Establishment and Characterization of Humanized Mouse NPC-PDX Model for Testing Immunotherapy

Article Establishment and Characterization of Humanized Mouse NPC-PDX Model for Testing Immunotherapy

Wai Nam Liu 1, Shin Yie Fong 1, Wilson Wei Sheng Tan 1 , Sue Yee Tan 1, Min Liu 1, Jia Ying Cheng 1, Sherlly Lim 1, Lisda Suteja 2, Edwin Kunxiang Huang 3, Jerry Kok Yen Chan 3,4, Narayanan Gopalakrishna Iyer 2 , Joe Poh Sheng Yeong 1, Darren Wan-Teck Lim 1,2,* and Qingfeng Chen 1,5,*

1 Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore 138673, Singapore; [email protected] (W.N.L.); [email protected] (S.Y.F.); [email protected] (W.W.S.T.); [email protected] (S.Y.T.); [email protected] (M.L.); [email protected] (J.Y.C.); [email protected] (S.L.); [email protected] (J.P.S.Y.) 2 Division of Medical Oncology, National Cancer Centre, Singapore 169610, Singapore; [email protected] (L.S.); [email protected] (N.G.I.) 3 Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore 229899, Singapore; [email protected] (E.K.H.); [email protected] (J.K.Y.C.) 4 Experimental Fetal Medicine Group, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore 5 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore * Correspondence: [email protected] (D.W.-T.L.); [email protected] (Q.C.); Tel.: +65-6586-9873 (Q.C.) Received: 22 March 2020; Accepted: 17 April 2020; Published: 22 April 2020

Abstract: Immune checkpoint blockade (ICB) monotherapy shows early promise for the treatment of nasopharyngeal carcinoma (NPC) in patients. Nevertheless, limited representative NPC models hamper preclinical studies to evaluate the efficacy of novel ICB and combination regimens. In the present study, we engrafted NPC biopsies in non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain-null (NSG) mice and established humanized mouse NPC-patient-derived xenograft (NPC-PDX) model successfully. Epstein–Barr virus was detected in the NPC in both NSG and humanized mice as revealed by Epstein–Barr virus-encoded small RNA (EBER) in situ hybridization (ISH) and immunohistochemical (IHC) staining. In the NPC-bearing humanized mice, the percentage of tumor-infiltrating CD8+ cytotoxic T cells was lowered, and the T cells expressed higher levels of various inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) than those in blood. The mice were then treated with nivolumab and ipilimumab, and the anti-tumor efficacy of combination immunotherapy was examined. In line with paired clinical data, the NPC-PDX did not respond to the treatment in terms of tumor burden, whilst an immunomodulatory response was elicited in the humanized mice. From our results, human proinflammatory cytokines, such as interferon-gamma (IFN-γ) and interleukin-6 (IL-6) were significantly upregulated in plasma. After treatment, there was a decrease in CD4/CD8 ratio in the NPC-PDX, which also simulated the modulation of intratumoral CD4/CD8 profile from the corresponding donor. In addition, tumor-infiltrating T cells were re-activated and secreted more IFN-γ towards ex vivo stimulation, suggesting that other factors, including soluble mediators and metabolic milieu in tumor microenvironment may counteract the effect of ICB treatment and contribute to the tumor progression in the mice. Taken together, we have established and characterized a novel humanized mouse NPC-PDX model, which plausibly serves as a robust platform to test for the efficacy of immunotherapy and may predict clinical outcomes in NPC patients.

Cancers 2020, 12, 1025; doi:10.3390/cancers12041025 www.mdpi.com/journal/cancers Cancers 2020, 12, 1025 2 of 19

Keywords: immune checkpoint blockade; nasopharyngeal carcinoma; patient-derived xenografts; humanized mouse model

