high grade glioma was examined in twelve patients with high grade malignant glioma after receiving 105 p.f.u. of HSV1716 by direct intratumoral injection 4 to 9 days prior to tumor resection. The tissue was examined for presence of HSV DNA by PCR, southern blotting, and immuno-histochemistry and for the presence of infectious viral particles by plaque assay. The results confirmed the presence of HSV1716 in tumor tissue 4 to 9 days after treatment without any obvious signs of toxicity [35].

In the third study to test the therapeutic efficacy of the HSV1716 in human glioma patients, a total of twelve patients diagnosed with high grade recurrent (6 patients) or 10 newly diagnosed (6 patients) glioma underwent gross tumor resection [36]. After the initial tumor debulking, 105 p.f.u. of HSV mutant 1716 was injected into 8-10 sites within the tissue surrounding the tumor cavity. Despite the lack of toxicity seen in the previous trials, due to paramount safety concerns, the therapeutic dose given to the patients was not increased as would have been done for other clinical trials with a chemotherapeutic agent. However, the lack of any virus-related toxicity in this study considerably strengthened the safety profile of this approach as the virus had been injected into tissue involving the functioning brain. The expected median survival for patients with GBM is one year from the time of initial diagnosis with resection and radiation and only 3-6 months following diagnosis of a recurrent tumor. In this study, one patient with a recurrent tumor and two patients diagnosed with primary GBM survived for more than

22, 18, and 15 months, respectively, following virus therapy. Indeed the patient diagnosed with a recurrent tumor was disease free at 22 months. This study demonstrated that HSV1716 can be injected into the normal brain of individuals without any ensuing toxicity. A causal relationship, however, between response and viral treatment could not be established.

HSV1716 is currently being pursued by Virttu Biologics, Glasgow, UK. The company has been granted orphan drug status in Europe for “the use of HSV mutant 1716 in the treatment of glioma” and has received regulatory approvals to initiate a Phase III trial for glioma patients [32]. For this trial, glioma patients with a first recurrence after surgery will be randomized into one of two treatment arms: HSV1716 or conventional 11 chemotherapy. Satisfactory results in this trial could lead to a license and marketing authorization in glioma research.

In order to minimize the chances of an oncolytic HSV-1 reverting back to a wild type virus G207, a doubly attenuated oncolytic HSV-1 that has deletions at both γ34.5 (RL1) loci as well as an in-frame, gene disrupting insertion of the E. coli β-galactosidase gene within the ICP6 gene, was created. Intraneoplastic administration of the virus resulted in slower tumor growth and/or prolonged survival [37, 38]. Furthermore, G207 maintained the attenuated neurovirulence, temperature sensitivity, and ganciclovir hypersensitivity

[37], and the insertion of β-galactosidase permitted easy detection of cells harboring the virus in infected tissue. Preclinical testing of this virus was performed in nude mice harboring subcutaneous or intracranial gliomas. Preclinical toxicity studies of G207 were performed by direct intracranial inoculation of mice and HSV-sensitive non-human primates revealing it to be safe [39]. Even after two years of inoculation of G207 in new world owl monkeys (Aotus nancymae), no infectious viral particles were recovered and viral DNA was found to be restricted to the brain. Absence of the viral DNA in tears, saliva, and vaginal secretions indicated a lack of viral shedding suggesting that strict biohazard management of G207 patients may not be required.

The safety in intracerebral inoculation of G207 in humans was determined by a dose escalation study with patients suffering from recurrent malignant glioma [40]. The

12 protocol was approved by the Recombinant DNA Advisory Committee of the National

Institute of Health and Food and Drug Administration. The trial was initiated with the administration of 106 p.f.u. of G207, into the brains of three patients post tumor resection.

After 28 days of observation to confirm lack of acute toxicity three patients were then recruited into the next six cohorts and were treated with the next higher dose of G207

(doses of each cohort were 107, 3 x 107, 108, 3 x 108, 109, and 3 x 109 p.f.u. respectively).

The three patients in the last cohort were treated with G207 inoculated into five sites after surgical resection of the brain tumor, while all other patients received the virus at a single locus post resection [40]. No toxicity which could be directly attributed to the infectious virus was noted in this study, suggesting that G207 can be safely inoculated into human brain tumors up to doses of 109 p.f.u. without any adverse events. This agent is currently being investigated for efficacy in clinical trials.

Section 5: Future developments in oncolytic viral therapy

All of the clinical trials that have tested the efficacy of the various oncolytic herpes simplex viruses in human patients with malignant glioma have revealed the safety of this approach, however significant evidence of efficiency has not been established. New methods of enhancing oncolytic viral therapy are therefore being developed. These include transcriptionally targeting viruses, arming viruses with tumor microenvironment modulating genes, and modulating host responses to viruses. Currently there are several studies that describe various ways to enhance the therapeutic efficacy of OV, many of which are being tested in preclinical animal models to evaluate effectiveness and toxicity

13 prior to being tested in clinical trials. In the following sections we will discuss some of these studies, which have arisen from research done to modulate the tumor angiotome, the antiviral host responses, and the tumor ECM proteins.

Section 5.1: Anti-angiogenic developments in oncolytic virus therapy

The impact of OV treatment on tumoral vasculature has been the focus of several studies as its ability to infect and replicate in proliferating endothelial cells, leading to their lytic destruction has been shown [41]. Consistent with these reports, treatment of human ovarian carcinoma and malignant peripheral nerve sheath tumors grown as xenografts in athymic nude mice with oncolytic HSV mutants (G207, hrR3, and 1716) resulted in infection of tumor vasculature, and an antiangiogenic effect [42, 43]. While these studies demonstrated the immediate direct antiangiogenic effect of OV, two recent studies have revealed increased angiogenesis of the residual tumor that grows after oncolysis. Aghi et al found a significant decrease in the production of antiangiogenic proteins TSP-1 and

TSP-2 in glioma xenografts treated with G207 which resulted in an increased microvessel density in tumors [44]. Further treatment of glioma bearing mice with a combination therapy regimen of G207 and 3TSR (a peptide containing the TSP-1 region) significantly reduced tumor microvessel density and enhanced antitumor efficacy compared to G207 alone. Additionally, Kurozumi et al have reported induction of the integrin activating and angiogenic protein CCN1 in gliomas treated with oncolytic HSV mutant viruses [45].

The authors tested the impact of Cilengitide (cRGD), an antagonist of CCN1-mediated integrin activation, on OV treatment. Angioreactors filled with glioma cells and then

14 treated with the OV in the presence of Cilengitide had significantly reduced angiogenesis compared to angioreactors filled with OV alone. Together, these studies indicate that while treatment with OV has an immediate antiangiogenic effect, OV induced changes in the tumor angiotome permit re-growth of tumor vasculature in the residual tumor after viral clearance. These studies underscore the potential therapeutic advantage of combining OV treatment with antiangiogenic agents in vivo. The apparent effect of oncolytic viral therapy on angiogenesis as well as the innate role angiogenesis plays in the tumor microenvironment has lent to the interest in combating both tumor cells and endothelial cells. The concept of combining at least one oncolytic virus and at least one antiangiogenic agent for use in tumor therapy has been described [45].

Combination treatment of an oncolytic adenovirus with an antiangiogenic agent has been observed to be more effective than treatment with either agent alone [46]. The oncolytic virus (vKH6) in this case was a transcriptionally driven conditionally replication competent adenovirus (CRAD), wherein the early promoters of adenovirus are driven by

APC or beta catenin genes to selectively replicate in cells with an activated Wnt signaling pathway. Additionally, the fiber of the virus was modified to include an integrin targeting sequence to increase the infection promiscuity. Combination of vKH6 with RAD001 (an angiogenic inhibitor) resulted in enhanced antitumor efficacy possibly by substantially delaying the re-growth of the tumor while still allowing the virus to spread within it. In a more recent study, pretreatment of tumors with a single dose of an antiangiogenic agent

15 prior to treatment with an oncolytic HSV-1 derived virus also resulted in an enhanced antitumor effect of the OV [47].

While the concept of combining various antiangiogenic agents with OV remains very exciting, careful preclinical studies are needed to identify dosing schedules and mechanisms to achieve maximal synergy. The significance of drug interactions with OV and a careful study of dosing schedule has been made more significant by recent observations wherein the combination therapy including OV and Bevacizumab did not significantly increase survival compared to animals treated with OV alone [48].

Nevertheless the concept of choking a tumor by attacking its vasculature in conjunction with oncolytic cancer cell killing has been exploited in the design of several novel oncolytic viruses armed with genes encoding for angiostatic factors. This has led to the development of several “dually armed viruses” which can kill cancer cells by oncolysis as well as deliver a transgene with potent antiangiogenic effects. For example, the regulatory cytokine IL-12 has been inserted into an oncolytic HSV virus, NV1023, to create an OV expressing IL-12 (NV1042) [49]. Matrigel plugs containing squamous cell carcinoma (SCC VII) cells were treated with NV1023, NV1042, or PBS and assayed for hemoglobin (Hb) content. Plugs with NV1042 treated cells had a significant reduction in

Hb, and treatment of mice bearing SCC VII flank tumors with NV1042 resulted in a smaller tumor as compared with NV1023 or saline treated mice. This study showed that an OV engineered to express the cytokine IL-12 exerts antiangiogenic effects and the resulting oncolytic virus incorporating a nucleic acid sequence encoding IL-12 into an

HSV vector has been described [50, 51]. Similarly, Ye et al patented the delivery of the

E1A gene by a recombinant oncolytic adenovirus (rAD-E1A) [52]. Treatment of experimental subcutaneous tumors with rAD-E1A oncolytic virus resulted in a significant reduction of tumor growth compared to control. It was determined that the inhibition of tumor growth could be partly attributed to the reduction in vascular density by rAD-E1A.

Another oncolytic virus exploiting the use of antiangiogenic gene therapy in conjunction with OV mediated tumor destruction integrates the expression of Platelet Factor 4 (PF4) within the backbone of the oncolytic HSV G47Δ [53]. The resulting OV, bG47Δ-PF4, was found to significantly delay tumor growth compared to control. Further investigation of the antiangiogenic effects of PF4 revealed a reduced number of vasculature structures in harvested tumor tissue compared to control [54].

The incorporation of angiostatic factors such as angiostatin and endostatin into oncolytic viruses has also been tested leading to several patents [55]. Inclusion of a fusion endostatin-angiostatin protein with the G207 backbone of oncolytic HSV was found to have potent antitumor and antiangiogenic effects [56]. Along the same lines, Mullen et al constructed an oncolytic HSV encoding for the murine endostatin gene and found that secreted endostatin, produced by tumor cells infected with this recombinant virus, retained its biological activity and was able to augment the antitumor efficacy of OV

[57]. Yoo et al sought to exploit the angiostatic effect of VEGF signaling interference by constructing an adenovirus expressing a short hairpin RNA expression system against

VEGF [57]. The conditionally replication competent Ad-ΔB7-shVEGF and the replication incompetent Ad-ΔE1-shVEGF were constructed and compared for their antiangiogenic efficacy. The viruses were found to successfully inhibit VEGF expression thereby enhancing the efficacy of oncolytic adenoviral therapy through an antiangiogenic mechanism. The idea to use small interfering RNAs specific for VEGF was described in

2006 [58]. Preclinical testing of combination oncolytic viral therapy with angiostatic gene delivery to combat both tumor cells and endothelial cells by way of an antiangiogenic mechanism has shown much promise in the field of cancer research.

Section 5.2: Host immunity to oncolytic virus infection

As the prospects of moving OV therapy into the clinic become increasingly tangible, additional attention has been directed towards the role of host immunity in the context of

OV administration. The increasing understanding of the very complex interplay between antiviral immunity and antitumor immunity, has led to the development of several viruses related to OV therapy and host immune responses which will be discussed next.

Host immunity can be simplified into two distinct components: the innate immune response and adaptive immunity. Mediators of the innate immune system function as the first responders to viral infection, clearing infected cells, and producing cytokines and chemokines leading to subsequent activation of the immune system. Consequently, innate immunity is critically important in limiting wild-type viral infections; however, in the context of OV therapy, this branch of the immune system appears to be a potent obstacle

18 for achieving OV replication and tumor destruction [59-62]. Following activation of innate immunity, the adaptive component of the immune system is recruited to the site of infection and participates in both the killing of virally infected cells and the production of antibodies against foreign antigens. As a result, stimulation of adaptive immunity has a positive impact on therapy through its promotion of a cytotoxic T cell response, which creates an antitumor vaccination effect [50, 54].

Although the innate immune system has a variety of functional components to protect the host from infection, its critical objectives are to limit viral propagation, signal for maturation of antigen presenting cells, and activate the adaptive immune response through antigen-specific T cell and B cell maturation [63]. Owing to its significant contribution towards limiting viral infection, it has been described as one of the critical limitations to effective OV therapy. Treatment of rat glioma with an oncolytic HSV has been shown to result in a significant increase in IFNγ accompanied by a rapid recruitment of macrophages, microglia, and natural killer cells to the site of administration [64-66].

Both the antitumor efficacies as well as the intratumoral viral titers were found to be significantly increased with the concurrent depletion of mononuclear cells and the elimination of antiviral cytokines. More interestingly, Kurozumi et al found that pretreatment of rats bearing orthotopic gliomas with an antiangiogenic agent prior to OV therapy limited the infiltration of antiviral host immune cells into the tumor. This resulted in reduced antiviral immune responses and increased viral propagation and efficacy [47].

With the elucidations of such pathways, the quest of combining novel pharmacological

19 approaches with OV therapy to enhance viral infection, replication, and propagation is being pursued [67].

While antiviral immune responses are considered detrimental to therapy, activation of an adaptive antitumor immune response upon viral infection has been shown to be beneficial for cancer therapy. Cytotoxic T cell lymphocytes (CTL) have been implicated as the critical responders to viral antigens presented on the surface of tumor cells. CTLs are subsequently redirected to tumor cell antigens, thereby enhancing the efficacy of oncolytic HSV-1 by inducing antitumor immunity [68, 69].

The inclusion of genes encoding for various cytokines into viral vectors to enhance antitumor immune responses has been also tested and found to be beneficial in several preclinical models of cancer. For example, the inclusion of IL-2 in advanced head and neck carcinomas resulted in the induction of immunotherapy, and subsequent tumor rejection [70]. Post et al found that the inclusion of IL-4 gene therapy in a hypoxia driven oncolytic adenovirus also had a rapid and maintained tumor regression in human glioma xenografts in mice [71]. Similar results were observed with the inclusion of IL-12 and

GM-CSF which were intended to enhance OV therapy by ultimately facilitating an adaptive immune response [49, 72, 73]. Based on these studies several novel OVs have been created encoding for T-cell co-stimulatory factors, pro-inflammatory cytokines, chemokines, and intercellular adhesion molecules designed to increase the autologous antitumor vaccination by OV [49, 51, 69, 74].

While the significance of apoptotic bodies produced upon viral infection and their subsequent engulfment by antigen presenting cells (APCs) to potentiate a cytotoxic T lymphocyte activity and antitumor vaccines has been described [75, 76], the potential to exploit this in conjunction with OV therapy to enhance antitumor immunity is only beginning to be realized. For instance, Endo et al demonstrated that H1299 human lung cancer and SW260 human colorectal cancer cells infected with the OBP-301 oncolytic adenovirus cause the release of dendritic cell (DC) maturation stimuli, leading to efficient tumor loading on these APCs and subsequent CTL potentiation [77]. Similarly, oncolytic infection of mesothelioma cells with the measles virus leads to the production of apoptotic bodies that efficiently induce spontaneous DC maturation and activation, proinflammatory cytokine production, and the amplification of tumor-specific CD8 T cells [78]. Taken together, these findings are the first reports to describe DC maturation in the context of OV-induced apoptosis and they will provide critical mechanistic cues that can be used to harness the combined potential of antitumor immunity with OV therapy.

Section 5.3: Enhancing viral spread throughout a solid tumor

Apart from efficient infection and host immune evasion, an OV needs to replicate and then spread efficiently through the tumor interstitium to effectively infect and eradicate all cancer cells within the solid tumor. Physical barriers within the tumor microenvironment are problematic to efficient viral dissemination and hence limit

21 antitumoral efficacy. The use of enzymes to selectively disrupt the extracellular matrix and surrounding tissue in order to enhance viral spread throughout the tumor has led to the development of several viruses which will next be discussed.