1. Introduction Nasopharyngeal carcinoma (NPC) is characterized by distinct geographical distribution, which is highly associated with chronic infection of Epstein–Barr virus (EBV) [1]. According to the latest statistics, there were around 130,000 newly reported NPC cases globally, with a higher prevalence found in Southern China and South-East Asia [2], and the incidence and mortality rates are higher in males than in females [3]. Some risk factors have been implicated for the NPC in endemic areas, including EBV infection and host genetics [4]. According to the World Health Organization, NPC can be classified into three pathological subtypes, namely non-keratinizing, keratinizing and basaloid squamous cell carcinoma. In those endemic areas, most NPC cases belong to the non-keratinizing subtype that is highly correlated with EBV infection, where the susceptibility to the infection is likely affected by dietary components and smoking [5]. NPC shows EBV type II pattern, where Epstein–Barr nuclear antigen 1 (EBNA1), latent membrane protein 1 (LMP1), LMP2A, LMP2B and Epstein–Barr virus-encoded small RNA (EBER) are expressed, and each of the EBV antigens contribute differentially to the development of NPC and modulation of immunosurveillance [6,7]. Of equal importance, it has been suggested that certain human leukocyte antigens (HLA), such as HLA-A*02:27 or HLA-A*11:01 increase NPC risk [8], while another report summarizes that individuals having HLA-A*02:07, HLA-A*33:03 or HLA-B*38:02 alleles result in higher risk towards NPC [7]. NPC is highly sensitive to chemotherapy and the five year-survival rates in Stages I and II NPC exceed 80%, whereas the rate drops to 10% for stage IV disease [9]. Concurrently, radiotherapy is applied to advanced NPC patients alongside with platinum-based chemotherapy, where the latter sensitizes tumors to the toxic effect of radiotherapy [10]. Due to dense infiltration of lymphocytes in tumor stroma and high expression of programmed death ligand 1 (PD-L1) on tumor cells, immune checkpoint blockades (ICB), particularly through programmed cell death protein 1 (PD-1)/PD-L1 axis, are involved for the treatment of NPC patients [11]. In recent years, combination therapies using nivolumab plus ipilimumab, or nivolumab plus chemoradiation have been evaluated in clinical trials [12], which shed light for the possibility of novel combination regimens. Yet, limited EBV-positive models, both in vitro and in vivo, have hampered the progress of NPC research. Over decades, attempts have been made to generate EBV-positive cell lines from NPC biopsies, such as CG1, NPC/HK1 and C666-1 [13–15]. It is speculated that the presence of large numbers of lymphoid cells, fibroblasts and connective tissues in the NPC biopsies may lower the successful rate to establish NPC cell lines [13,16]. In addition, the cell lines may lose their EBV episomes upon prolonged passages [17]. In past decades, several patient-derived xenografts (PDX) from primary or metastatic NPC have been established and characterized in athymic nude mice [18,19], while more recently another few EBV-positive PDX have been successfully propagated in non-obese diabetic-severe combined immunodeficiency (NOD scid) mice [20]. Nonetheless, these in vivo models provide limited information to immune-oncology research due to the absence of human immune system. Therefore, the development and characterization of humanized mouse cancer models are essential to further research and understanding of NPC biology. For this purpose, CD34+ hematopoietic stem cells (HSC) are engrafted into immunocompromised mice by different approaches, followed by inoculation or transplantation of human cancer cell lines or PDX to generate diverse humanized mouse cancer models [21,22]. In the presence of humanized immune system, different immunological characteristics, including circulating and tumor-infiltrating immune cell profile, as well as cytokine profile can be studied in relation to the tumor. Till now, a broad spectrum of cancers, including human gastric carcinoma [23], hepatocellular carcinoma [24], melanoma [25], prostate cancer [26] and renal cell carcinoma [23,27] has been used to generate different humanized mouse cancer models. Nevertheless, humanized mouse NPC model has not yet been established, which Cancers 2020, 12, 1025 3 of 19

Cancers 2020, 12, x 3 of 20 hinders preclinical studies to evaluate the efficacy and safety of therapeutic agents for NPC patients. Hence,pressing there need is to a pressingdevelop needand characterize to develop anda reliable characterize and robust a reliable human and-in- robustmouse human-in-mouseNPC platform for NPCscreening platform and for testing screening next-generation and testing combination next-generation immunotherapy. combination immunotherapy. InIn the the present present study, study, we we established established and and validated validated a a humanized humanized mouse mouse NPC-PDX NPC-PDX model, model, and and examinedexamined the the modulation modulation of of the the humanized humanized immune immune system system after after NPC NPCtransplantation. transplantation. Furthermore,Furthermore, thethe anti-tumor anti-tumor effi efficacycacy of established of established checkpoint checkpoint inhibitors inhibitors nivolumab nivolumab and ipilimumab and ipilimumab was evaluated, was andevaluated the phenotypes, and the phenotyp of tumor-infiltratinges of tumor T-infiltrating cells were T compared cells were with compared paired clinicalwith paired data. clinical data.