One of the earlier research papers published with interest in breaking down the physical barriers impeding viral transduction was that by Maillard, et al in 1998. It was observed that the endothelium and internal elastic lamina (IEL) were the main barriers to adenovirus-mediated gene transfer to medial smooth muscle cells (SMC). Treatment of rabbit iliac endothelium with elastase disrupted these barriers leading to increased adenoviral vector gene transfer [79]. This study provided evidence that the surrounding connective tissue and ECM may serve as potential barriers to virus transduction in tumor tissue, and these barriers may be the cause of uneven penetration and distribution of oncolytic viruses. A more recent study found that pretreatment of human glioma xenografts in mice with proteases such as trypsin resulted in a significant increase in virus-mediated gene transduction, possibly due to digestion of the tumor extracellular matrix [80]. Similarly it has been observed that HSV virions injected into human melanoma tumors grown in mice, did not spread efficiently in collagen-rich areas. This was tested by co-injecting HSV viral vectors with bacterial collagenase, and resulted in an increase in viral distribution throughout the entire tumor. The combination therapy also significantly increased the antitumor efficacy of the oncolytic virus [81]. Ganesh et al examined the effects of treating tumors with recombinant human hyaluronidase enzyme (rHuPH20), to enhance the spread of recombinant oncolytic adenoviral vectors

[82]. Athymic nude mice bearing PC-3 (human prostate carcinoma) xenografts were treated with oncolytic adenovirus Ad5/35GFP (an E1 deleted, non-replicating virus with a GFP reporter), rHuPH20, or a combination of the two. Histological examination of tumors excised from animals which were co-treated with hyaluronidase revealed a significantly greater distribution of virus within tumors compared to tumors treated with virus alone. The combination therapy also resulted in a significant reduction in tumor growth compared to control. The effect of treating tumors with matrix metalloproteinases

(MMPs) (Zn-dependent proteases that can efficiently break down components of the

ECM [83]) was tested by comparing viral spread in xenografts derived from control or cells stably expressing MMP-1 and MMP-8 in SCID mice. Immunostaining for HSV in tumor sections seven days following treatment, revealed that tumors treated with either

MMP-1 or MMP-8 contained HSV particles throughout the entire section while control tumors contained HSV particles only in the periphery [84].

All of these results collectively indicate the significance of modulating the tumor ECM to enhance viral spread. The effect of relaxin, (a peptide hormone that can induce the expression of MMPs) on viral spread was evaluated in two independent studies. First,

Kim et al engineered a replication incompetent and a replication competent oncolytic adenovirus that expressed relaxin Ad-ΔE1B-RLX [85]. Tumor spheroids transduced with relaxin expressing virus permitted a better penetration of virus compared to control virus transduced spheres in vitro and in vivo. Treatment of mice with subcutaneous tumors revealed a potent antitumor effect of adenovirus Ad-ΔE1B-RLX compared to control

23 virus and histological examination of the tumor revealed an apparent lack of collagen and wide spread viral presence in tumors with Ad-ΔE1B-RLX.

In the second study, two oncolytic adenoviruses, OV-5 and OV-5T35H, based off of the

Ad5 virus and the Ad5/35 chimeric virus respectively, were engineered to express relaxin

[86]. The chimeric virus recognizes the CD46 receptor on tumor cells, significantly improving viral entry and antitumor efficacy [87] and was described by authors

Wickham, Roelvink, and Kovesdi in 1998 [88]. Both OVs expressing relaxin were found to be significantly more invasive than their non-relaxin-expressing counterparts and were found to have a positive correlation between degree of infection and antitumor efficacy.

Based on these results inventors Yun and Kim tested and patented an oncolytic adenovirus expressing relaxin [89].

These findings reveal the significant role that physical barriers, such as the ECM, and host cellular antiviral immunity have on the infectability and spread of oncolytic viruses.

As increasing methods to effectively disrupt these barriers, while maintaining a degree of safety, come into view, the closer we get to enhancing overall OV therapeutic efficacy.

Next we will discuss the extracellular matrix protein CCN1, which we have found to play a role in inhibiting virus therapy.

Section 6: Cysteine-rich 61 (CCN1)

Cysteine-rich 61 (CCN1) is the first member of the growth factor inducible immediate early family CCN, named as such for its first three members: Cysteine-rich 61,

Connective tissue growth factor (CTGF) and Nephroblastoma-overexpressed (Nov) [90].

It is most commonly found in association with the extra-cellular matrix and the cell surface of a wide variety of cell types [91], and has also been found to localize intracellularly within both the cytoplasm and nucleus of smooth muscle cells [92]. CCN1 is composed of four primary domains, three of which mimic the insulin-like growth factor binding protein, the Von Willebrand factor, and the thrombospondin type 1 protein, and the final contains a cysteine-knot, lending to CCN1‟s designation (figure 2).

Binding regions contained within these domains enable CCN1‟s adherence to cell surface heparan-sulfate proteoglycan (HSGP) moieties as well as to a variety of integrin receptors

[93]. Acting on these receptors enables the activation of cellular signal transduction pathways and modulation of a variety of cellular functions including cell adhesion, migration, proliferation, angiogenesis, and tumorigenicity [94].

Overexpression of CCN1 protein in a variety of cancers, including those of the breast, prostate, colon, rectum, ovary, and brain, correlates with poor prognosis [95-98].

Conversely its expression in lung, endometrial, and gastric cancer has been associated with a better prognosis and outcome [99-101]. Apart from affecting direct tumor cell growth it has also been associated with accelerated tumor cell invasion and vascularization in athymic nude mice in vivo [98, 102]. CCN1 is overexpressed in 68%

(27/40) of glioblastoma multiforme specimens and in glioma cell lines derived from high- grade gliomas [103]. Its increased expression in the mucosa of patients with colorectal cancer has also been implicated in “priming for carcinogenesis” [95] and its oncogenic potential is largely accredited to activation of integrin-linked kinase-mediated βcatenin-

TCF/LEF and the AKT pathway [103]. CCN1 has been shown to promote a dose- dependent attachment and chemotaxis of endothelial cells in vitro [104], and promote angiogenesis in chick chorioallantoic membrane assay and in rodent corneal angiogenesis assays [105]. Its inhibition therefore may have a positive impact on the tumor microenvironment leaving cells less invasive and less angiogenic. In addition, we describe below evidence that CCN1 inhibition enhances OV therapeutic efficacy.

CCN1 is rapidly induced in response to mechanical stress or injury and has been implicated as a major player in the regulation of angiogenesis, matrix remodeling, and wound healing [106-108]. Interestingly, CCN1 overexpression in breast cancer and colon cancer cells has also been shown to increase resistance to paclitaxel (taxol) and 5- fluorauracil (5-FU) [109] through the activation of the PI3-kinase/AKT pro-survival, antiapoptotic pathway.

Our group has previously shown that CCN1 gene expression and protein is strongly upregulated following oncolytic HSV-1 therapy in glioma [110]. Consistent with this,

CCN1 has also been found to be dys-regulated in HeLa cells infected with

Coxsackievirus B3 (CVB3) and downregulated in adenovirus type 12 (Ad12)-infected cells [111, 112].

Additionally, an emerging role for CCN1 as a pro-inflammatory molecule is being uncovered [113]. It has been demonstrated that purified CCN1 protein induces a pro- inflammatory genetic program in murine macrophages characterized by upregulation of cytokines TNF-α, IL-1α, IL-1β, IL-6, and IL-12b and chemokines MIP-1α and MCP-3 and down-regulation of anti-inflammatory factor TGF-β1 [114]. Furthermore, recent studies have shown that CCN1 plays a role in the regulation and modulation of several inflammatory cytokines, including Fas ligand, and TRAIL, in normal human skin fibroblasts [115, 116]. Chapters 3-5 of this dissertation will focus on the role of CCN1 in the tumor microenvironment on OV therapy for glioma.

Section 7: Hypothesis and Overview

The current dissertation examines the interplay between the tumor microenvironment and the efficacy of oncolytic virus therapy for glioma. We have previously reported on the significant increase in vascular permeability, leukocyte infiltration, and IFNγ production following OV therapy for glioma. Inhibition of these events was achieved by a single dose of angiostatic cRGD peptide treatment before oncolytic virus, and this led to enhanced antitumor efficacy of oncolytic virus [47]. This discovery led to the construction of RAMBO (Rapid Antiangiogenesis Mediated By Oncolytic virus), an

27 oncolytic virus which expresses the anti-angiogenic protein Vstat120 within the rHSVQ1 backbone [22, 117]. Vstat120 contains a cyclic RGD binding domain and its insertion within rHSVQ1 led to a reduction in OV-induced angiogenesis and an enhancement in

OV efficacy. To further enhance the efficacy of RAMBO, we describe in Chapter 2 the creation and testing of the novel oncolytic virus 34.5ENVE which has been created from the combination of viruses rQNestin34.5 [118] and RAMBO, thus containing Vstat120 and harboring greater specificity due to its rQNestin34.5 background.

Our report describing an enhancement in OV efficacy by angiostatic cRGD also prompted our group to investigate the contributing factors of the increase in angiogenesis following OV therapy. We reported in 2008 on the significant induction of the extracellular matrix protein Cysteine-rich 61 (CCN1) following OV therapy and its contribution to the increase in angiogenesis [110]. Interestingly, in this report, we found

CCN1 gene expression and protein is induced within hours following OV infection. This observation led to the question asking why a dying cell would produce such copious amounts of a protein and prompted our hypothesis that CCN1 acts as an inhibitor to virus infection. In Chapter 3 we describe the cloning and characterization of tetracycline- inducible glioma cell lines containing CCN1 in a tet-on regulatory manner. In Chapter 4 we describe the novel role for CCN1 in activating and enhancing an innate antiviral type

1 IFN response by binding integrin α6β1 on the cell surface, inhibiting OV infection and replication. Given CCN1‟s ability to bind the monocyte and macrophage cell surface receptor integrin αMβ2 [119] and activate a pro-inflammatory cascade in murine 28 macrophages [114] we further examined CCN1‟s effect on OV therapy by investigating its role in the infiltration and activation of macrophages following infection; the methods and results from this study are described in Chapter 5. Taken together, the novel results described in this doctoral thesis collectively suggest there is a woven interplay between changes in the tumor microenvironment induced by oncolytic virus therapy and the subsequent effects these changes then have on subsequent virus infection and replication.

The main hypothesis presented here spans not only oncolytic virus therapy, but indeed all therapies which have been approved for the treatment of malignant glioma in addition to all therapies which are currently in clinical and pre-clinical trials to combat this disease.

This thesis is a small look into the importance of understanding the changes which occur within the tumor microenvironment following therapy with the hope to reduce subsequent tumor cell therapeutic resistance.

Section 8: Figures and Tables

A B C

Figure 1: Classification of Astrocytomas

A. Diffuse astrocytoma, WHO Grade II, characterized by cytologic atypia. B. Anaplastic astrocytoma, WHO Grade III, characterized by cytologic atypia and mitotic activity. C. Glioblastoma multiforme, WHO Grade IV, characterized by cytologic atypia, mitotic activity, tumor cell necrosis and/or angiogenesis (Adapted from [120]).

Figure 2: A modular representation of the human CCN1 protein A description of i) the signal sequence; ii) the insulin-like growth factor-binding protein homology domain (IGFB, blue); iii) the von Willebrand factor type C domain (VWC, green); iv) the thrombospondin type 1 homology domain (TSP1, orange); and the cysteine knot-containing C-terminal domain (yellow). Semitransparent colored bands around the domains indicate sites that interact with integrins (e.g., integrin αvβ3, α6β1, and αMβ2); also shown are the two binding sites for heparin (purple C terminal bands). The dashed line indicates the hinge region; numbers represent amino acid positions in the protein sequence. The dendrogram reflects sequence conservation among the human CCN proteins (Adapted from [121]).

Table 1: List of completed and on-going oncolytic virus trials for patients with GBM

Standard Font indicates completed trials; italic font indicates initiated or ongoing trials. HSV indicates herpes simplex virus, AdV indicates adenovirus, ReoV indicates reovirus, NDV indicates Newcastle Disease Virus, H1 indicates rat H1 parvovirus. Adapted from [122].

Chapter 2: Anti-tumor efficacy of 34.5ENVE: a transcriptionally retargeted and

“Vstat120” expressing oncolytic virus

Introduction

The poor prognosis associated with Glioblastoma multiforme (GBM) underscores the urgent need to seek out novel strategies to improve patient outcome [1]. Oncolytic virus

(OV) therapy is one of such strategies, as it utilizes viruses with a propensity to replicate and destroy cancer cells as an anti-neoplastic agent. First generation OVs have been tested in patients and have been proven safe, yet have revealed limited efficacy [40, 123,

124].

We have previously created and tested the therapeutic efficacy of a first generation OV armed with the anti-angiogenic gene Vstat120 (RAMBO) [117]. While this virus showed significant antitumor efficacy compared to control rHSVQ, single treatment of mice with established intracranial tumors resulted in only 20% of the mice showing a complete response. It is likely that the highly attenuated rHSVQ viral backbone of RAMBO limited oncolysis and anti-tumor efficacy. Several innovative approaches are currently under investigation to enhance OV therapeutic efficacy, and transcriptional retargeting of viral replication is one of such exciting approaches [125-127]. rQnestin34.5 is one such virus which is enhanced by transcriptional retargeting as it expresses viral ICP34.5 under the

32 regulation of a glioma-specific nestin promotor in an ICP34.5 deleted viral backbone

[118, 125, 128, 129]. This virus has shown significant antitumor efficacy against glioma and neuroblastoma tumors [118, 129]. Thus we hypothesized that incorporation of

Vstat120 within the rQnestin34.5 backbone would show enhanced anti-tumor efficacy compared to RAMBO or rQnestin34.5.

Based on these results we engineered and tested the therapeutic efficacy of arming rQnestin34.5 with the anti-angiogenic gene Vstat120. Here we describe the construction of a novel OV, 34.5ENVE (viral ICP34.5 Expressed by Nestin promotor and Vstat120

Expressing). This virus showed significant glioma specific killing and anti-angiogenic effects in vitro and in vivo. In addition to the nestin driven specificity of 34.5ENVE, we also observed a highly significant increase in anti-tumor efficacy, even at a single intratumoral injection, in intracranial glioma bearing mice. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) revealed increased blood brain barrier permeability (increased Ve) in tumors treated with OV compared to control PBS treated tumors indicating OV mediated increased blood brain barrier (BBB) disruption.

Additionally, both histology and calculated DCE-MRI parameters revealed a significant increase in necrotic areas in 34.5ENVE-treated tumors compare to rQnestin34.5-treated tumors.

Results

Generation and characterization of recombinant 34.5ENVE

34.5ENVE, an OV expressing Vstat120 within the backbone of rQnestin34.5, was engineered using HSVQuik technology as described [22]. Figure 3a shows the genetic structures of wild type HSV-1 and 34.5ENVE along with the first and second generation

OVs used in this study. rHSVQ is a first generation OV deleted for both copies of

ICP34.5 and disrupted for ICP6; rQnestin34.5 is a transcriptionally driven OV, expressing ICP34.5 under the control of the glioma specific nestin promotor in an rHSVQ backbone; RAMBO is a Vstat120-expressing OV within the rHSVQ backbone; and

34.5ENVE expresses Vstat120 within the rQnestin34.5 viral backbone [117, 118]. Figure

3b shows a schematic of the PKR activated cellular defense response and the role of viral

ICP34.5 in this process. Activation of cellular PKR, post viral infection, results in phosphorylation of cellular eIF2α and subsequent shut down of protein synthesis [130].

Viral ICP34.5 activates cellular phosphatases, leading to de-phosphorylation of eIF2α thus permitting protein synthesis and viral replication. The phosphorylation state of cellular eIF2α in infected cells was evaluated to investigate the expression of functional

ICP34.5. As expected, figure 3c demonstrates increased phosphorylation of eIF2α (P- eiF2α) in glioma cells infected with control ICP34.5 deleted OVs (rHSVQ or RAMBO).

Reduced P-eiF2α in glioma cells infected with 34.5ENVE or rQnestin34.5 was evident in cells positive for nestin expression (U251 and X12-V2), but not in glioma cells with low nestin expression (T98G) (Figure 3c,d). The ability of 34.5ENVE to produce Vstat120 was evaluated in U251 glioma cells infected with control (rHSVQ or rQnestin34.5) or

Vstat120 expressing (RAMBO or 34.5ENVE) OV. Western blot analysis revealed efficient production and secretion of Vstat120 from glioma cells infected with RAMBO or 34.5 ENVE (Figure 3e).

Oncolysis of 34.5ENVE in nestin expressing glioma cells

Viral ICP34.5 is driven by the nestin promotor in rQnestin34.5 and it has been shown to have increased virus replication compared to rHSVQ in nestin expressing glioma and neuroblastoma cells, but not in low nestin expressing cells [118, 129]. To test if insertion of the Vstat120 gene expression cassette alters the specificity of rQnestin34.5 replication, we compared the viral replication of rQnestin34.5 and 34.5ENVE in twelve different glioma cell lines and primary normal cells. As glioma cells are varyingly susceptible to virus infection, we normalized the fold increase in replication of 34.5ENVE and rQnestin34.5 to ICP34.5 deleted rHSVQ. Figure 4a shows the scatter plot in Log2 function for the fold change in replication of rQnestin34.5 and 34.5ENVE relative to rHSVQ in twelve glioma and normal cell lines. There was a statistically significant positive correlation between the replication of rQnestin34.5 and 34.5ENVE (Pearson correlation coefficient = 0.95091, p<0.0001), suggesting that Vstat120 expression did not alter specificity of rQnestin34.5 replication in different cell lines. To determine if

34.5ENVE retained glioma cell specific oncolysis, we compared the cytotoxic ability of the four different viruses in cells with high and low nestin expression (Figure 5).