2.2. ResultsResults

2.1.2.1. EstablishmentEstablishment and and Characterization Characterization of of NPC-PDX NPC-PDX in in Humanized Humanized Mice Mice InIn the the present present study, study, we we first first attempted attempted to to engraft engraft NPC-PDX NPC-PDX in in non-obesenon-obese diabetic-severe diabetic-severe combinedcombined immunodeficiency immunodeficiency interleukin-2 interleukin-2 receptor receptor gamma gamma chain-null chain-null (NSG) (NSG) mice mice using using fresh fresh NPC NPC biopsiesbiopsies obtainedobtained fromfrom stagestage IVIV metastaticmetastatic andand/or/or recurrentrecurrent patients. Seven Seven NPC NPC xenografts xenografts from from a atotal total of of 37 37 biopsies biopsies exhibited exhibited subcutaneous subcutaneous growth growth in the in NSG the NSG mice, mice, with witha successful a successful rate of rate 18.9%. of 18.9%.One of One the ofNPC the-PDX NPC-PDX,, propagated propagated from fromthe biopsy the biopsy,, was further was further transplanted transplanted into intohumanized humanized mice micefor immunological for immunological characterization characterization.. Of note Of, note,the HLA the HLAof the of NPC the- NPC-PDXPDX and immune and immune system system in the inhumanized the humanized mice w miceere werenot identical, not identical, but a butminimum a minimum of two of alleles two alleles on HLA on HLA-A*-A* and HLA and HLA-B*-B* were werematched matched [24]. [Tumor24]. Tumor growth growth in the inhuman the humanizedized mice and mice NSG and counterparts NSG counterparts was monitored was monitored weekly, weekly,and the andtumor thes were tumors harvested were harvested eight weeks eight after weeks transplantation after transplantation (Figure 1A). (Figure From1 A).our Fromresults, our the results,NPC-PDX the NPC-PDXgrew slower grew in slowerthe humanized in the humanized mice and mice the tumor and the weight tumor was weight significantly was significantly reduced reduced(Figure (Figure 1B,C), 1suggestingB,C), suggesting that humanthat humanizedized immune immune system system might might play play a a role role in modulating modulating the the progressionprogression of of a a tumor. tumor. We We further further confirmed confirmed that that the the PDX PDX remained remained the the undi undifferentiatedfferentiated NPC NPC type type (similar(similar to to the the original original tumor) tumor) by by hematoxylin hematoxylin and and eosin eosin (H&E) (H&E) staining, staining, and and the the continued continued presence presence ofof EBV EBV was was revealed revealed by by EBER EBER in in situ situ hybridization hybridization (ISH) (ISH) and and immunohistochemical immunohistochemical (IHC) (IHC) staining staining usingusing LMP1 LMP1 and and LMP2A LMP2A antibodies antibodies across across di differentfferent passages. passages.Remarkably, Remarkably, these these EBV EBV proteins proteins were were conservedconserved in in the the tumor tumor from from both both NSG NSG and and humanized humanized mice mice (Figure (Figure1D,E). 1D,E).

A B C NSG mice Humice 1400 4 NSG mice ***

) 1200 Humice

) m

g 3

1000 (

(

e g

800 i

e u

l 2

600 r

m o

400 u m

T 1 u T 200

0 0 0 2 4 6 8 e e ic ic Weeks post transplant m m G u S H N

D 5x 20x Figure 1. ECont. 5x 20x

H&E

EBER

LMP1 LMP2A A B C NSG mice Humice 1400 4 NSG mice ***

) 1200 Humice

) m

g 3

1000 (

(

e g

800 i

e u

l 2

600 r

m o

400 u m

T 1 u T 200

0 0 Cancers 2020, 12, 1025 0 2 4 6 8 e e 4 of 19 ic ic Weeks post transplant m m G u S H Cancers 2020, 12, x N 4 of 20