Consistent with nestin promotor enhanced virus replication, both rQnestin34.5 and

34.5ENVE showed statistically significant increased cytotoxicity (relative to rHSVQ) in

35 nestin-expressing cells (U251, U87ΔEGFR, LN229, and X12-V2) but not in cells with low or negative nestin expression (T98G, Gli36Δ5, human normal astrocyte and human normal hepatocyte) (Figure 4b-c).

34.5ENVE inhibits endothelial cell migration and tube formation in vitro and in vivo

Next, we tested the effect of 34.5ENVE on endothelial cell migration and tube formation in vitro. To investigate this, we evaluated tube formation ability of endothelial cells cultured with conditioned media (CM) derived from OV infected glioma cells (HSV-1 neutralizing antibody was added to the CM to avoid secondary infection of endothelial cells by contaminating virus particles). Figure 6a shows representative images of the formation of elongated tube-like structures in endothelial cells treated with CM derived from U251 cells treated with PBS, or the indicated virus; structured tube number/view is quantified in figure 6b. Note the significant reduction in the number of tubes obtained from the cells treated with 34.5ENVE and RAMBO compared to rHSVQ or rQnestin34.5 infected cells (p<0.001). We also investigated the effects of 34.5ENVE on the migration of HDMECs in a transwell chamber. Quantitative analysis showed that CM collected from U251 cells infected with 34.5ENVE or RAMBO significantly reduced the migration of endothelial cells by 33 and 34.1%, respectively, relative to rHSVQ (Fig. 6c).

Collectively, these findings demonstrate that 34.5ENVE effectively inhibits endothelial cell tube formation and migration in vitro. To determine the impact of 34.5ENVE on tumoral angiogenesis in vivo, we compared microvessel density (MVD) in a U251T3 subcutaneous tumor xenograft model as described by Weidner et al [131]. Figure 7a

36 shows representative images of adjacent tumor sections stained with H&E, anti-HSV-1, and anti-CD31 antibodies at high and low magnification. Figure 7b shows quantification of CD31 positive blood vessels in viable tumor tissue adjacent to HSV-1 positive staining tumor area. Quantification revealed 3.5, 2.1, and 1.8 fold reduction in MVD in tumors treated with 34.5ENVE compared to rHSVQ, rQnestin34.5, or RAMBO respectively (P value = 0.0001, 0.0002 and 0.0033, respectively).

Anti-tumor effects of 34.5ENVE in vivo:

We next investigated the anti-tumor effects of 34.5ENVE in vivo in four different glioma models. Mice with subcutaneous tumors (U251T3 glioma cells) were treated with PBS, rQnestin34.5, RAMBO, or 34.5ENVE. Figure 8a shows that PBS treated tumors grew rapidly and animals had to be sacrificed by day 17 due to tumor burden. In marked contrast, 7/7 mice treated with 34.5ENVE showed complete tumor regression by day 25, while on a long term follow up to day 49, only 4/7 mice treated with RAMBO or rQnestin34.5 showed a complete response with the rest of the mice showing progressive disease (as measured by increasing tumor volume). We next compared the anti-tumor efficacy of 34.5ENVE in mice bearing an intracranial U87ΔEGFR (Figure 8b), X12-V2

(Figure 8c), and Gli36Δ5 (Figure 8d) glioma. Figure 8b shows Kaplan-Meier curves for survival of mice (with U87ΔEGFR glioma) in each group (n=8/group). Control mice treated with PBS died of tumor burden (median survival: 20 days), while mice treated with rHSVQ, rQnestin34.5, RAMBO or 34.5ENVE showed increased median survivals

(median survival = 33, 34 53, and > 80 days respectively). Consistent with our previous

37 results, mice treated with RAMBO showed a significant increase in median survival compared to rHSVQ treated mice [117]. Significantly, 75% of the mice treated with

34.5ENVE lived longer than 80 days at which point they were sacrificed and found to be tumor free (p<0.001 between 34.5ENVE and rHSVQ, p=0.002 between 34.5ENVE and rQnestin 34.5, p=0.077 between 34.5ENVE and RAMBO). To evaluate if anti-tumor efficacy of 34.5ENVE was dependent on nestin status of cells, we compared antitumor efficacy of 34.5ENVE and RAMBO in glioma with high (X12-V2) and low nestin

(Gli36Δ5) expression (Figure 5). Consistent with nestin driven specificity of 34.5ENVE, we observed a significant increase in anti-tumor efficacy of 34.5ENVE compared to

RAMBO in high nestin expressing X12-V2 intracranial glioma bearing mice (p value =

0.026 between RAMBO and 34.5ENVE) with no difference in low nestin expressing

Gli36Δ5 glioma bearing mice (Figure 8c,d).

Increased Necrosis in Tumors treated with 34.5ENVE

In a parallel experiment to measure the impact of Vstat120 expressed by 34.5 ENVE on the tumor microenvironment, we used dynamic contrast-enhanced MRI (DCE-MRI) to non-invasively measure the antitumor response to rQnestin34.5 and 34.5ENVE treatment in intracranial glioma (U87ΔEGFR) bearing mice. Mice were treated with PBS

(n=3/group), rQnestin34.5 (n=4/group), or 34.5ENVE (n=4/group) on day 10 post tumor cell implantation. Figure 9 shows representative coronal T2-weighted MRI images from one mouse/group pretreatment and on days 3 and 6 post treatment, respectively.

Assessment of tumor volumes in all mice showed that while PBS treated tumors grew

38 rapidly, all of the OV treated tumors showed initial increase in apparent tumor volume

(comparing day 3 post treatment to pretreatment), followed by tumor regression on day 6 post treatment. Interestingly, despite a better survival, we did not observe a significant difference in tumor volume shrinkage measured by T2-weighted MRI between rQnestin34.5 and 34.5ENVE at these time points. Since tumoral necrosis and edema can contribute to apparent tumoral volume, we investigated changes in tumoral perfusion and necrosis in animals treated with rQnestin34.5 and 34.5ENVE. Figure 10 shows contrast enhancement of all tumor bearing mice imaged three days after the indicated treatment immediately following gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA) administration. Contrast enhancement was evident in all of the OV treated animals at the site of OV injection. However, in the 34.5ENVE-treated animals immediately post Gd-

DTPA administration, there was a central un-perfused area (yellow arrow) at the site of virus injection accompanied by increased leakiness surrounding this core (Figure 10), giving the appearance of a halo. We utilized Gd-DTPA-based DCE-MRI to calculate

trans changes in K and ve between rQnestin34.5 and 34.5ENVE treated animals (Figure 11).

Ktrans refers to increased leakage of contrast agent and suggests increased endothelial permeability facilitating the entry of the contrast agent from the blood plasma, and ve refers to the volume of contrast in the extravascular and extracellular space per unit volume of tissue [132]. We found a significant increase in ve, in animals treated with

34.5ENVE compared to rQnestin34.5, (p=0.0009, n=4/group) without a significant

trans change in K (Figure 11). Spatially, the increased ve in all the 34.5ENVE treated animals was localized to the non-contrast enhancing core visualized immediately after

Gd-DTPA administration (Figure 12a). Such a pattern of increase of ve in the non- contrast enhancing area has been shown to indicate tumoral necrosis [133]. We thus investigated the non-contrast enhancing core further using histological analysis. Figure

12b shows representative H&E stained images of tumor bearing brain sections, showing increased necrosis (asterisks) in the intracranial tumor core of animals treated with

34.5ENVE compared to rQnestin34.5. Collectively, these findings indicate that

34.5ENVE treatment results in increased tumor necrosis and enhanced antitumor efficacy compared to rQnestin34.5 [133].

Discussion

Oncolytic virus (OV) treatment is a promising biological therapy currently being evaluated in human patients for safety and efficacy. Rapid viral clearance along with reduced viral replication in tumor cells is thought to be one of the major factors responsible for limited efficacy [62]. Transcriptional retargeting of OV by utilizing glioma specific nestin enhancer driven ICP34.5 expression has been described and has shown efficacy in preclinical models of glioma and neuroblastoma [118, 128, 129].

Nestin was initially described as a marker for neuronal stem cells, however its expression has been reported in several malignancies including brain, gastrointestinal, pancreatic, prostate, breast, malignant melanoma, and thyroid tumors [134]. Thus, nestin driven oncolytic viruses have therapeutic significance for many different types of cancer [118,

128, 129]. Apart from virus replication, the tumor microenvironment also presents a barrier for OV therapy, and arming of OV with anti-angiogenic genes has shown promise

40 in several preclinical studies [135]. Here we describe the construction and anti-tumor efficacy of 34.5ENVE, an OV transcriptionally driven to have increased virus replication in nestin-positive tumor cells and armed with the anti-angiogenic Vstat120 gene to modulate the tumor microenvironment.

Vstat120 is an extracellular fragment of Brain angiogenesis inhibitor 1 (BAI1), whose expression has been shown to be reduced in several malignancies [136-139]. Given the anti-angiogenic role attributed to Vstat120, the reconstitution of its expression in BAI1 null tumors may enhance anti-cancer therapeutic efficacy [117, 135, 140-142]. The antiangiogenic effects of Vstat120 are attributed to five Thrombospondin type 1 domains within its N terminal sequence, and an integrin antagonizing RGD motif [141, 143, 144].

Oncolytic HSV-1 therapy has been shown to reduce TSP-1 protein, and also increase integrin-activating CCN1 protein in the tumor extracellular matrix, resulting in an increased angiogenesis in the residual tumors after OV therapy [44, 110]. Thus we hypothesized that Vstat120 gene delivery in conjunction with transcriptionally retargeted oncolysis would reduce OV induced vascular permeability and prolong OV propagation and efficacy in tumors.

Here we describe significant anti-tumor efficacy of 34.5ENVE in both subcutaneous and intracranial glioma models. Changes in vascular permeability have been correlated with both increased and reduced OV efficacy [47, 145]. Here we utilized DCE-MRI of mice with intracranial tumors to investigate the impact of Vstat120 on vessel leakiness. While

41 there was no change in absolute value of Ktrans between rQnestin34.5 and 34.5ENVE, analysis of the parametric images of ve along with histologic analysis of intracranial tumors showed increased tumoral necrosis in 34.5ENVE treated animals. Future studies evaluating its safety and biodistribution in HSV-1 sensitive BalbC mice will be needed prior to its clinical investigation in patients.

In conclusion, this study reports on the construction and anti-tumor efficacy of a novel, transcriptionally driven OV with unsurpassed anti-tumor efficacy and encourages its further development as a potent therapeutic agent for patients.

Materials and methods

Cell lines and viruses

Human normal astrocytes, hepatocytes, human umbilical vein endothelial cells

(HUVEC), and human dermal microvascular endothelial cells (HDMEC) were purchased from ScienCell (Sandiego, CA). Vero cells were obtained from ATCC. U251, LN229,

T98G, Gli36Δ5, have been cultured in our laboratory and U251T3 cells were obtained as a tumorigenic clone of U251 cells by serially passaging these cells three times in mice.

U87ΔEGFR cell line expresses a truncated, constitutively active, mutant form of epidermal growth factor receptor (EGFRvIII), and has been previously described [146].

X12 primary tumor derived cells were obtained from Dr. Sarkaria, and were sub-cloned to express GFP to generate X12-V2 (Mayo Clinic, MN) [110]. All the cells were evaluated for their nestin expression by RT-PCR as described (Supplementary Figure S1)

[118]. The construction and efficacy of RAMBO, a Vstat120-expressing OV within the context of rHSVQ, a first generation OV deleted for both copies of ICP34.5 and disrupted for ICP6, and rQnestin34.5, a transcriptionally driven OV expressing ICP34.5 under the control of glioma specific nestin promotor, have been previously described [117, 118].

To generate 34.5ENVE, the expression cassette encoding for Vstat120 gene under the control of the viral IE4/5 promotor and ICP34.5 under the regulation of nestin enhancer driven promotor was inserted into fHSVQ using HSVQuik technology as previously described [22]. All viruses were propagated in Vero cells. Three days after infection, secreted virus and virus-infected Vero cells were harvested and subjected to three cycles of freeze-thaw to release the viruses completely, and cell debris was cleared by centrifugation (4,000xg, 20 min). Virus was filtered to remove cell debris and pelleted by centrifugation at 13,000g for 1 hrs. The titer (plaque forming units per ml, PFU/ml) of the resulting virus was determined by plaque forming unit assay in Vero cells [22].

Cell viability and virus replication assays

To measure OV mediated cytotoxicity in 96-well plates, cells were infected with the indicated virus at the indicated MOI. 72 hr post infection, viable cells were measured by a standard crystal violet assay as described [117]. For virus replication assays, the indicated glioma cells were infected at an MOI of 0.01 for 2 hours, washed and media replaced. Three days following infection, cells and supernatant were harvested and the number of infectious virus particles present was determined by performing a standard plaque forming unit assay on Vero cells.

In vitro endothelial cell assays

U251 glioma cells were infected with the indicated virus at an MOI of 2. After 14 hrs,

CM was harvested and cellular debris and free floating virions were removed by centrifugation (27,700 xg for 1 hrs). The CM was then concentrated 100 fold in Amicon

Ultra centrifuge tubes (Millipore, Billerica, MA), and 0.4% of IgG was added to neutralize contaminated OV. Endothelial cell migration assays were performed using transwell chambers (8-μm pore size, from Corning Costar, (Cambridge, MA) coated with

0.1% fibronectin as previously described [117]. Tube formation assay was performed as described previously [57]. Briefly, HDMECs were grown on growth factor-reduced

Matrigel (Collaborative Biomedical Products, Bedford, MA). Cells were then allowed to form tubes for 4 hr at 37°C, and photographed (X200). Pictures of the formed tubes (200

μm or larger, and connected at both ends) were quantified by counting 10 microscopic view/well, and the data presented as the averages of four wells.

Antibodies

For western blot analysis: anti BAI1 antibodies were raised as described previously

[140], anti-human GAPDH (ab9484) and anti-ICP4 (ab6514) were purchased from

Abcam (Abcam, Cambridge, MA), and anti-eIF2α (9722) and anti-phosphor-eIF2α

(9721) were obtained from Cell Signaling Technology (Cell Signaling Technology,

Beverly, MA). To observe microvessel density (MVD), tumor sections were treated with purified rat anti-mouse CD31 (Pharmingen, San Jose, CA) to visualize endothelial cells lining the blood vessels (n = 3 mice/group), and then with biotin-conjugated goat anti-rat

IgG (BD Biosciences Pharmingen, San Diego, CA). The three most vascularized areas within the tumor ("hot spots") were chosen at low magnification, and vessels were counted under a representative high magnification (X200) field in each view field [147].

Mean MVD was calculated as the average of counts/view field in three hot spot areas as described by Dr. Folkman [131]. Vessels at the periphery of the tumor were disregarded in the MVD counts. The MVD for each group was then averaged together (n = 2–4 sections/tumor, and n = 3 tumors/group) to get the final count ± SEM.

Animal surgery

All animal experiments were performed in accordance with the Subcommittee on

Research Animal Care of The Ohio State University guidelines and have been approved by the Institutional Review Board. 6-8 week-old Female athymic nu/nu mice (Charles

River Laboratories, Frederick, MD), were used for all tumor studies.

For subcutaneous tumors, nude mice were implanted with 1.5 x 107 U251T3 glioma cells into the rear flank. When tumors reached an average size of 250 mm3, mice were administered PBS, or the indicated virus by direct intra-tumoral injection (5 x 105 pfu) on days 1 and 3. Tumor volume was calculated using the following formula: volume =

0.5LW2 as described [148].

For intracranial tumor studies, anesthetized nude mice were fixed in a stereotactic apparatus, and a burr hole was drilled at 2 mm lateral to the bregma, to a depth of 3 mm.

U87ΔEGFR (1 × 105), X12-V2 (3 × 105), or Gli36Δ5(1 × 105) glioma cells were implanted. On day seven, ten, and seven after U87ΔEGFR, X12-V2 or Gli36Δ5 glioma cell implantation respectively, the mice were anesthetized again and stereotactically inoculated with 5 × 104 pfu of the indicated virus at the same location. Animals were observed daily and were euthanized at the indicated time points or when they showed signs of morbidity.

DCE MRI imaging

Mice bearing intracranial tumors were treated with PBS or the indicated virus on day 10 after tumor cell implantation. Anatomic imaging was done on day 9 (pre treatment), on day 13 (3d post treatment), and on day 16 (6d post treatment), using T2-weighted RARE imaging sequence (TR=2500 ms, TE=12 ms, Rare Factor=8, navgs=4). The imaging was performed using a Bruker Biospin 94/30 magnet (Bruker Biospin, Karlsruhe

Germany). For dynamic contrast enhanced imaging (DCE-MRI), anaesthetized mice were injected with 0.5 mmol/kg Magnevist TM (Bayer Health Care Pharmaceuticals,

Wayne NJ) Gd-DTPA contrast agent. A 2.0 cm diameter receive-only mouse brain coil was placed over the head, and the mouse bed with surface coil was placed inside a 70 mm diameter linear volume coil. DCE data were collected using a FLASH sequence

(TR=135.8 ms, TE=2.4, flip angle = 50o). Several baseline images were collected before the bolus of Gd-DTPA was injected through the tail vein catheter. Images were collected post-Gd-DTPA injection for approximately 30 mins. The acquisition parameters for both

46 the T1- and T2-weighted multi-slice scans were as follows: FOV = 20 mm x 20 mm, slice thickness = 1.0 mm, matrix size = 256 x 256.