D 5x 20x E 5x 20x

H&E

EBER

LMP1 LMP2A

FigureFigure 1. Establishment 1. Establishment of humanizedof humanized mouse mouse nasopharyngealnasopharyngeal carcinoma carcinoma (NPC (NPC)-patient-derived)-patient-derived xenograft (PDX) model. NPC-PDX were transplanted in non-obese diabetic-severe combined xenograft (PDX) model. NPC-PDX were transplanted in non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain-null (NSG) mice (n = 14) and humanized mice immunodeficiency interleukin-2 receptor gamma chain-null (NSG) mice (n = 14) and humanized mice (n = 11) subcutaneously. (A) Representative images of tumor from NSG mice (Left) and humanized (n = 11)mice subcutaneously. (Right) after eight (A weeks) Representative of transplant. images The tumor of tumor volume from (B) NSGand tumor mice weight (Left) and(C) from humanized the mice (Right)mice are after shown. eight *** weeks p < 0.001. of transplant. Representative The tumor photomicrographs volume (B) and showing tumor hematoxylin weight (C) fromand eosin the mice are shown.(H&E),*** Epsteinp < 0.001.–Barr Representative virus-encoded photomicrographs small RNA (EBER showing) in situ hematoxylin hybridization and (ISH eosin) and (H&E), Epstein–Barrimmunohistoch virus-encodedemical (IHC small) staining RNA on (EBER) NPC- inPDX situ from hybridization the NSG mice (ISH) (D) and and humanized immunohistochemical mice (E). (IHC)The staining results on of NPC-PDXH&E staining from confirmed the NSG that mice the PDX (D) belongs and humanized to the undifferentiated mice (E). The NPC results type, ofand H&E stainingthe presence confirmed of Epstein that the–Barr PDX virus belongs (EBV) was to theindicated undi ffbyerentiated the expression NPCs of type, EBER, andlatent the membrane presence of Epstein–Barrprotein ( virusLMP)1(EBV) and LMP2A. was indicated Bars: 100 μm by. the expressions of EBER, latent membrane protein (LMP)1 and LMP2A. Bars: 100 µm. 2.2. Activation of the Immune Response in NPC-transplanted Humanized Mice 2.2. ActivationTo investigate of the Immune the phenotypic Response changes in NPC-transplanted of immune cells Humanized after NPC Miceengraftment, peripheral blood Tomononuclear investigate cells the (PBMC) phenotypic from humanized changes ofmice immune were examined cells after by flow NPC cytometric engraftment, analysis peripheral. The bloodgating mononuclear strategy is cells shown (PBMC) in Figure from S1. humanizedIn the presence mice of NPC, were there examined was minimal by flow effect, cytometric if any, on analysis. the chimerism of the mice (Figure 2A), whilst there was a gradual increase in the percentage of CD3+ T The gating strategy is shown in Figure S1. In the presence of NPC, there was minimal effect, if any, cells (Figure 2B). The increase in the CD3+ T cells was contributed by both CD4+ and CD8+ T cells on the(Figure chimerism 2C,D). In of contrast, the mice the (Figure percentage2A), of whilst CD19+ there B cells was was areduced gradual after increase NPC transplant in the percentage (Figure + + + of CD32E). TOther cells immune (Figure 2cellsB)., Theincluding increase CD14 in+ themacrophages CD3 T, CD56 cells+ was natural contributed killer (NK) by cells both and CD4 their and + + CD8 subsetsT cells were (Figure also2 detectedC,D). In in contrast, our model the (Figure percentage S2A–G).of From CD19 our results,B cells there was were reduced fewer afterclassic NPC transplantmacrophages (Figure and2E). cytokine Other- immuneproducing cells, NK cell includings in the NPC CD14-engrafted+ macrophages, mice at experimental CD56+ naturalendpoint killer. (NK)Intriguingly, cells and their the CD8 subsets+ T cells were showed also detectedan augmented in our level model of HLA (Figure-DR expression S2A–G). (Figure From our2F) and results, theredisplayed were fewer an classic effector macrophages memory phenotype and cytokine-producing (Figure S2H), indicating NK cellsthat in the the humanized NPC-engrafted immune mice at experimentalresponse was endpoint. elicited after Intriguingly, tumor engraftment. the CD8+ CTirculating cells showed cytokine an and augmented chemokine level profile of HLA-DR was examined by LEGENDplex and enzyme-linked immunosorbent assay (ELISA), and plasma expression (Figure2F) and displayed an e ffector memory phenotype (Figure S2H), indicating that concentrations of interferon-gamma (IFN-γ), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte the humanized immune response was elicited after tumor engraftment. Circulating cytokine and chemoattractant protein-1 (MCP-1) and transforming growth factor-beta 1 (TGF-β1) were chemokineupregulated profile (Figure was examined 2G–K). Spleen by LEGENDplex was harvested and at enzyme-linkedexperimental endpoint immunosorbent and the immune assay (ELISA),cell and plasmaprofile was concentrations investigated. ofConcordant interferon-gamma with the imm (IFN-unomodulationγ), interleukin-6 observed (IL-6), in blood interleukin-8, there was an (IL-8), monocyteelevation chemoattractant in the percentage protein-1 of splenic (MCP-1) CD3+ T andcells, transformingaccompanied by growth a reduction factor-beta in CD19 1+ (TGF-B cells βafter1) were upregulatedtumor transplant (Figure2, G–K).and the Spleen increase was in the harvested splenic T cells at experimental was dominantly endpoint contributed and by the CD8 immune+ T cells cell profile was investigated. Concordant with the immunomodulation observed in blood, there was an elevation in the percentage of splenic CD3+ T cells, accompanied by a reduction in CD19+ B cells after tumor transplant, and the increase in the splenic T cells was dominantly contributed by CD8+ T Cancers 2020, 12, 1025 5 of 19