For data analysis, a region-of-interest (ROI) that included the tumor was manually outlined using the T2-weighted images. Tumor volumes were calculated from the outlined ROI. Gd-DTPA concentrations were measured for all voxels within the ROI using the method described [149]. A fixed value of T1(0) was chosen as 2029 based on

T1 measurements made in normal mouse brain at 9.4T [150]. C(t) was calculated for each voxel within the ROI and the average for the entire ROI was calculated. The integrated area under the curve (IAUC) was calculated from the mean Gd-DTPA C(t) curve post- injection and the cumulative IAUC (CIAUC) was calculated from the IAUC. The mean

C(t), IAUC, and CIAUC curves were evaluated and compared for the rQnestin34.5,

34.5ENVE, and PBS-treated mice at each imaging time point. The vascular input function required for the pharmokinetic modeling was obtained by measuring signal intensity in the superficial temporal vein within the same image slices as those of the tumor. A two-compartment general kinetic model was used for analyzing the distribution and flow of gadolinium in the tumor. The rate constant, Ktrans , and the extravascular exctracellular space, ve, were determined by nonlinear curve fitting the Gd concentration,

trans C(t), over a 20 minute time period post Gd injection. Color-coded maps for K and ve were created to visualize the spatial distribution of Gd in the tumor. Histograms of Ktrans and ve for the entire tumors were calculated and median values were compared between the treatment groups.

Statistical Analysis

Student's t-test was used to analyze changes in cell killing, HDMEC transwell migration, tube formation assay data, and changes in MVD. A P value <0.05 was considered statistically significant. Kaplan–Meier curves were compared using the log rank test for survival analysis. Spearman‟s rank correlation coefficient was calculated for the correlation analysis on the fold change (Log2) in virus replication of rQnestin34.5/34.5ENVE compared to rHSVQ. The scatter plot of fold increase (Log2) in rQnestin34.5 (x axis) and 34.5ENVE (y-axis) replication relative to rHSVQ in each cell line was plotted against relative nestin expression. Each dot represents one of the twelve cell lines used in this study. Holm‟s procedure was used to correct the P value for multiple comparisons. A P value <0.05 was considered statistically significant. All statistical analyses were performed with the use of SPSS statistical software (version

14.0; SPSS, Chicago, IL), or SAS (version 9.2; SAS Institute, Cary, NC).

Figures and Tables

Figure 3: Structure and Characterization of 34.5ENVE

49 a) Genetic map of wild type HSV-1, and the various oncolytic viruses (OVs) used in this study. b) Activation of PKR upon OV infection causes phosphorylation of eIF2α, and subsequent shutoff of protein synthesis. Viral ICP34.5 activates protein phosphatase 1α, to reverse phosphorylation of eIF2α and the subsequent protein translation. c) Western blot analysis of phosphorylated and total eIF2α in high nestin expressing U251 and X12- V2 glioma cells treated with PBS (lane 1) or infected with rHSVQ (lane 2), rQnestin34.5 (lane 3), RAMBO (lane 4) or 34.5ENVE (lane 5) (MOI = 0.1), 24 hrs post infection. d) Western blot analysis of phosphorylated and total eIF2α in low nestin expressing T98G glioma cells treated similarly (MOI=0.1), 24 hrs post infection. e) Western blot analysis of U251 glioma cells treated with PBS (lane 1), rHSVQ (lane 2), rQnestin34.5 (lane 3), RAMBO (lane 4) or 34.5ENVE (lane 5) at an MOI=0.1. The cells were harvested at 6, and 12hrs post infection and analyzed for expression of secreted and cellular Vstat120 and ICP4. Note the presence of Vstat120 in cells infected with RAMBO and 34.5ENVE.

Figure 4: Increased virus replication and cytopathic effect of 34.5ENVE in high nestin expressing cells.

Twelve different cell lines (Glioma and normal) were infected with rHSVQ, rQnestin34.5, or 34.5ENVE (MOI = 0.01). 72hrs post infection the amount of rHSVQ, rQnestin34.5, and 34.5ENVE in each cell line was evaluated by plaque assay. a) Scatter plot of relative virus yield of Log2 fold increase in 34.5ENVE to rQnestin34.5 relative to rHSVQ in each cell line. Each dot represents one of the twelve glioma/primary cells evaluated. b-c) Quantification of cell viability in the indicated cells infected with rHSVQ, rQnestin34.5, RAMBO, or 34.5ENVE (MOI=0.05) relative to PBS three days post infection.

Figure 5: Real Time-PCR analysis

Real time PCR analysis of the indicated glioma cell lines for relative nestin expression. GAPDH was used as internal control.

Figure 6: Anti-angiogenic effect of 34.5ENVE in vitro

53 a) Images of endothelial cell tube formation after being cultured on Matrigel. Endothelial cells were incubated with conditioned medium (CM) derived from U251 cells infected with the indicated virus or PBS. b) Quantification of the average number of tubes/view field (n=4/group) observed above. c) Inhibition of endothelial cell migration: HDMECs were incubated with CM derived from U251 cells treated with PBS, or the indicated virus. Cells were plated in the upper chamber of transwell and allowed to migrate toward CM used as a chemo-attractant in the bottom chamber. The migrated cells on the bottom side of the filter were quantified as described (n = 4/group) [140]. Data are presented as mean ± SEM of number of cells/view field.

Figure 7: Reduced angiogenesis in tumors treated with 34.5ENVE 55 a) Representative images of immuno-histochemistry for H&E, CD31, and HSV-1 staining of adjacent tumor sections treated with PBS, or the indicated virus. Necrotic area is marked by asterisks and black dotted lines. b) Quantification of micro-vessel density (MVD) in tumors treated with PBS, or the indicated virus. Data shown are mean MVD ± SEM for each group (n = 6 sections/tumor and n = 3 tumors/group). All scale bars are 100 μm for 10X magnification images and 500 μm for 4X images respectively.

Figure 8: Anti-tumor effects of 34.5ENVE in vivo 56 a) Antitumor efficacy of 34.5ENVE against U251T3 subcutaneous glioma model. Mice with U251T3 subcutaneous tumors were treated with PBS or 5 × 105 pfu every other day for a total of two times (Q2Dx2) once tumors reached an average volume of 250 mm3. Mean tumor growth of mice after treatment with PBS, rQnestin34.5, RAMBO, or 34.5ENVE is shown as a function of time. CR: complete response shown in mice by day 49. b) Kaplan–Meier survival curve of mice implanted with U87ΔEGFR intracranial glioma treated with PBS or 5 × 104 pfu of rHSVQ, rQnestin34.5, RAMBO or 34.5ENVE 7 days after tumor cell implantation. c) Kaplan–Meier survival curve of mice implanted with X12-V2 intracranial glioma treated with PBS or 5 × 104 pfu of RAMBO or 34.5ENVE 10 days after tumor cell implantation. d) Kaplan–Meier survival curve of mice implanted with Gli36Δ5 intracranial glioma treated with PBS or 5 × 104 pfu of RAMBO or 34.5ENVE 7 days after tumor cell implantation.

Figure 9: OV treatment induced tumor regression T2-weighted MRI images of coronal sections of a representative tumor bearing mouse one day before (left panel), three days post (middle panel), and six days post (right panel) treatment with rQnestin34.5 (top), 34.5ENVE (middle) or PBS (bottom).

Figure 10: Contrast enhancement of PBS and OV treated tumors Contrast enhanced images of coronal sections of mice three days after treatment with PBS (n=3), rQnestin34.5 (n=4), or 34.5ENVE (n=4). Diffusion of the contrast agent immediately post Gd-DTPA administration results in contrast enhancement appearing as area of high signal intensity. Arrow indicates the central non-enhancing core observed in all four of the mice treated with 34.5ENVE, which is not evident in any mice treated with rQnestin34.5 or PBS.

trans Figure 11: K and Ve for rQnestin34.5 and 34.5ENVE treated tumors

trans (a-d) Color coded parametric images K (a-b) and ve (c-d) of coronal sections of mice three days after treatment with rQnestin34.5 (a, c), or 34.5ENVE (b, d). Mice with intracranial tumors were treated with direct intratumoral injection of rQnestin34.5 or 59

34.5ENVE (10 days post tumor cell implant). Shift towards red indicates higher values. e) changes in mean ve between rQnestin34.5 and 34.5ENVE treated animals.

Figure 12: Effect of 34.5ENVE on tumor necrosis a) Inverse spatial correlation of ve and contrast enhancing tumor area in 34.5ENVE treated mice. Color coded parametric images of ve in each of the four 34.5 ENVE treated mice (top) show increased ve (arrow head) in tumoral area that initially lacked contrast 60 enhancement (arrow) immediately after Gd-DTPA administration in each of the four mice treated with 34.5ENVE. b) Histologic analysis of necrosis in rQnestin34.5 and 34.5ENVE treated brain tumors. Representative H&E stained sections of rQnestin34.5 and 34.5ENVE treated animals. Large necrotic area evident in 34.5ENVE treated tumor (white dotted line and asterisks) is surrounded by viable tumor area (black dotted line).

Chapter 3: Cloning and characterization of Cy-1 tetracycline-inducible cells

Introduction

Tetracycline-inducible systems enable researchers to selectively and reversibly turn on and off gene transcription in the presence of the antibiotic tetracycline to more specifically evaluate gene function. Here we used a tet-on system to specifically control the expression of CCN1 in the parental LN229 glioma cell line. In this system, the tetracycline derivative doxycycline (dox) acts as an effector molecule in that it‟s binding the reverse tet trans-activator protein (rtTA) enables rtTA‟s recognition of a tet „O‟ operator sequence located on our gene of interest, and thus stimulates transcription

(Figure 13). In this section we describe the characterization of the glioma cell line Cy-1 expressing myc-tagged CCN1 under a tetracycline-inducible promoter.

Results

Cy-1 cells express CCN1 following dox treatment

To verify Cy-1‟s tetracycline-inducibility we used both a dose-response assay and a time course induction assay (Figure 14A). Cells were treated with increasing concentrations of doxycycline (0.2, 0.4, 0.6, 0.8ug/ml) for 24 hours and analyzed by western blot for protein expression. As indicated in figure 14A, with increasing concentrations of

62 doxycycline there is increased expression of CCN1 protein. To understand the kinetics of

CCN1 protein expression following dox incubation we treated cells with 1ug/ml doxycycline and harvested lysates at the indicated time points. Western blot analysis revealed a significant induction in CCN1 in cells treated with doxycycline as early as 4 hours following treatment (Figure 14B). Later, it was confirmed that CCN1 protein is expressed within minutes of dox treatment (see figure 27I).

To confirm CCN1 induction in our tet-on system in vivo, we implanted athymic nude mice subcutaneously in the rear right flank with 4x106 Cy-1 cells. When tumors reached an average size of 100mm3 mice were split into two groups; half received 1mg/ml doxycycline in 5% sucrose drinking water while the other half received 5% sucrose only as control. Tumors were harvested 48 hours later and analyzed by western blot. Figure

14C shows that tumors from mice fed dox positively express CCN1 protein tagged with myc, while control mice showed no sign of CCN1 induction.

CCN1 inhibits glioma cell proliferation

The effect of CCN1 on the proliferation of cancer cells is unclear and has remained controversial in the literature [115, 151-154]. We thus were interested in understanding the effect CCN1 has on glioma cell migration and proliferation in our model. To elucidate the direct effect of CCN1 on glioma cell proliferation, Cy-1 cells were treated with 1ug/ml dox and allowed to grow over the course of 5 days. Growth was assessed

63 daily by standard crystal violet assay and LN229 glioma cells were run in parallel to control for dox treatment alone. Figure 15A shows Cy-1 cells expressing CCN1 stop proliferating almost immediately. We next asked if CCN1 would have a similar effect on

Cy-1 cell growth in vivo as it did in vitro. Athymic nude mice were implanted subcutaneously with Cy-1 cells and administered dox in their drinking water when the tumor sizes became 100mm3. As indicated in figure 15B, dox significantly suppressed the growth of subcutaneous Cy-1 gliomas. To examine if this effect occurred due to secreted

CCN1 acting on the cell surface, we co-cultured Cy-1 cells with U87∆EGFR-luc glioma cells in the presence or absence of dox. Quantifying luciferase activity as a determinant for U87∆EGFR-luc glioma cell proliferation, we show in figure 15C, secreted CCN1 from Cy-1 glioma cells had no effect on the proliferation of neighboring cells. Next we used flow cytometry to examine if CCN1 was playing a distinct role in the mitotic cycle resulting in the reduction in cell proliferation. Figure 16 shows CCN1 expression in Cy-1 cells inhibits proliferation by imposing a block on the G1-S transition of the cell cycle.

CCN1 enhances glioma cell migration and invasion

The results presented thus far have shown that CCN1 expression leads to reduced glioma cell proliferation, yet the literature has often placed CCN1 in a more tumorigenic category rather than a tumor suppressive one [102, 103, 155]. Following the “go no- grow” hypothesis, we questioned if CCN1‟s effect on proliferation were dichotomous to its effect on cell migration and invasion. Using a scratch assay with Cy-1 cells treated ± dox, we used time-lapse microscopy to observe the migration patterns of these two

64 groups. Figure 17A shows by 8 hours cells treated with dox have migrated significantly more than cells treated without dox. To investigate the effect CCN1 has on invasion, we used a hanging drop assay and then embedded our spheres in matrigel in the presence or absence of dox. As shown in figure 17B, 24 hours following sphere embedding, Cy-1 spheres treated with dox invaded the surrounded matrix much more greatly than Cy-1 spheres not treated with dox; LN229 glioma cells were used to control for dox alone.

Collectively, these results indicate CCN1 has a significant biological effect on the glioma cell within which it is expressed.

Discussion

CCN1 has been shown to play a variety of cellular functions, often based on the cell type within which it is expressed. While it has been shown to increase cell migration and proliferation in osteoblasts, CD34+ progenitor cells, gastric cancer AGS cells, chondrosarcoma cells, and breast cancer cells, it has also revealed itself as a tumor suppressor in hepatocellular carcinoma, melanoma, non-small cell lung cancer, and endometrial cancer cells [101, 156-163]. The opposing effect of CCN1 is likely due to its ability to bind a variety of cellular receptors, which may differ tissue to tissue. Here we describe the construction and characterization of tetracycline-inducible CCN1 expression in LN229 glioma cells. We show that CCN1 expression in these glioma cells results in a slowing of cell proliferation due to the arrest of mitosis from the G1 to S phase.

Additionally, we reveal CCN1 expression results in an enhanced migratory and invasive

65 phenotype in these cells. The results from this study indicate that CCN1‟s expression alone can lead to phenotypic changes in the cell and lends to the notion that when cells are actively migrating, they slow their proliferation as described in the “go, no-grow” hypothesis [164].

The effect of CCN1 on the glioma cell line U343 was explored by Xie et al in 2004.

Here, authors found that exogenous CCN1 protein resulted in increased cell growth and indeed, stable expression of CCN1 in this glioma cell line resulted in an increase in the S phase of mitosis along with an increase in tumorigenicity [103]. Though contrasting our results presented above, it is likely that CCN1 is acting on different receptors in these two glioma cell types resulting in the varying effects. In addition, Xie et al found that CCN1 relayed its effects in U343 cells through interacting with the integrin receptor αvβ3. Thus, future studies examining the receptor which CCN1 interacts with to mediate its effects in

LN229 glioma cells will be important to pursue.

In conclusion, we have shown that tetracycline-inducible CCN1 expression in LN229 glioma cells enhances their migration and invasion, while at the same time slowing their proliferation by inhibiting the G1 to S phase transition in mitosis. The results from these studies indicate a significant role for CCN1 in the biology of glioma cells. Future studies describing the mechanisms behind its effects will be necessary to truly understand the role of this protein in human glioma.

Materials and Methods

Cell lines and transfections

Human LN229 and Cy-1 glioma cell lines were maintained in Dulbecco‟s modified minimal essential medium (DMEM) supplemented with 2% fetal bovine serum, 100U/ml penicillin, and 100ug/ml streptomycin as described [165]. The open reading frame of

Cyr61 (amino acids 1-382) was cloned into pcDNA3.1+myc-His (Invitrogen) for constitutive expression (wt Cyr61) or into pTRE2 (Clontech) for tet-regulated expression

(Cyr61mycHispTRE2) using standard molecular biology techniques. Transient

Transfections were carried out with Lipofectamine Reagent supplemented with Plus

Reagent (Invitrogen). The tet-regulated system was established by stably transfecting

LN229 cells with an rtTA expression vector. The isolated tet-responsive clone L2 was then transfected with Cyr61mycHispTRE2 and Cy-1 and Cy-2 were generated by selecting for neomycin-resistant clones (900ug/ml) and tested for their ability to induce

Cyr61 in response to dox (1ug/ml for 24 h). Expression of Cyr61 RNA following transfections was determined by using Titan One Tube RT-PCR Kit (Roche Applied

Sciences), per manufacturer‟s instructions. A total of 10ng RNA was run for each sample and expression levels were normalized to endogenous GAPDH.