Cancers 2020, 12, x 5 of 20 cells that exhibited an eﬀector memory phenotype (Figure S3A–E). Moreover, there was a decrease in the percentagethat exhibited of classic an effector and memory non-classic phenotype macrophages, (Figure S3A–E). andMoreover cytokine-producing, there was a decrease in NK the cells in the NPC-bearingpercentage mice (Figure of classic S3F–L). and non Taken-classic together,macrophages, our and results cytokine suggested-producing NK that cells the inhumanized the NPC- immune bearing mice (Figure S3F–L). Taken together, our results suggested that the humanized immune + system was activatedsystem was activated after NPC after transplant,NPC transplant, as as reﬂected reflected by by the the increase increase in the proportion in the proportion of CD3+ T of CD3 T cells and thecells activation and the activation of CD8 +of TCD8 cells+ T cells in thein the humanized humanized mice. mice.

A chimerism B CD3+ C CD4+

100 100 100 +

Without NPC + Without NPC Without NPC

5 4

With NPC 4 With NPC With NPC D

80 D 80 80

i 60 60 * 60

l l

h ** e

40 e 40 40

3 D

20 D 20 20

% % 0 0 0 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 Weeks post transplant Weeks post transplant Weeks post transplant D E F CD8+ CD19+ HLA-DR

100 100 400

+ +

Without NPC 5 Without NPC

5 ***

4 4

With NPC D With NPC D 80

C 80 C

h 300

)

60 e 60

t L

t 200

H a

l *

(

e I

40 r 40

+ ***

M 8

1 ** 100 D

20 D 20

C h *** * h

% * 0 % 0 0 0 2 4 6 8 0 2 4 6 8 C C P P N N Weeks post transplant Weeks post transplant t u th o i h W it W G H I IFN-γ IL-6 IL-8 300 25 5000

Without NPC Without NPC Without NPC

)

l l l With NPC With NPC With NPC

20 4000

/ /

/ *

g g



 



- (

200 (

(

n F

n 15 3000

a a

a **

t a

10 a 2000

n n

n *

l *

P P

e e

e 100

c c

c ***

o o

o 5 1000

c c c

0 0 0 * 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 Weeks post transplant Weeks post transplant Weeks post transplant J K MCP-1 TGF-β 3000 400

Without NPC **

)

) l

With NPC l

m /

/ 300





P (

2000 (

t a

a 200

l e

1000 ** e

P n

n 100

o c ** c

0 0 0 2 4 6 8 C C P P N N Weeks post transplant t u th o i h W it W Figure 2. ActivationFigure 2. ofActivation humanized of human immuneized immune system systemafterNPC after transplant. NPC transplant. NPC-PDX NPC-PDX were weretransplanted transplanted in humanized mice subcutaneously. Blood samples from humanized mice with or in humanized mice subcutaneously. Blood samples from humanized mice with or without tumor (n = 6 from each group) were collected at the indicated weeks post-transplant. The chimerism (A), percentages of CD3+ T cells (B), CD4+ T cells (C), CD8+ T cells (D) and CD19+ B cells (E) were analyzed by ﬂow cytometry. (F) The expression of human leukocyte antigens (HLA)-DR on the circulating CD8+ T cells from the mice (n = 4 from each group) was examined eight weeks after transplant. Cytokines and chemokine levels in plasma, including interferon-gamma (IFN-γ)(G), interleukin (IL)-6 (H), IL-8 (I), monocyte chemoattractant protein-1 (MCP-1) (J) and transforming growth factor-beta 1 (TGF-β1) (K) were analyzed by LEGENDplex and ELISA (n = 4 from each group). Data are expressed as means standard error of mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001. ± Cancers 2020, 12, 1025 6 of 19