Animals

All animal experiments were performed in accordance with the Subcommittee on

Research Animal Care of the Ohio State University guidelines. Athymic nude mice 6-8

67 weeks of age were purchased from NCI/NIH. Mice were anesthetized and injected into the rear right flank with 4x106 Clone 16 cells. At 100mm3 mice were administered 5% sucrose water ± dox (1mg/ml). Tumors were measured weekly and mice were sacrificed when tumor volumes reached 2000mm3.

Antibodies

Antibodies used in this study were obtained from the following sources: anti-CCN1

(Novus Biologicals), anti-myc tag and anti-GAPDH (Abcam), anti-mouse HRP (GE

Healthcare), anti-rabbit HRP (DAKO).

Western Blot Analysis

Immunoblots were performed on cell lysates (lysed in RIPA buffer: 150mM NaCl, 1%

Nonidet P-40, 0.5% sodium Deoxycholate, 0.1% SDS, 150mM Tris) from indicated cells.

Equal amounts of protein were resolved on a 10% SDS-PAGE followed by transfer to

PVDF membranes. Blots were probed for the indicated proteins using the appropriate antibodies and visualized by enhanced chemiluminescence (GE Health).

Cell proliferation, migration, and invasion assays

To measure CCN1‟s effects on cell proliferation, cells were plated in 96-well dishes and treated with doxycycline (1ug/ml) for 5 days. Cell proliferation was quantified daily as

68 measured by standard crystal violet assay as described [166]. To measure CCN1‟s effects on cell migration, cells were plated to confluence on glass chambers overnight. Using a pipette tip a scratch was made through the middle of the cells which were then incubated in media ± dox. Migration was monitored using time-lapse microscopy for 16 hours. To measure CCN1‟s effects on glioma cell migration, cells were plated up-side-down in a hanging drop to form a sphere. Spheres were then embedded in matrigel basement membrane matrix (BD Biosciences) and incubated in media ± dox. Pictures were taken at

10x, 24hours following dox administration.

Flow Cytometry

For flow cytometry analyses of CCN1‟s effect on cell cycle, cells were arrested at the beginning of S phase by a double thymidine block. Following the second block cells were incubated with media ± dox and allowed to progress through G2- and mitosis. Cells were then fixed in PFA, pelleted and resuspended in cold 70% EtOH overnight. Cells were then resuspended in propidium iodine and measured at 488nm light to induce fluorescence.

Statistical Analysis

Results are presented as mean values ± standard error of the mean (SEM). Statistical analysis was carried out by unpaired Students‟ t-test using GraphPad Prism® 5.01 software. P values <0.05 were considered statistically significant.

Figures and Tables

Figure 13: Schematic showing tetracycline-inducible tet-on system In the tet-on system, the presence of doxycycline (dox) allows a conformational change to occur for the rtTA enabling its activation of the tet operon (tetO) sequence and transcription of the indicated gene of interest (X).

Figure 14: Induction of CCN1 in vitro and in vivo Western blot analysis of Cy-1 cells in the presence or absence of doxycycline (dox). (A) Cells treated with increasing concentrations of dox were analyzed by western blot and normalized to GAPDH. (B) Cells treated with dox and monitored for CCN1 expression at

71 the indicated time points. (C) Tumor tissue harvested from mice bearing subcutaneous Cy-1 gliomas and fed 5% sucrose water ± dox.

Figure 15: CCN1 inhibits proliferation in vitro and in vivo

(A) Cy-1 and LN229 cells treated ± doxycycline for five days. Results presented as mean absorbance normalized to untreated control. (B) Athymic nude mice implanted 72 subcutaneously with Cy-1 glioma cells were fed sucrose water ± dox every other day beginning when tumors reached 100mm3. Tumor size was measured weekly and is presented as mean ± SEM. (C) U87∆EGFR-Luc glioma cells co-cultured with Cy-1 or LN229 glioma cells ± dox. Cell growth was determined by luciferase activity and is presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 16: CCN1 blocks glioma cell mitosis at G1-S phase 74

Cell mitosis of Cy-1 cells following double thymidine block in the presence or absence of dox. Following double thymidine block, cells were released for 24 hours ± dox stained with propidium iodine and analyzed by flow cytometry. (A) Cells immediately following release. (B) Cells 24 hours following release in the absence of dox. (C) Cells 24 hours following release in the presence of dox.

Figure 17: CCN1 increases glioma cell migration and invasion (A) Scratch assay of Cy-1 glioma cells ± dox at 0 and 8 hours following scratch. (B) Cy-1 and LN229 hanging spheres embedded in matrigel. Embedded spheres were incubated in DMEM ± dox and pictures were taken 24 hours later. Black circles depict sphere growth.

Chapter 4: Extracellular matrix protein CCN1 limits oncolytic efficacy in glioma

Introduction

Glioblastoma multiforme (GBM) is the most common primary brain tumor and despite aggressive therapy involving tumor resection, chemotherapy and radiation treatment, median survival of patients remains less than 15 months from diagnosis [167]. Oncolytic viruses (OVs) are biological therapeutics that selectively replicate in and kill tumor cells.

These viruses have shown promising results in preclinical models [135], and their safety and efficacy is currently being investigated in clinical trials. Despite these advances, the impact of changes in the tumor microenvironment on OV therapeutic efficacy has not been very well studied.

We have previously described a dose dependent and rapid induction of the secreted angiogenic inducer Cysteine rich 61 (CCN1) in the tumor microenvironment following

OV therapy [110]. CCN1 is a member of the growth factor inducible immediate early family CCN, named as such for its first three members Cysteine rich 61, connective tissue growth factor (CTGF), and Nephroblastoma-overexpressed (Nov) [90]. It is a secreted protein which typically localizes in the extracellular matrix (ECM) and on the cell surface [91], where it binds integrin receptors to modulate a variety of cellular functions including adhesion, migration, and proliferation [94]. In brain tumors CCN1 is 77 overexpressed in 68% (27/40) of GBM specimens and in cell lines derived from high- grade gliomas [102]. Its increased expression in the mucosa of patients with colorectal cancer has also implicated it in “priming for carcinogenesis” [95] and its oncogenic potential is largely accredited to its activation of integrin-linked kinase-mediated

βcatenin-TCF/LEF and AKT [103].

Apart from its induction in glioma cells infected with herpes simplex virus-1 (HSV-1) derived OVs, CCN1 has also been found to be dysregulated in cells after infection with

Coxsackievirus B3 (CVB3) and Adenovirus type 12 (Ad12), suggesting that it may play a role in viral infection of mammalian cells [111, 112]. Here we evaluated the impact of

CCN1 expression on OV efficacy. Our findings indicate that CCN1 limits OV replication and cytotoxicity due to its significant activation and enhancement of the innate antiviral type-I interferon (IFN) response in cells. Furthermore, our studies reveal that this IFN response is activated by CCN1 binding to integrin α6β1 on glioma cells, which results in the rapid and early secretion of IFNα and activation of the Jak/Stat signaling pathway.

The results from this study demonstrate a novel role for CCN1 and integrin α6β1 in regulating cellular innate defense responses against viral infection and indicate a need for patient selection based on gene expression profiling for therapeutic interventions.

Results

CCN1 gene expression is upregulated by virus but not by chemotherapy or radiation treatment

Apart from increased CCN1 gene expression in glioma cells post OV infection, its induction has also been described in H19-7 cells after treatment with etoposide, in UV irradiated human skin fibroblasts, and in HeLa cells infected with Coxsackievirus B3 virus [112, 168, 169]. Here we tested if induction of CCN1 in glioma cells infected with oncolytic HSV-1 represents a general response to glioma cell killing. Figures 18A and B show that while LN229 glioma cells infected with rHSVQ1 led to a significant increase of CCN1 mRNA, its expression was not increased after radiation or temozolomide treatment. To determine if this response could be generalized to other viruses, we examined changes in its expression in LN229 cells infected with three different viruses in addition to wild type HSV-1: Vesicular stomatitis virus (VSV), Adenovirus (Ad), and

Newcastle Disease virus (NDV). Figure 1C shows a significant induction of CCN1 in glioma cells after infection with all the viruses tested indicating that its induction may represent a general response of glioma cells to viral infection.

Extracellular CCN1 expression inhibits viral transgene expression, replication, and oncolysis

In order to investigate the impact of induction of CCN1 gene expression on viral therapy we analyzed its effect on OV gene expression in glioma cells transiently expressing

CCN1 (Gli36ΔEGFR-H2B-RFP and U251T2 cells) and in tet-inducible glioma cells (Cy-

1 and Cy-2). Figures 19A & B and Figure 20 show a significant reduction in viral transgene expression upon both transient and tet-inducible induction of CCN1 gene expression (Figure 20B & C, Table 2) and this reduction is dose-dependent (Figure 21A).

No change was observed in parental LN229 glioma cells treated with dox (Figure 21B).

To evaluate if the reduction in OV infection/replication was a result of secreted CCN1 in the ECM, we seeded U251T2 and LN229 glioma cells on CCN1/BSA coated plates prior to infection with rHsvQ1-IE4/5-Luc virus. Confocal fluorescent microscopy revealed reduced GFP positive cells when seeded on purified CCN1 compared to BSA (Figure

19C-D). Quantification of OV expressed luciferase indicated a significant reduction of viral transgene expression in cells seeded on CCN1 matrix compared to control (Figure

22A-B). To examine the role of endogenous CCN1 on OV replication, we infected glioma cells in the presence or absence of CCN1 neutralizing antibody. Figure 19E shows that inhibition of physiological levels of CCN1 enhances viral transgene expression in three different glioma cell lines. Furthermore, CCN1 mediated reduction in viral transgene expression in dox induced Cy-1 cells was rescued in the presence of CCN1 neutralizing antibody, indicating that CCN1 acting on the cell surface of glioma cells mediates the OV inhibition (Figure 19F).

We next evaluated the impact of CCN1 expression on viral replication by measuring the total amount of infectious viral particles released by Cy-1 glioma cells in vitro. Figure

23A shows a significant reduction in viral titers in cells upon CCN1 induction. Consistent with reduced virus replication, we also found a reduction in the ability of OV to kill glioma cells expressing CCN1 (Figure 23B). To test the in vivo relevance of these findings, we examined the impact of CCN1 induction on virus replication in subcutaneous tumors. Mice bearing Cy-1 tumors were fed sucrose water±dox to induce

CCN1 expression, two days prior to infection with rHSVQ1. Two days post OV infection, viral progeny was isolated and quantified. We found tumoral expression of

CCN1 led to a significant reduction viral progeny by 5.6 fold (Figure 23C); a difference which reduces viral anti-tumor efficacy in vivo (Figure 24). Collectively, these results demonstrate reduced virus replication and reduced killing of glioma cells with increased levels of CCN1 both in vitro and in vivo.

Transcript profiling uncovered CCN1 mediated induction of type-I IFN response

The ECM has been shown to influence cellular gene expression through its interaction with cell surface receptors [170]. Transcript profiling of Cy-1 glioma cells induced to express CCN1 revealed a significant induction of the anti-viral type-I IFN pathway

(Figure 25A). To identify functional networks and gene ontologies, we analyzed the upregulated gene expression data using Ingenuity Pathway Analysis software.

Investigating key biological functions linked to CCN1 gene expression, we found the main functions of genes upregulated with CCN1 were as follows: interferon signaling, 81 activation of interferon regulatory factor (IRF) by cytosolic pattern recognition receptors, and recognition of bacteria and viruses by pattern recognition receptors (Figure 25B).

Ingenuity‟s Top Network Analysis revealed a highly significant relationship between the genes differentially expressed by CCN1 induction and regulation of the antimicrobial response, inflammatory response, and infection mechanism in glioma cells (Figure 25C).

Interestingly both IPA and a detailed PubMed analysis did not reveal a published link between type-I IFN activation and CCN1 expression in ECM.

Real time quantitative PCR analysis was utilized to verify induction of a subset of the type-I IFN responsive genes involved in the antiviral defense response in these cells

(Table 3). Statistically significant induction of IFNs α and β along with downstream regulatory genes such as signal transducers and activators of transcription 1 and 2 (Stat1 and Stat2), double stranded RNA-dependent protein kinase (PKR), interferon regulatory factors (IRF) 1, 3, and 7, and 2‟,5‟-oligoadenylate synthetase 2 (OAS2) was observed.

These genes were further upregulated in Cy-1 cells expressing CCN1 following infection with rHSVQ1 suggesting an enhanced activation of the type-I IFN response by CCN1

(Table 3). Consistent with this, western blot analysis of Cy-1 cell lysates revealed increased phosphorylation of both Stat1 and Stat2 in cells induced to express CCN1 in the presence and absence of OV infection, suggesting CCN1 both activates and exacerbates the innate cellular antiviral response. No difference was found in phosphorylation status of Stat1 or Stat2 in control LN229 cells treated with dox (Figure

26A-B). 82

To test if the observed CCN1-mediated antiviral effects were dependent on activation of the type-I IFN pathway, we compared viral transgene expression in cells expressing

CCN1 in the presence of valproic acid (VPA), an HDAC inhibitor known to interfere with the transcriptional activation of type-I IFN responsive genes [66, 171]. Figure 26C shows that VPA treatment rescued CCN1 mediated inhibition of viral transgene expression.

CCN1 mediated OV inhibition is dependent on α6β1 integrin receptor-mediated IFNα secretion

CCN1 is a multifunctional, secreted ECM protein that has been shown to bind to multiple cell surface receptors including integrins αvβ3, αvβ5, and α6β1. In order to determine the cell surface receptor through which CCN1 is mediating its antiviral effects, we investigated the potential contribution of these receptors. We first evaluated the ability of cRGD (Cilengitide; αvβ3 antagonist) and LM609 (a function-blocking monoclonal antibody against αvβ3) to rescue virus inhibition in dox induced Cy-1 cells. Figures 27A-

B show that neither agent could rescue CCN1 mediated OV repression. Consistent with this result, LN229 glioma cells plated on fibronectin coated plates (a known αvβ3 activating ligand) also had no effect on OV transgene expression (Figure 27C).

We next assessed the potential role of integrin αvβ5 in CCN1 mediated OV inhibition.

Figure 27D shows that treatment of glioma cells with P1F6 (an αvβ5 function blocking

83 antibody) did not rescue CCN1 mediated reduction of OV. Moreover, activation of cell surface αvβ5 by vitronectin (a known αvβ5 activating ligand), also did not affect OV transgene expression (Figure 27E).

CCN1 binds to and activates integrin α6β1 on fibroblasts, vascular smooth muscle cells, and vascular endothelial cells [107, 172, 173]. More recently, glioblastoma stem cells were also found to express the integrin α6 chain of this heterodimeric receptor [174]. To investigate if CCN1 mediated OV inhibition was due to the activation of integrin α6β1 on glioma cells we measured the impact of function-blocking monoclonal antibodies against

α6 and β1 on viral infection. Figure 6F shows that the inhibition in OV transgene expression observed when Cy-1 cells express CCN1 is rescued in the presence of function-blocking monoclonal antibodies against either α6 or β1. Consistent with this, glioma cells plated on laminin (a known α6β1 activating ligand) leads to a significant inhibition of OV transgene expression (Figure 27G). This ability of laminin to inhibit viral transgene expression is rescued in the presence of a function-blocking antibody against integrin α6, indicating that CCN1 mediated activation of integrin α6β1 on glioma cells leads to the induction of an anti-viral defense response (Figure 27H). Figure 28A shows presence of integrin α6 on all glioma cell lines tested.

Integrin mediated cell-matrix interactions are known to play a role in protein secretion

[175-177], and among these, integrin α6β1 has been shown to mediate insulin secretion in

84 primary rat β-cells [178, 179]. In order to further delineate the underlying mechanism behind integrin α6β1 activation of the type-I IFNs we performed an ELISA looking for changes in the IFNα secretion pattern in the presence of CCN1. A time course analysis with Cy-1 cells induced to express CCN1 indicated that this protein is induced within minutes after treatment with dox (Figure 27I). Interestingly, minutes after protein induction, we observed a rapid burst in the secretion of IFNα (Figure 27J) independent of its gene expression (Figure 28B) and independent of dox treatment (data not shown). This suggests that CCN1 protein induction mediates a rapid type-I IFN secretion in glioma cells. Additionally, CCN1 mediated OV transgene inhibition was rescued by IFNα2 receptor blocking antibody indicating that secreted IFNα was required for this antiviral effect in vitro (Figure 27K). Consistent with this, CCN1 did not have an antiviral effect on U87ΔEGFR cells, which have a homozygous deletion of the entire IFNA/IFNW gene cluster and of the IFNB1 gene (Figure 28C) [180-182].

To examine if CCN1 induced by OV infection could activate this antiviral response in adjacent uninfected cells, we cultured JiEGFR cells, which are resistant to HSV infection

[183] (Figure 29), in the presence of LN229 cells infected with OV. Figure 27L-M shows increased phosphorylation of Stat1 in JiEGFR cells. More significantly, this increased phosphorylation is rescued in the presence of CCN1 neutralizing antibodies (Figure 27N) indicating that endogenous CCN1 induced after OV infection could activate Jak/Stat signaling in adjacent uninfected cells.

Collectively, these results indicate that increased expression of CCN1 in the tumor microenvironment leads to the activation of integrin α6β1 on glioma cells, resulting in the secretion of IFNα and activation of an antiviral response in the tumor microenvironment which ultimately limits OV infection and replication (Figure 30).

Discussion

CCN1 is a pleiotropic ECM molecule which binds several cell surface receptors, and modulates cell signaling events affecting diverse cellular functions including proliferation, adhesion, and migration. In the current study, we report the induction of

CCN1 gene expression in glioma cells infected with several different viruses. We further show that CCN1 in the tumoral ECM binds to cell surface α6β1 integrin receptors to activate an innate anti-viral defense response by the secretion of IFNα. Collectively, these results suggest that secretion of CCN1 upon infection orchestrates an “alarm signal” in the tumor microenvironment which activates an antiviral state in adjacent uninfected cells leading to increased resistance to viral infection/replication (Figure 6L-N). To our knowledge, this is the first report linking integrin binding and activation by extracellular

CCN1 to secretion of IFNα and activation of the antiviral type-I IFN response. Although

CCN1‟s role as a pro-inflammatory molecule is beginning to be realized [113], its effect on the type-I IFN response is quite novel and this is the first study linking an inhibitory role of CCN1 to OV therapy. This study has several implications for biological therapies and viral infections.

CCN1‟s role in tumor biology has been extensively studied, and depending on the tissue type has been found both pro- and anti-tumorigenic [184]. Apart from negatively modulating the cellular response to OV therapy, the expression of CCN1 protein in breast, prostate, and ovarian cancer correlates with a poor prognosis [96, 160, 185].

Conversely, its expression in lung, endometrial, and gastric cancer has been associated with a better prognosis and outcome [99-101]. Though the reason underpinning CCN1‟s opposing effect in different tissue has not been elucidated, it may depend in part on the context in which CCN1 is expressed differing by the presence of co-activators and repressors, and the receptor expression profiles present in different tissues.

Here we show that CCN1 activates a type-I IFN pro-inflammatory cascade in glioma cells by binding to and activating the α6β1 integrin receptor and inducing secretion of

IFNα. We show that CCN1 expression not only upregulates the type-I IFNs α and β, but also several downstream mediators of the type-I IFN response known to play key roles in the cellular antiviral defense response such as PKR and OAS [186, 187]. These results suggest that while expression of CCN1 leads to increased angiogenesis and invasion in the tumors, it also interferes with oncolytic viral therapy and inhibition of this pathway may provide opportunities to enhance OV anti-tumor efficacy.

Recently, integrin α6 has been recognized as an enrichment marker for glioblastoma stem cells (GSCs) [174], and was found to be coexpressed with CD133 (a widely accepted

87 glioma stem cell marker) in GBM biopsies. Apart from increased tumorigenicity, glioma stem cells have been shown resistant to both radiation and chemotherapy [188]. In U87 glioma cells, it has been shown that stable cell surface expression of integrin α6β1 leads to both enhanced proliferation and decreased apoptosis in vitro and in vivo [189]. The results from our study indicate that CCN1 mediated activation of integrin α6 contributes to the reduced efficacy of viral oncolytic therapy and it will be interesting to understand how the CCN1-integrin α6 interaction plays a role in glioma therapeutic resistance.

In conclusion, this is the first study to reveal the effect of a secreted matricellular integrin binding protein on the initiation of an innate type-I IFN cellular defense response to virus infection. This study suggests that therapeutic interventions which inhibit the CCN1- integrin α6 interaction may sensitize glioma to chemo and radiation therapies and viral oncolysis. Future studies will evaluate the extent to which expression of CCN1 and/or integrin α6 receptor on tumors can serve as a predictor of patient response to oncolytic viral therapy.

Materials and Methods

Cell lines and viruses

Human LN229, U343, Gli36ΔEGFR-H2B-RFP, U251T2, and U251T3 glioma cell lines are maintained as described [110]. EGFR-transduced baby hamster kidney JiEGFR cells are maintained as described [183]. Tet-regulated CCN1 expressing clones Cy-1 and Cy-2

88 were established as described [140]. For radiation studies, cells were irradiated at 10gy, using RS-2000 Biological Irradiator. HSV-1-derived OVs, rHSVQ1, rHsvQ1-IE4/5-Luc, and ENVE, have been previously described [22, 166, 190].

Animals

All animal experiments were performed in accordance with the Subcommittee on

Research Animal Care of The Ohio State University guidelines. Six to eight week old female athymic nude mice were used for all studies.

For CCN1 effects on viral progeny, mice were implanted subcutaneously with 4x106 Cy-

1 or LN229 cells into the rear flank and monitored for tumor growth. When tumors reached 100mm3 mice were randomized and fed sucrose±dox (1mg/ml) in drinking water. 2 days post dox treatment initiation, mice were administered rHSVQ1 (1x106 pfu) by direct intratumoral injection and sacrificed 48h post-infection; tumors were harvested for the number of infectious virus particles and analyzed by a standard plaque assay.

For effects of viral progeny on tumor cell growth, mice were implanted subcutaneously with 1.5x107 U251T3 cells into the rear flank. When tumors reached an average of

250mm3 mice were administered ENVE virus by direct intratumoral injection with the indicated dose. Tumor volume was calculated using the following formula: volume=0.5LW2 as described [148].

Antibodies and Reagents

Reagents used in this study were obtained from the following sources: Cilengitide

(Merck), Valproic acid & Laminin (Sigma), Fibronectin (Calbiochem), Vitronectin

(Promega), CCN1 protein (Cell Sciences). Antibodies were obtained from the following sources: CCN1 (Novus Biologicals), GAPDH & ITGA6 (Abcam), STAT1 & PSTAT1

(Cell Signaling), STAT2, PSTAT2, LM609, P1F6, GoH3, P5D2 & IFNαR2 (Millipore), sheep anti-mouse HRP (GE Healthcare), goat anti-rabbit HRP, IgG negative control

(DAKO). IFNα levels were measured from cell supernatants using PBL Interferon

Verikine Human IFNα ELISA Kit.

Real Time-PCR

RNA was isolated using RNeasy Mini Kit (Qiagen). For Quantitative Real-Time PCR, cDNA was made using Superscript First-Strand Synthesis System (Invitrogen). Real time continuous detection of PCR product was achieved using Sybr Green (Applied

Biosystems). GAPDH was used as an internal control. Primers were designed using the

Primer Express Program (Applied Biosystems) (Table 4).

Microarray

Total RNA from Cy-1 cells incubated±dox for 24h was isolated using RNeasy Mini Kit

(Qiagen). Samples were then submitted to The Ohio State University Microarray Shared

Resource Center for microarray analysis using the Affymetrix GeneChip Analysis. The 90 microarray data from this publication have been submitted to the GEO database

(accession number GSE29384).

Statistical Analysis

Results are presented as mean values±standard error of the mean (SEM). Statistical analysis was carried out by unpaired Student‟s t-test using GraphPad Prism® 5.01 software. P values <0.05 were considered statistically significant. Affymetrix GeneChip was used for gene expression study. Signal intensities were quantified by Affymetrix software.

Figures and Tables

Figure 18: CCN1 gene expression is upregulated by virus but not by chemotherapy or radiation therapy.

Real time PCR analysis of CCN1 gene expression in LN229 glioma cells treated with (A) rHSVQ1 at MOI=1, (B) 10gy radiation or 0.5mM temozolomide, (C) Vesicular Stomatitis Virus (ts 45) at MOI=0.1, Adenovirus (type 5) at MOI=100, Newcastle Disease Virus at MOI=2, or WtHSV-1 at MOI=1 24h after treatment. Data shown are the mean CCN1 gene expression relative to endogenous GAPDH and error bars are standard error of the mean of at least three replicates and represent at least three independent experiments. ns=not significant, *P<0.05, ***P<0.001

Figure 19: Extracellular CCN1 expression inhibits viral transgene expression, replication, and cell killing. (A) U251T2 glioma cells and Gli36ΔEGFR-H2B-RFP glioma cells transiently transfected with pcDNA3.1myc-hisB+CCN1 (CCN1) or pcDNA3.1myc-hisB+empty (control), 24h prior to being infected with rHsvQ1-IE4/5-Luc (MOI=1). 24h post infection, virus encoded luciferase activity (relative light units) was measured in infected cell lysates. Data shown are %RLU/mg±SEM relative to control. (B) OV encoded luciferase activity (relative light units: RLU) of Cy-1 tetracycline-inducible glioma cells treated±dox for 24h, prior to infection with rHsvQ1-IE4/5-Luc (MOI=1). Results presented are %RLU/mg±SEM relative to uninduced cells, 6 and 24h post-infection. (C & D) Confocal fluorescent and bright field images of GFP positive infected (C) U251T2 and (D) LN229 glioma cells seeded on plates coated with CCN1/BSA (5ug/ml) infected 93 with rHsvQ1-IE4/5-Luc. (E) Inhibition of endogenous CCN1 increases OV transgene expression in three different glioma cell lines. Quantification of OV encoded luciferase activity of U251T3, LN229, and Gli36∆EGFR-H2B-RFP glioma cells infected with rHsvQ1-IE4/5-Luc±CCN1 mAb measured 24h post infection. Data shown are %RLU/mg±SEM relative to control. (F) Rescue of CCN1 mediated viral inhibition by CCN1 neutralizing monoclonal antibody. Cy-1 glioma cells treated±dox were infected with rHsvQ1-IE4/5-Luc±CCN1 mAb. Virus encoded luciferase activity was quantified 24h post infection. Results presented are the %RLU/mg±SEM relative to control of at least three different experiments. Scale Bar = 100um, ns=not significant, *P<0.05, **P<0.01, ***P<0.001

Figure 20: CCN1 inhibition of viral transgene expression & CCN1 expression

(A) OV encoded luciferase activity (relative light units: RLU) of Cy-2 tetracycline- inducible glioma cells treated ± dox for 24h, prior to infection with rHsvQ1-IE4/5-Luc 94

(MOI=1). Results presented are %RLU/mg ± SEM relative to uninduced cells, 6 and 24h post-infection. (B) U251T2 and Gli36ΔEGFR-H2B-RFP cells transiently transfected with pcDNA3.1myc-hisB+CCN1 (CCN1) or pcDNA3.1myc-hisB+empty (control) were analyzed for changes in CCN1 mRNA expression by semi-quantitative PCR as described in methods. (C) Western blotting analysis of Cy-1 cells ±dox compared to LN229, U251T3 and GBM169 glioma cells ±rHSVQ1 showing similar CCN1 induction.

Figure 21: Doxycycline dose response & effect on LN229 glioma cells. (A) OV encoded luciferase activity (relative light units: RLU) of Cy-1 cells treating with increasing concentrations of dox for 24h, prior to infection with rHsvQ1-IE4/5-Luc (MOI=1). (B) OV encoded luciferase activity of LN229 treated ± dox for 24h, prior to

95 infection with rHsvQ1-IE4/5-Luc (MOI=1). Results presented are %RLU/mg ± SEM relative to uninduced cells, 24h post infection. ns=not significant

Figure 22: Quantification of virus expressed luciferase

(A) U251T2 and (B) LN229 glioma cells seeded on CCN1/BSA coated plates (5ug/ml) for 16h prior to infection with rHsvQ1-IE4/5-Luc. Data shown are %RLU/mg ± SEM relative to control, 24h post infection. *P<0.05, ***P<0.0001

Figure 23: CCN1 in the ECM limits OV replication and cytotoxicity

(A) Total infectious virus particles obtained 24h post-infection of Cy-1 or control LN229 cells±dox for 24h prior infection with rHSVQ1 (MOI=2.5). Data shown are fold change in number of virus particles±SEM between control and dox treated cells. (B) Percent surviving cells in infected Cy-1 cells (±dox) relative to uninfected (±dox) cells on days 1, 2, and 3 post infection with rHSVQ1 (MOI=1). Data shown are percent cell survival in infected Cy-1 cells±dox at different time points relative to uninfected cells (day 0). (C) Reduced viral replication in tumors induced to express CCN1. Mice implanted with Cy-1 or LN229 cells fed sucrose±dox were infected intra-tumorally with rHSVQ1 as described in methods. 48h post infection the number of virus particles in each tumor was measured by a standard plaque assay. Data shown are fold change in number virus particles±SEM between control and dox treated cells. ns=not significant, *P<0.05, ***P<0.001.

Figure 24: Survival data of athymic nude mice implanted subcutaneously with U251T3 1.5x107 glioma cells following ENVE virus therapy.

Mice with U251T3 subcutaneous tumors (250 mm3) were treated with 5x104 pfu or 1x105 pfu plaque-forming units (pfu) of ENVE virus on days 1 and 3 (n = 10/group) by intratumoral injection. Mean tumor growth of mice is shown as a function of time.

Figure 25: Transcript profiling of Cy-1 cells induced to express CCN1:

(A) Heat map representing hierarchical clustering of a subset of the differentially regulated genes, plotted using the log 2 values of the genes with p<0.05 (unpaired t test) that are involved in the type-I IFN response. Each column represents a sample plotted in triplicate and each row in the heat map represents a gene that is differentially regulated in that particular comparison of samples. The color scale represents the degree of expression of the gene, green being the lowly expressed (below -3.0) and red being the highly expressed (above +3.0) genes in the sample sets, with black as the center of the scale at „0‟. (B) Biological functions associated with genes significantly changed by the induction of CCN1. The significance of each canonical pathway is determined based upon the p- values determined using right tailed Fisher‟s exact test and with a threshold less than 0.05. The top 9 possible canonical pathways of the genes induced by CCN1 induction are shown. The ratio of number of genes in a given pathway satisfying the cutoff and total number of genes present in that pathway was determined by IPA. (C) Ingenuity Pathway Analysis (IPA) generated pathway associated with type-I interferon responsive genes expressed upon induction of CCN1. Solid lines represent a direct interaction; dotted lines represent an indirect interaction.

Figure 26: Functional activation of a type-I IFN response by CCN1 mediates OV inhibition.

(A & B) Representative western blots of (A) Cy-1 and (B) LN229 cells treated±dox at 0, 4, and 12h post infection with rHSVQ1 probed for phosphorylated Stat1 and Stat2 in cells in response to CCN1 induction. Total Stat1, Stat2, and GAPDH protein levels were utilized as controls. (C) Cy-1 cells±dox were incubated with valproic acid (VPA) for 16h prior to infection with rHsvQ1-IE4/5-Luc (MOI=1). Virus expressed luciferase activity was quantified. Data shown are %RLU/mg±SEM in dox treated cells relative to uninduced cells. ns=not significant, *P<0.05

100

Figure 27: CCN1 mediated OV inhibition is dependent on its interaction with cell surface α6β1 integrin independently of its ability to bind to αvβ3 and αvβ5.