2.3. Tumor-Infiltrating T Cells Display Exhausted Phenotypes Due to the influence of tumor microenvironment, it is anticipated that the phenotypes and functions of tumor-infiltrating lymphocytes (TIL) are distinguishable from the immune cells in periphery and organs. To characterize the immune profile of TIL in our model, NPC was harvested from humanized mice at the experimental endpoint. In the tumor-bearing humanized mice, the percentages of tumor-infiltrating CD3+, CD4+ and CD8+ T cells were lower than those in blood and spleen (Figure3A–C), while there were no significant changes in the percentage of CD19 + B cells (Figure3D). In addition, the percentages of various CD14 + macrophage subsets in the tumor were lowered (Figure3E–H), whereas the cytokine-producing NK cells, but not the cytotoxic NK cells, were increased statistically (Figure3I–K). Similarly, the percentages of the tumor-infiltrating T cells were also reduced, as demonstrated using another NPC-PDX (Figure S4A–K). Consistent with the primary tumor, T cells were also the major immune cell subset in the NPC-PDX, albeit the tumor-infiltrating macrophages and NK cells accounted for around 5% in our model [28,29]. Hence, the expressions of immune checkpoint receptors on the cytotoxic T lymphocytes were further examined. By flow cytometric analysis, it was found that the tumor-infiltrating CD8+ T cells from both PDX expressed higher levels of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), PD-1, lymphocyte-associated gene 3 (LAG3) and T-cell immunoglobulin and mucin domain 3 (TIM3) when compared to those in the circulation and spleen (Figure4A–D; Figure S4L–O), revealing that the T cells exhibited exhausted phenotype in the immunosuppressive tumor milieu [30]. Cancers 2020, 12, x 7 of 20

A CD3+ B CD4+ C CD8+ D CD19+

100 100 100 100

*** +

+ +

* * 5

5 5

4 4 *** 4

* * D *

D D 80 D 80

80 80 C

C C

h h

o o

t t

60 60 60 e 60

e e

v v

i i

t t

a a

l l

e e

40 40 40 r 40

r r

+ +

4 8

D D

20 20 20 D 20

C C

h h

% % % 0 0 0 0 d n r d n r d n r d n r o e o o e o o e o o e o lo le m lo le m lo le m lo le m B p u p u p u p u S T B S T B S T B S T E CD14+ F CD14++CD16- G CD14++CD16+ H CD14+CD16+

20 20 10 10

e 5

*** ** ** e ***

e D

*** *** ** a **

+ h

C 8 8

5 p

h 15 15

r a

e 6 6

t t

10 t 10

v v

i 4 4

e e

5 5 r

D e

2 2

% % 0 0 % 0 0 d n r d n r d n r d n r o e o o e o o e o o e o o le m o le m lo le m lo le m l p u l p u p u p u B S T B S T B S T B S T

I CD56+ J CD56+CD16- K CD56+CD16+

10 s 10 10

l 5

* * e **

* ** s *

+ l

C 8 8 8

e c

6 6 N 6

r v

4 4 v 4

r D

2 o 2 2

0 % 0 0 d n r d n r d n r o e o o e o o e o lo le m lo le m lo le m B p u B p u B p u S T S T S T Figure 3. PresenceFigure and 3. Presence modulation and modulation of human of human immune cells cells in tumor. in tumor.NPC-PDX were NPC-PDX transplanted were in transplanted humanized mice subcutaneously. After eight weeks post-transplant, blood samples, spleens and in humanized micetumors subcutaneously.were collected from the mice After (n = eight 15, combined weeks from post-transplant, 3 experiments). The percentages blood samples, of CD3+ spleens and tumors were collectedT cells (A), from CD4+ theT cells mice (B), CD8(n =+ T15, cells combined (C), CD19+ B from cells (D 3) experiments)., CD14+ macrophages The (E), percentages classic of CD3+ + macrophages+ (F), intermediate+ macrophages (G), non-classic+ macrophages (H), CD56+ natural killer T cells (A), CD4(NK)T cells cells (I), ( cytokineB), CD8-producingT cells NK cells(C), (J CD19) and cytotoxicB cells NK (cellsD), (K CD14) in thesemacrophages organs were (E), classic macrophages (Fexamined), intermediate by flow cytometric macrophages analysis. Data ( areG), expressed non-classic as means macrophages ± SEM. * p < 0.05; ** ( Hp <), 0.01; CD56 *** p + natural killer (NK) cells (I), cytokine-producing< 0.001. NK cells (J) and cytotoxic NK cells (K) in these organs were examined by ﬂow cytometric analysis. Data are expressed as means SEM. * p < 0.05; ** p < 0.01; *** p < 0.001. ± Cancers 2020, 12, 1025 7 of 19 Cancers 2020, 12, x 8 of 20