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(A&B) Dox induced Cy-1 cells were infected with rHsvQ1-IE4/5-Luc (MOI=0.1), in the presence of (A) Cilengitide (cRGD, 50ug/ml) an αvβ3 antagonist or (B) LM609 a function blocking antibody against anti-αvβ3 (50ug/ml). Viral transgene expression was determined by measuring luciferase activity. Data shown are %RLU/mg relative to control treated cells. (C) LN229 glioma cells were seeded on fibronection (a known αvβ3 agonist) coated plates (5ug/ml) or control plates and infected with rHsvQ1-IE4/5-Luc at MOI=0.1 for 24h. Viral transgene expression was determined by luciferase quantification, normalized to mg protein and represented as % RLU/mg relative to control cells. (D) Dox induced Cy-1 cells were infected with rHsvQ1-IE4/5-Luc (MOI=0.1), in the presence of P1F6, a function blocking antibody against anti- αvβ5 (50ug/ml). Viral transgene expression was determined by luciferase quantification, normalized to mg protein and represented as %RLU/mg relative to control cells. (E) LN229 glioma cells seeded on control or vitronectin (a known agonist for integrin αvβ5) coated plates (5ug/ml) were infected with rHsvQ1-IE4/5-Luc at MOI=0.1 for 24h. Viral expressed luciferase activity was measured and is represented as %RLU/mg relative to control cells. (F) Dox induced Cy-1 cells were infected with rHsvQ1-IE4/5-Luc (MOI=0.1), in the presence of function blocking antibody GoH3 against integrin α6 or P5D2 against integrin β1 (50ug/ml). Viral expressed luciferase activity was measured and is expressed as %RLU/mg relative to un-induced cells. (G) LN229, Gli36ΔEGFR-H2B- RFP, and U251T2 glioma cells were seeded on laminin (a known agonist for α6β1) coated plates (5ug/ml) or non-coated plates and infected with rHsvQ1-IE4/5-Luc at MOI=0.1 for 24h. Viral expressed luciferase activity was measured and is expressed as %RLU/mg relative to control cells. (H) LN229 glioma cells seeded on laminin coated plates (5ug/ml) were infected with rHsvQ1-IE4/5-Luc in the presence or absence of function blocking antibody GoH3 against integrin α6. Viral expressed luciferase activity was quantified, and represented as %RLU/mg relative to control cells. Cy-1 cells were incubated in the presence of dox and harvested at the indicated time points. (I) Representative western blot of Cy-1 cells treated±dox for two minutes indicating CCN1 protein induction at a very early time point. GAPDH protein level was utilized as a control. (J) Supernatants were concentrated and analyzed for changes in IFNα secretion relative to non-treated control cells by ELISA. Data are shown as the mean±SEM of at least three replicates and represent at least three different experiments. (K) Cy-1 cells±dox were incubated with an antibody against the IFNα receptor chain 2 (50μg/ml) prior to infection with rHsvQ1-IE4/5-Luc (MOI=1). Virus expressed luciferase activity (RLU) was quantified. Data shown are %RLU/mg±SEM in dox treated cells relative to uninduced cells. (L) Schematic of experimental setup for 6M-N, showing the culture of JiEGFR cells with infected or uninfected LN229 cells. (M) Western blot for Stat1 phosphorylation of JiEGFR cells, cultured in the presence of secreted medium from infected or uninfected LN229 cells. Total Stat1 and GAPDH were used as controls. (N) Western blot for Stat1 phosphorylation of JiEGFR cells, cultured in the presence of secreted medium from infected LN229 cells, cultured in the presence or absence of CCN1 neutralizing antibodies. Total Stat1 and GAPDH were used as controls. „=minute, ns=not significant, *P<0.05, **P<0.01, ***P<0.001

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Figure 28: ITGA6 expression, IFNα expression, & CCN1 effect on U87∆EGFR glioma cells

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(A) Western blot analysis showing integrin α6 receptor (ITGA6) expression in a panel of cell lines, compared to GAPDH. (B) Total cellular RNA was isolated and IFNα gene expression relative to endogenous GAPDH was evaluated by quantitative real-time PCR. „=minute, h=hour, ns=not significant. (C) U87ΔEGFR glioma cells, deleted for IFN (chromosome 9p22), were transiently transfected with pcDNA3.1myc-hisB+CCN1 (CCN1) or pcDNA3.1myc-hisB+empty (control), 24h prior to being infected with rHsvQ1-IE4/5-Luc (MOI=1). Twenty four hours post infection, viral transgene expression was determined by measuring the amount of virus expressed luciferase in infected cell lysates. Data shown are percentage relative light units per milligram of total protein ± SEM (%RLU/mg) relative to control. ns=not significant.

Figure 29: Confocal fluorescent and bright field images of GFP positive infected LN229 glioma cells and JiEGFR HSV-resistant cells infected with rHsvQ1 MOI=5.

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Figure 30: Model for CCN1 mediated OV inhibition. Initial infection with oncolytic HSV-1 results in the induction and secretion of CCN1 protein [110]. Secreted CCN1 in the extracellular matrix then binds cell surface receptor integrin α6β1. Binding and activation of the integrin α6β1enables the secretion of type I IFNα. Secreted IFNα then binds to interferon α receptor 1 and 2, resulting in the dimerization, activation, and subsequent phosphorylation of Stat1 and Stat2 proteins within the cytoplasm. Upon nuclear translocation, IRF9 associates with the heterodimer forming the heterotrimeric complex ISGF3, which then binds ISREs initiating transcription of IFNα/β-regulated genes such as: PKR, OAS1 and 2, STAT1 and 2, and IRF1 and 7. The expression of these genes sets up an antiviral response in cells and thus limits oncolytic viral efficacy.

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Table 2: CCN1 fold induction

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Table 3: CCN1 induces expression of type-I interferon response gene in the presence and absence of OV Abbreviations: IFN, Interferon; STAT, signal transducer and activator; IRF, interferon regulatory factor; PKR, double-stranded RNA-dependent protein kinase; OAS, 2‟5‟- oligoadgenylate synthetase; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; Dox, doxycycline.

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Table 4: Primer Sequences Abbreviations: IRF, interferon regulatory factor; OAS, 2‟5‟-oligoadenylate synthetase; PKR, double stranded RNA-dependent protein kinase; STAT, signal transducer and activator; IFN, Interferon; CCN1, Cysteine-rich 61; GAPDH, glyceraldehyde-3- phosphate dehydrogenase

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Chapter 5: Inhibition of CCN1 enhances oncolytic virus therapy by reducing macrophage

mediated viral clearance

Introduction

Glioblastoma multiforme (GBM) is a destructive cancer of the central nervous system, leaving patients with less than a 15 month median survival despite treatment with radiation therapy and chemotherapy [1]. Novel therapeutic options are therefore necessary to combat this disease. Oncolytic herpes simplex viruses (OV) depend on an initial infection followed by subsequent tumor cell lysis and represent a new biological therapy currently being evaluated in patients for safety and efficacy [36, 40, 191, 192].

Though efficacy has been shown in a laboratory setting, the innate immune response to virus infection presents a barrier to OV propagation in vivo and hence reduces efficient tumor cell destruction [62, 63, 193, 194]. A significant infiltration of microglia/macrophages and NK cells to the tumor site has been shown to increase virus clearance and their depletion enhances viral replication and oncolysis [64, 65, 195].

Efforts to understand changes in the tumor microenvironment which enhance these responses will lead to a better understanding of how to attain the most of this promising therapeutic strategy.

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We have previously described the induction of Cysteine-rich 61 (CCN1) in the tumor microenvironment following OV therapy [110]. CCN1 is a secreted protein primarily found within the extracellular matrix [91]. First identified as a regulator of vascular development [172, 173], CCN1‟s role in cell migration and inflammation is beginning to be understood [116, 157, 158, 196-200]. For example, CCN1 is upregulated during chronic states of inflammation such as in patients with Crohn‟s disease and ulcerative colitis [201], and more recently CCN1 was shown to activate a proinflammatory genetic program in murine macrophages characteristic of the classically activated M1 phenotype

[114]. We have recently demonstrated that CCN1 orchestrates a robust intracellular antiviral response to infection by interacting with integrin α6β1 and subsequently inducing a type I interferon (IFN) response in glioma cells [165]. However the relationship between CCN1 induction following OV infection of glioma cells and its impact on macrophage infiltration and activation following OV therapy for glioma is not understood. Here we investigated the impact of CCN1 on macrophage mediated viral clearance. CCN1 neutralization led to increased OV persistence and reduced macrophage infiltration in tumors in mice. CCN1 increased the migratory potential of macrophages towards infected glioma cells both directly and indirectly by increasing macrophage chemotactic factor secretion from glioma cells. Further it also enhanced the pro- inflammatory activation towards infected glioma cells, and increased virus clearance in co-cultures of infected glioma cells with macrophages. The results from this study indicate a novel role for CCN1 and specify a need for targeting CCN1 to improve OV propagation in vivo.

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Results

CCN1 increases macrophage infiltration toward oncolytic HSV-1 infected tumors in vivo

In order to test if function neutralizing CCN1 antibodies could improve OV persistence in vivo, mice bearing subcutaneous tumors were treated with a function neutralizing anti-

CCN1 antibody [202] or control IgG serum. Mice were then injected intra-tumorally with rHsvQ1-IE4/5-Luc and virus encoded luciferase was monitored by IVIS imaging. Figure

31a shows increased luciferase activity in tumors of all mice treated with anti-CCN1 antibody compared to control IgG treated mice, suggesting increased OV in mice treated with function neutralizing CCN1 antibody. Consistent with this, immunohistochemistry for HSV-1 showed increased virus in tumor sections derived from animals treated with anti CCN1 (Figure 31b). To assess the impact of CCN1 on macrophages, tumors were harvested and stained for the macrophage marker CD68. Figure 31c shows reduced staining for CD68 in mice treated with anti-CCN1 antibody, indicating that increased virus propagation in vivo observed after inhibition of CCN1 correlated with a reduction in macrophage infiltration following OV therapy.

CCN1 increases macrophage migration toward OV infected glioma cells

To directly test if OV induced CCN1 is involved in macrophage migration toward OV infected glioma cells we measured the chemotaxis of RAW264.7 macrophage cells in vitro towards uninfected or infected cancer cells. Consistent with previous observations of increased macrophage infiltration in tumors treated with OV [64, 65], we found a significant increase in macrophage migration towards infected LN229 glioma cells

111 compared to uninfected cells (Figure 32). More importantly this increased migration of macrophages towards infected cells was rescued by neutralizing CCN1 antibody indicating a significant role for OV induced CCN1 in macrophage migration toward OV infected glioma cells (Figure 32).

CCN1 induces MCP-1 and MCP-3 gene expression by infected glioma cells.

We tested if secreted endogenous CCN1 acts on glioma cells to induce the expression of macrophage chemotactic factors resulting in increased macrophage migration. A significant induction of monocyte chemotactic proteins 1 and 3 (MCP-1 and MCP-3) was observed in cells infected with OV (Figure 33a). More importantly MCP-1 and MCP-3 induction by OV was partially rescued when glioma cells were treated with a function neutralizing CCN1 antibody (Figure 33a), indicating that CCN1 played a significant role in the induction of these chemokines.

We have previously shown that CCN1 binds to integrin α6β1 on glioma cells to activate an innate intracellular type 1 IFN antiviral defense response [165]. To test if the induction of MCP-1 and MCP-3 by glioma cells involved CCN1 binding and activation of integrin

α6β1, we used glioma cells knocked out for integrin α6 (Figure 33b). KD4 is a lentivirus which delivers shRNA directed against integrin α6 [174]. Consistent with our previous studies, knock down of integrin α6 with KD4 resulted in a significant reduction in glioma cell produced IFNα and IFNβ along with an increase in viral transgene expression

(Figures 33c, d). However, quantitative real time PCR analyses of changes in gene

112 expression revealed that levels of MCP-1 and MCP-3 induced by OV infection remained

-/- un-changed in α6 glioma cells compared to control, indicating that CCN1 mediated induction of MCP-1 and MCP-3 is independent of its interaction with integrin α6β1

(Figure 33e).

CCN1 directly effects macrophage migration by binding integrin αMβ2

The above results show that CCN1 can induce glioma cells to increase secretion of MCP-

(Figures 34a, b).

To identify the receptor responsible for CCN1 induced macrophage chemotaxis towards infected glioma cells we measured the migration of macrophages treated with the various function-neutralizing antibodies towards infected glioma cells using a transwell assay.

Figure 34c shows that treatment of macrophages with blocking antibodies against integrins αM and β2 partially rescued the increased migration of macrophages towards infected cells.

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These results indicate that CCN1 influences macrophage chemotaxis towards infected cells by both directly increasing macrophage migration and also by acting on glioma cells to induce the secretion of macrophage attracting chemokines.

Enhanced activation of macrophages by CCN1 leads to increased viral clearance

Collectively these results show that CCN1 produced in tumor microenvironment increases the chemotaxis of macrophages to the site of infection by both directly acting on macrophage cell surface αMβ2 receptors and also by acting on glioma cells to increase the secretion of macrophage chemotactic proteins such as MCP-1 and MCP-3.

To test if CCN1 expression also affected macrophage response towards infected glioma cells we co-cultured RAW264.7 murine macrophages with rHSVQ1 infected human glioma cells overexpressing CCN1 (Cy-1) or with rHSVQ1 infected control wild type

LN229 human glioma cells. Using murine specific primers we analyzed changes in macrophage gene expression 12 hours following infection. In figure 35 we show macrophages co-cultured with infected glioma cells overexpressing CCN1 have a significant induction in IL-1β, MCP-1, IP-10, MCP-3, and IFNγ whose expression indicates classical macrophage activation [204-207].

To test if CCN1 affected macrophage-mediated oncolytic virus clearance we co-cultured infected glioma cells with macrophages in the presence or absence of CCN1 function- blocking antibodies and measured viral ICP4 gene copy. Addition of macrophages to

114 infected glioma cells significantly reduced viral ICP4 expression indicative of macrophage-mediated viral clearance. Interestingly, however, pre-incubation of macrophages with an anti-CCN1 antibody significantly rescued the macrophage- mediated viral clearance (figure 36a). We have previously shown that CCN1 activates type I IFN signaling in glioma cells, leading to reduction in viral replication [165]. To discriminate between the direct effects of CCN1 on glioma cells and the impact of CCN1 on macrophage mediated virus clearance we used KD4 to knock down integrin α6 on glioma cells. Figure 36b demonstrates a significant reduction in viral ICP4 gene expression when both non-target transfected cells (NT) and integrin α6KD cells (KD4) were co-cultured with macrophages, indicating CCN1‟s effect on macrophage mediated viral clearance is independent of its effect on induction of type I IFN signaling in glioma cells.

Lastly, we investigated if CCN1‟s interaction with integrin αMβ2 led to an increase in macrophage-mediated viral gene copy reduction. Using function-neutralizing antibodies, we show in figure 36c when macrophages are pre-incubated with neutralizing antibodies against integrin αMβ2 prior to culturing with infected glioma cells there is a significant inhibition in the macrophage-mediated viral gene copy reduction.

Collectively, these results indicate that increased CCN1 expression in the tumor microenvironment following OV therapy enhances macrophage infiltration to the tumor bed, and ultimately reduces the efficacy of OV therapy by increasing viral clearance.

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Discussion

We have previously shown rapid and dose dependent induction of CCN1 in the glioma microenvironment following OV infection [110]. Increased levels of CCN1 induced by

OV can then bind to integrin α6β1 on glioma cells and activate the innate intracellular antiviral type I IFN responsive pathway resulting in reduced virus replication in vitro and in vivo [165]. In this study, we examined the relationship between OV induced CCN1 and its effect on macrophage-mediated virus clearance in vitro and in vivo. Here we found that CCN1 induced upon OV infection of glioma cells increased chemotaxis of macrophages towards infected cells, by both directly stimulating migration of macrophages and also by inducing the secretion of macrophage chemotactic factors from infected glioma cells. Additionally, we found that CCN1 enhances the pro-inflammatory activation of macrophages leading to increased macrophage-mediated virus clearance. To our knowledge, this is the first study linking CCN1 mediated induction of macrophage migration and pro-inflammatory activation to a reduction in OV propagation. This study shows that CCN1 can enhance the innate macrophage-mediated antiviral immune response to oncolytic virus infection by directly interacting with effector macrophages and by enhancing chemokine production by infected glioma cells.

CCN1 is a secreted extracellular matrix molecule whose emerging role as a pro- inflammatory molecule is being uncovered [208]. While CCN1 is well known as an angiogenic inducer and has been shown to be involved in migration of various different cancer cells its role in modulating immune responses if just beginning to be explored. In

116 human fibroblasts, CCN1 induces high levels of the reactive oxygen species (ROS) H2O2 and unmasks the cytotoxic potential of TNF-α by binding integrins αvβ5, α6β1, and HSPG syndecan-4 leading to ROS induction independent of NFkB [199], suggesting a pro- apoptotic synergism between TNFa and CCN1. In gastric cancer AGS cell lines, CCN1 enhances the expression of chemokine receptors CXCR1 and CXCR2 [158] implying its role in IL-8 mediated chemotaxis. CCN1 has also been shown to enhance expression of

MCP-1 in osteoblastic cells [209], and induce the release of multiple growth factors and chemokines in CD34+ progenitor cells [157]. Additionally, Bai et al recently identified

CCN1 as a regulator of pro-inflammatory gene expression in murine macrophages [114].

In this report, treatment of macrophages with purified CCN1 for 1, 6, and 24 hours, resulted in enhanced inflammatory gene expression, with expression peaking at 6 hours.

Here we examined the impact of secreted endogenous CCN1 from infected glioma cells on macrophage function. Our studies show increased production and secretion of CCN1 in the tumor microenvironment enhances macrophage migration towards the site of inflammation by CCN1 acting directly on macrophages through integrin αMβ2, and also by CCN1 acting on glioma cells leading to an increased production of MCP-1 and MCP-

3 macrophage chemotactic factors. We show that in addition to increasing macrophage migration toward virally infected tissue, CCN1 also enhances the activation status of effector cells, ultimately resulting in reduced viral replication in vitro and in vivo. These results show for the first time that CCN1 can enhance virus clearance in tumors and suggest it plays a significant role in pathogen clearance.

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Chemokines regulate leukocyte trafficking and play a key role in orchestrating the host defense response to HSV-1 infection. Studies examining the effects of macrophage depletion on wild type or oncolytic HSV-1 infection have indicated a significant role for macrophages in viral clearance [65, 210]. In response to local inflammation, peripheral circulating monocytes, intravasate into tissues via a multi-step process involving rolling, adhesion and migration, and finally diapedesis, [211]. Following transendothelial migration, monocytes pass across the endothelial basement membrane and into the tissues

[212] and integrins β1 and β2 on the effector cell surface interact with ECM proteins during this final step of migration [213]. Here we show purified CCN1 protein coated on transwells increases the migratory capacity of monocytes and macrophages implying

CCN1 as a novel ECM mediator of macrophage transendothelial migration toward sites of inflammation.

Interestingly, a recent study showed CCN1 expression in animal models of cardiomyopathy led to a reduction in disease severity due to the suppression of cardiac immune cell infiltration, while having no effect on chemokine or cytokine secretion

[200]. However, in vitro they show that while long-term exposure to CCN1 (24 hours) diminished immune cell migration, in agreement with our data presented here, short-term exposure enhanced effector cell migration. Along with this, pre-incubation with either

CCN1 or cRGD was found to abrogate CCN1, MCP-1 and SDF-1α induced migration.

These data indicate that prolonged exposure to CCN1 may be resulting in a negative feedback mechanism and also that CCN1‟s interaction with one of the αv- integrins is

118 mediating its effect. In the context of oncolytic virus therapy for glioma, this study implies that long term exposure to CCN1 may confer resistance to the adaptive antitumor immune response. Here we show CCN1‟s interaction with integrin αMβ2 mediates its effect on macrophage migration and activation, independently of integrins αVβ3 and αVβ5.

Taken together these two studies reveal the very real effect of tissue specificity in relation to CCN1 function.

In conclusion, this is the first study to investigate the effect of CCN1 on the inhibition of oncolytic virus propagation in the context of infiltrating immune cells. We show CCN1 reduces OV efficacy by both increasing macrophage infiltration and increasing macrophage pro-inflammatory activation. This study suggests that therapeutic interventions which inhibit the CCN1-integrin αM interaction may sensitize glioma cells to viral oncolysis. Future studies will evaluate the effect of CCN1 inhibition on oncolytic virus therapy in patients with glioblastoma.

Materials and Methods

Cell lines and viruses

Human LN229, U251T3, and Cy-1 [165] glioma cell lines were maintained in

Dulbecco‟s modified minimal essential medium (DMEM) supplemented with 2% fetal bovine serum, 100U/ml penicillin, and 100ug/ml streptomycin. We used rHSVQ1 and rHSVQ1-IE4/5-Luc, HSV-1 derived recombinant oncolytic viruses, which are disrupted in the UL39 locus and deleted for both copies of the γ34.5 gene; rHSVQ1-IE4/5-Luc also 119 contains the luciferase transgene under the HSV-1 IE4/5 promoter [22]. Viral stocks were generated in Vero African green monkey kidney cells (American Type Culture

Collection, Manassas, VA) as previously described [190].

Animals

All animal experiments were performed in accordance with the Subcommittee on

Research Animal Care of the Ohio State University guidelines. Six- to eight-week-old female athymic nu/nu mice (Charles River Laboratories, Frederick, MD) were used for all tumor studies. Mice were anesthetized and injected into the rear right flank with

1.5x107 U251T3 glioma cells. When tumors reached an average size of 200mm3 mice were injected intraperitoneally with control anti-rabbit IgG or anti-CCN1 antibody for 5 days. On the third day, mice were injected intratumorally with rHSVQ1-IE4/5-Luc at

1x106 pfu. On day 6 tumors were excised, fixed in 4% paraformaldehyde, and dehydrated in 30% sucrose.

Antibodies and Reagents

Reagents used in this study were obtained from the following sources: Pure recombinant

CCN1 protein (Cell Sciences, Canton, MA), D-Luciferin (Caliper Life Sciences,

Hopkinton, MA), lentiviral constructs KD4 and NT were kindly provided by Dr. Justin

D. Lathia (Cleveland Clinic, Cleveland, OH). Antibodies were obtained from the following sources: For in vivo imaging: Rabbit IgG & anti-CCN1 (Santa Cruz, Santa

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Cruz, CA); for function-inhibition assays: anti-CCN1 (Novus Biologicals, Littleton, CO),

[M1/70] anti-CD11b (Abcam, Cambridge, MA), rat anti-mouse integrin αv, mouse anti- human integrin αvβ3, mouse anti-human integrin αvβ5, mouse anti-human integrin β2

(Millipore, Billerica, MA), rat anti-mouse integrin β2 (BD Pharmigen, San Jose, CA), hamster anti-mouse integrin β3 (AbD Serotec, Raleigh, NC), anti-integrin β5

(eBioscience, San Diego, CA), for western blotting: anti-integrin α6 & anti-GAPDH

(Abcam, Cambridge, MA), Amersham ECL anti-mouse IgG HRP (GE Healthcare,

Pataskala, OH), poly-clonal goat anti-rabbit IgG (Dako, Carpenteria, CA); for immunohistochemistry: rat anti-mouse CD31 (BD Pharmingen, San Jose, CA), polyclonal rabbit anti-herpes simplex virus 1 (Dako, Carpenteria, CA), rat anti-mouse

CD68 (AbD Serotec, Raleigh, NC), peroxidase conjugated donkey anti-rabbit & biotin- conjugated goat anti-rat (Jackson ImmunoResearch Laboratories, West Grove, PA).

Cell Migration Assay

Cell migration of RAW264.7 murine macrophages and THP-1 human monocytes was evaluated using a 24-well chemotaxis chamber equipped with a polycarbonate filter with

5 um pores (Costar, Corning, NY). For CCN1 direct effect on migration, transwells were coated with CCN1 protein or BSA. Serum starved cells were plated in the upper chamber and left to migrate for 6 hours. For THP-1, cells that traversed the membrane were counted by hemacytometer. For RAW264.7 cells, following incubation cells that traversed the membrane were fixed and stained with crystal violet; non-migrated cells were removed with a cotton-tipped applicator. Cells were quantified by averaging the cell 121 count of 5 different view fields at 20x magnification. For CCN1 inhibition experiments,

LN229 glioma cells were plated in the bottom chamber and infest for 30 minutes. Cells were incubated for 4 hours after which serum starved RAW264.7 cells were plated in the top chamber in the presence of IgG, or one of the various antibodies, and left to migrate for 6 hours. Cells were quantified as stated above.

Real time-PCR

Cells were harvested with 0.5% trypsin-EDTA, centrifuged for 5 min at 2,000rpm, and cell pellets frozen. Cell pellets were homogenized using a QIAshredder (Qiagen,

Valencia, CA) and RNA was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA).

Real time continuous detection of PCR product was achieved using Sybr Green (Applied

Biosystems, Carlsbad, CA). GAPDH was used as an internal control with relative quantification being expressed as a ratio of the difference in the number of cycles needed for expression of a gene. To measure viral gene copy LN229 glioma cells were infected for 30 minutes and replaced with media containing macrophages pre-incubated with IgG or one of the various function-blocking antibodies. Total DNA from the co-cultures was purified using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) per manufacturer‟s instruction. Viral gene copy present in the co-cultures was measured by determining the total number of copies of the HSV specific ICP4 gene using quantitative real-time PCR analysis. Total HSV gene copy was determined by generating a linear regression curve using a plasmid containing ICP4 of HSV-1 viral gene (kindly provided by Dr. Deborah

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Parris, The Ohio State University, Columbus, OH). Primers were designed using the

Primer Express Program (Applied Biosystems, Carlsbad, CA) (Table 5).

IVIS Imaging

On days 4, 5, and 6, mice were given a single intraperitoneal injection (100ul, 25mg/ml) of D-Luciferin (Caliper Life Sciences, Hopkington, MA) in PBS. Approximately 5 minutes post-injection, mice were anesthestized using isofluorane and placed in the imaging chamber of a Xenogen IVISTM 100 (Xenogen, Alameda, CA) in the Department of Veterinary Sciences, The Ohio State University. Images were acquired every 10 minutes following injection, and the peak luminescence was analyzed with the

LivingImage software (Xenogen, Alameda, CA).

Western Blot Analysis

Immunoblots were performed on cell lysates (lysed in RIPA buffer: 150mM NaCl, 1%

Nonidet P-40, 0.5% sodium Deoxycholate, 0.1% SDS, 150mM Tris) from indicated cells.

Equal amounts of protein were resolved on a 10% SDS-PAGE followed by transfer to

PVDF membranes. Blots were probed for the indicated proteins using the appropriate antibodies and visualized by enhanced chemiluminescence (GE Health, Pataskala, OH).

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Luciferase Assay

Cells were lysed in 1X Cell Lysis Buffer (Promega Corp., Madison, WI) and measured for luciferase activity using Luciferase Assay System (Promega Corp, Madison, WI) per manufacturer‟s protocol. Protein concentration was determined using the RC-DC Protein

Assay (Bio-Rad, Hercules, CA).

Statistical Analysis

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Figures and Tables

Figure 31: CCN1 inhibition decreases macrophage infiltration toward oncolytic HSV-1 infected tumors, increasing OV activity, in vivo

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(a) Representative luciferase images of athymic nude mice bearing subcutaneous tumors following daily treatment with either IgG or anti-CCN1. Once tumors reached 200mm3, mice were treated i.p. with either IgG or anti-CCN1 (50ug/mouse/day). Images were taken 2 days following a single intratumoral injection with rHSVQ-IE4/5-Luc (1x106 pfu). (b) Quantitative analysis of luciferase activity in athymic nude mice bearing subcutaneous tumors following daily treatment with either IgG or anti-CCN1. Once tumors reached 200mm3, mice were treated i.p. with either IgG or anti-CCN1 (50ug/mouse/day). Average bioluminescence signal intensity of each region of interest (ROI) is shown, n=5 mice. (c) Representative images of OV treated tumor sections immunostained for HSV-1 from mice after treatment with IgG or anti-CCN1. (d) Representative images of OV treated tumor sections immunostained for CD68 from mice after treatment with IgG or anti-CCN1.

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Figure 32: CCN1 increases RAW cell migration towards infected glioma cells (a) Schematic describing basic experimental set-up using modified Boyden Chamber Assays shows migration of macrophages treated with control IgG or anti-CCN1 IgG (placed in upper chamber) towards glioma cells treated with/without OV placed in the

127 bottom chamber. (b) Quantification of migrated RAW264.7 cells in the presence or absence of anti-CCN1 antibody averaging 5 view-fields per assay. (c) Representative bright field images of migrated RAW264.7 cells in B. Values are presented as mean +/- SEM of at least three replicates. *P<0.05

Figure 33: CCN1 induces chemokine expression in glioma cells 128

(a) Real time quantitative PCR analysis of MCP-1 and MCP-3 gene expression in LN229 glioma cells in the presence of anti-CCN1 antibodies or control IgG serum 12 hours following infection with rHSVQ1 at MOI=3. (b) Western blotting analysis for integrin alpha 6 of LN229 glioma cells following lentiviral infection with a non-target construct (NT) and an integrin α6 knock-down (KD) construct. (c) Real time PCR analysis for IFNα and β gene expression in control (NT) and integrin α6 knocked down (KD4) LN229 glioma cells. (d) LN229 cells transfected with NT or KD4 were infected with rHSVQ- IE4/5-Luc (MOI=1). Virally expressed luciferase activity was measured and is expressed as luciferase/mg protein. (e) Real time PCR analysis of MCP-1 and MCP-3 gene expression in LN229 glioma cells transfected with NT or KD4. Data shown are the mean target gene expression relative to endogenous GAPDH and error bars are standard error of the mean of at least three replicates. *P<0.05, **P<0.01, ***P<0.001

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Figure 34: CCN1 protein increases migration of macrophages and monocytes by directly binding integrin αMβ2 130

(a, b) RAW264.7 murine macrophages and THP-1 human monocytic cells migration in response to purified CCN1 or BSA was detected using a modified Boyden Chamber assay. (a) Quantification of migrated RAW264.7 cells averaging 5 view-fields per assay. (b) Quantification of migrated THP-1 cells as determined by hemocytometer. (c) Quantification of migrated RAW264.7 cells on CCN1 coated transwells pre-incubated with control IgG or function-neutralizing antibodies against integrins αM, β2, αv, β3, or β5 averaging 5 view-fields per assay. Values are presented as mean +/- SEM of at least three replicates. ns: not significant *P<0.05, **P<0.01, ***P<0.001

Figure 35: CCN1 increases macrophage pro-inflammatory gene expression Real time quantitative PCR analysis of IL-1β, MCP-1, IP10, and MCP-3 gene expression in murine macrophages co-cultured with CCN1 expressing glioma cells or control glioma cells following a 30 minute infection of glioma cells with rHSVQ1 at MOI=3. Cells were harvested 12 hours following infection. Values are presented as mean +/- SEM of at least three replicates. *P<0.05, ***P<0.001

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Figure 36: CCN1 increases macrophage mediated viral clearance by binding integrin αMβ2

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(a) LN229 glioma cells were infected with rHSVQ1 at MOI=3 for 20 min. RAW264.7 cells, in the presence of anti-CCN1 antibodies or control IgG serum, were overlayed for 12 hours and HSV-1 ICP4 viral gene copy was quantified by real time PCR. (b) LN229 glioma cells transfected with non-target control (NT) or ITGA6 knock-down (KD4) lentivirus constructs were infected with rHSVQ1 at MOI=3 for 20 min. RAW264.7 cells were overlayed for 12 hours and HSV-1 ICP4 viral gene copy was quantified by real time PCR. (c) LN229 glioma cells were infected with rHSVQ1 at MOI=3 for 20 min. RAW264.7 cells, in the presence of anti-integrin αM or β2 antibodies or control IgG serum, were overlayed for 12 hours and HSV-1 ICP4 viral gene copy was quantified by real time PCR. Values are presented as mean +/- SEM of at least three replicates. ns: not significant, *P<0.05

Table 5: Primer Sequences Abbreviations: IFN, Interferon; MCP, Monocyte Chemotactic Protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, Interleukin; IP, Inflammatory Protein; HSV, Herpes Simplex Virus; ICP4, Infected Cell Polypeptide 4.

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Conclusions and Future Directions

The devastating nature of glioblastoma and the little progress which has been made in terms of survival benefit over the last decade indicates a great need for radical and novel therapeutics to become clinically available. The approval of oncolytic virus therapy for head and neck cancer in China [214] was a major achievement, yet its approval in subsequent countries and for other cancers stills remains to be seen. Clinical trials in human patients have highlighted the very low toxicity associated with oncolytic viral therapy. Current preclinical research is now focusing on ways to better understand host responses, enhance viral spread, and combine tumor microenvironment modulation with oncolysis in order to design strategies to improve therapeutic efficacy. Recent advances in molecular biology have facilitated the cumbersome process of constructing novel recombinant viruses with additional features. As these viruses are being made and tested, it is of paramount importance to always keep in mind the safety of the recombinant virus being generated. As more studies are being done to investigate the factors that limit viral infection, spread, and propagation in vivo, newer generation OVs will be able to replicate better and more specifically in cancer cells. Dosage studies investigating the impact of combining OV with the current standard of care will need to be carefully evaluated to determine optimal dosage schedules to enhance therapeutic efficacy. There is also a significant effort being made to improve existing technology for producing clinical grade virus. Improvements in virus production will facilitate the ability to treat patients with 134 larger doses than those currently feasible. Future clinical trials will reveal the efficacy of the various preclinical treatment strategies being tested in translational laboratories across the world. As the field has grown immensely over the past twenty years, the discovery of novel viruses and the advancement in knowledge of current oncolytic viruses have helped elucidate the strengths and weaknesses of this therapy.

In this thesis, we describe some of the limitations to effective oncolytic virus therapy brought on by an ever-changing tumor microenvironment facilitating cellular resistance.

In addition, and perhaps more importantly, we elucidate the transition from basic research to clinical application, which is at the heart of scientific research. Firstly, we describe the use of sophisticated genetic engineering to create a third generation oncolytic virus which combines tumor tissue specific re-targeting with an ability to combat the self-induced increase in angiogenesis by it being driven by a nestin-specific promoter and secreting the anti-angiogenic protein Vstat-120, respectively. Treatment of gliomas with this third generation virus, 34.5ENVE, led to reduced angiogenesis and increased tumor tissue necrosis ultimately resulting in enhanced animal survival.

Next, we describe the induction of the secreted extracellular matrix protein CCN1 in the tumor microenvironment following OV therapy and its subsequent effects on virus infection. CCN1 upregulation was revealed to be a rapid and specific effect of virus infection playing a significant role in the host cellular innate immune response to virus infection. Endogenously upregulated CCN1 was found to interact with integrin α6β1, in

135 both an autocrine and paracrine fashion, leading to the secretion of IFNα and subsequent activation of the JAK/STAT signaling pathway resulting in reduced viral infection and replication.

Lastly, we describe a second effect of CCN1 in the tumor microenvironment, that which increases the infiltration and activation of circulating monocytes and macrophages to the infected tumor bed resulting in increased virus clearance. Here we show endogenously upregulated CCN1 following OV infection of glioma increases macrophage migration and activation by binding integrin αMβ2 the macrophage cell surface and also by increasing chemokine production by the glioma cells. In vivo, treatment with anti-CCN1 antibodies resulted in increased viral expression and reduced CD68+ cell infiltration.

These results collectively indicate a need for CCN1 targeting and silencing to enhance

OV therapy.

The complexity of the tumor microenvironment poses a significant challenge to efficiently treating glioblastoma. In this thesis we describe some of the ways in which the tumor microenvironment essentially “fights back” following a course of oncolytic virus therapy, ultimately reducing the therapeutic efficacy of the virus. As in nature, however, there is a constant evolution of design and with a greater understanding of how this therapy negatively effects the tumor microenvironment we are able to reciprocate, creating more specific and more efficient viruses to not only attack the tumor cells, but also the changes which ensue within the tumor microenvironment. Future studies

136 examining the basic science behind the failure of current therapies for glioblastoma are necessary to create more efficient means for tumor cell destruction.

137

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