A B C D CTLA-4 PD-1 LAG3 TIM3

Unstained

Blood

Spleen

Tumor ** ** *** * 500 * 5000 ** 1000 * 1000 *

400 4000 800 800

)

- A

300 3000 G 600 600

(

F I

200 2000 F 400 400

M M 100 1000 200 200

0 0 0 0

d n r d n r d n r d n r o e o o e o o e o o e o lo le m lo le m lo le m lo le m B p u B p u B p u B p u S T S T S T S T + Figure 4. Presence of exhausted CD8+ TT cells cells in tumor. NPC NPC-PDX-PDX were transplanted in humanized mice subcutan subcutaneously.eously. After eight weeks post post-transplant,-transplant, tumors were collected from the mice ( n = 66).). The expression expression levels levels of of several several inhibitory inhibitory receptors, receptors, such such as cytotoxic as cytotoxic T-lymphocyte-associated T-lymphocyte-associated protein protein4 (CTLA-4) 4 (CTLA (A), programmed-4) (A), programmed cell death cell protein death 1 (PD-1)protein ( B1), (PD lymphocyte-associated-1) (B), lymphocyte-associated gene 3 (LAG3) gene (C 3) (andLAG3 T-cell) (C immunoglobulin) and T-cell immunoglobulin and mucin domain and mucin 3 (TIM3) domain (D) 3 were (TIM3 determined.) (D) were Datadetermined. are expressed Data are as means SEM. * p < 0.05; ** p < 0.01; *** p < 0.001. expressed± as means ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001. 2.4. Examination of Anti-Tumor Efficacy Using ICB in Humanized Mouse NPC-PDX Model 2.4. Examination of Anti-Tumor Efficacy Using ICB in Humanized Mouse NPC-PDX Model In recent years, ICB, with monoclonal antibodies such as pembrolizumab, nivolumab and In recent years, ICB, with monoclonal antibodies such as pembrolizumab, nivolumab and camrelizumab have been used to treat advanced stage NPC patients in multiple clinical trials [31–33]. camrelizumab have been used to treat advanced stage NPC patients in multiple clinical trials [31–33]. In our humanized mouse NPC-PDX model, the tumor-infiltrating CD8+ T cells expressed higher levels In our humanized mouse NPC-PDX model, the tumor-infiltrating CD8+ T cells expressed higher of PD-1 and CTLA-4, as well as LAG3, and these exhausted phenotypes might hinder the anti-tumor levels of PD-1 and CTLA-4, as well as LAG3, and these exhausted phenotypes might hinder the anti- efficacy of ICB by suppressing their cytotoxicity and cytokine-producing capability. Hence, it is of tumor efficacy of ICB by suppressing their cytotoxicity and cytokine-producing capability. Hence, it great interest to examine whether ICB are able to restore the functions of the tumor-infiltrating T cells is of great interest to examine whether ICB are able to restore the functions of the tumor-infiltrating and reduce tumor burden in humanized mice. These humanized mice were transplanted with the T cells and reduce tumor burden in humanized mice. These humanized mice were transplanted with NPC-PDX and upon formation of visible tumors, which takes usually 2 to 3 weeks after transplantation, the NPC-PDX and upon formation of visible tumors, which takes usually 2 to 3 weeks after the mice were then injected with a cocktail of nivolumab (anti-PD-1 antibody; 3 mg/kg) and ipilimumab transplantation, the mice were then injected with a cocktail of nivolumab (anti-PD-1 antibody; 3 (anti-CTLA-4 antibody; 1 mg/kg) on a weekly basis. Four weeks after treatment, the tumors were mg/kg) and ipilimumab (anti-CTLA-4 antibody; 1 mg/kg) on a weekly basis. Four weeks after harvested for downstream analysis (Figure S5A). From our results, the NPC-PDX did not respond to treatment, the tumors were harvested for downstream analysis (Figure S5A). From our results, the the ICB treatment in terms of the tumor weight (Figure S5B,C). NPC-PDX did not respond to the ICB treatment in terms of the tumor weight (Figure S5B,C). 2.5. Modulation of Immune Cell and Cytokine Profile after Treatment 2.5. Modulation of Immune Cell and Cytokine Profile after Treatment Next, we evaluated the modulation of immune responses in the NPC-bearing humanized mice afterNext, combination we evaluated therapy. the In modulation the ICB-treated of immune mice, there responses were no in significant the NPC-bearing changes humanized in the chimerism mice after(Figure combination5A). Intriguingly, therapy there. In was the an ICB increase-treated in the mice percentage, there were of CD3 no + significantand CD8+ T changes cells observed in the + + chimerismat later time (Figure point, 5A). resulting Intriguingly, in a decrease there in was CD4 an/CD8 increase ratio in (Figure the percentag5B–E). Ine addition,of CD3 and the CD8 expression T cells observedlevel of HLA-DR at later time on the point, CD8 resulting+ T cells in was a decrease up-regulated in CD4/CD8 (Figure 5ratioF) and (Figure immediate 5B–E). increasesIn addition, in thethe + expressionplasma IFN- levelγ and of IL-6 HLA were-DR found on (Figurethe CD85G,H), T cells which was indicated up-regulated that the (Figure immune 5 systemF) and was immediate further increaseactivateds in upon the theplasma treatment. IFN-γ and IL-6 were found (Figure 5G,H), which indicated that the immune system was further activated upon the treatment. Cancers 2020, 12, 1025 8 of 19 Cancers 2020, 12, x 9 of 20

A B C chimerism CD3+ CD4+

100 100 100 +

Saline + Saline Saline

5 4

Nivo+Ipi Nivo+Ipi 4 Nivo+Ipi D

80 80 D 80

i 60 60 60

h e

40 40 e 40

+ +

4 D

20 20 D 20

% % 0 0 0 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 Weeks post transplant Weeks post transplant Weeks post transplant D E F CD8+ CD4/CD8 HLA-DR 100 8 300

+ Saline Saline 5 ** 4 Nivo+Ipi Nivo+Ipi D 80

C 6

)

R t

o 200

r -

L t

D 4

(

e I

40 4

D 100

M C 8 2

D 20 C

h * * % 0 0 0 0 2 4 6 8 0 2 4 6 8 e i n Ip li + Weeks post transplant Weeks post transplant a o S iv G H N IFN-γ IL-6 800 200

Saline Saline

)

) l

l Nivo+Ipi Nivo+Ipi

m /

/ 600 150





(

a t

t 400 100

P c

c ** n

n 200 50

o c c * * 0 0 * 0 2 4 6 8 0 2 4 6 8 Weeks post transplant Weeks post transplant

Figure 5. Modulation of humanized immune system in blood after combination immunotherapy. Figure 5. Modulation of humanized immune system in blood after combination immunotherapy. NPC-transplanted humanized mice were injected with saline or nivolumab plus ipilimumab. Purple NPC-transplanted humanized mice were injected with saline or nivolumab plus ipilimumab. Purple arrows indicate the injection time point. Blood samples (n = 8 from each group) were collected at the arrows indicate the injection time point. Blood samples (n = 8 from each group) were collected at the indicated weeks post-transplant. The chimerism (A), percentages of CD3+ T cells (B), CD4+ T cells indicated weeks post-transplant. The chimerism (A), percentages of CD3+ T cells (B), CD4+ T cells (C), (C), CD8+ T cells (D), CD4/CD8 ratio (E) and the expression of HLA-DR on the CD8+ T cells at the CD8+ T cells (D), CD4/CD8 ratio (E) and the expression of HLA-DR on the CD8+ T cells at the experimental endpoint (F) were analyzed. Cytokines levels in plasma, including IFN-γ (G) and IL-6 (H) experimental endpoint (F) were analyzed. Cytokines levels in plasma, including IFN-γ (G) and IL-6 were determined (n = 4 from each group). Data are expressed as means SEM. * p < 0.05; ** p < 0.01. (H) were determined (n = 4 from each group). Data are expressed as means ± SEM. * p < 0.05; ** p < 0.01. 2.6. Presence of Re-Activated Tumor-Infiltrating T Cells after Treatment 2.6. Presence of Re-Activated Tumor-Infiltrating T Cells after Treatment Apart from PBMC, the immune cell profile in tumor was analyzed after ICB treatment. From our results,Apart there from were PBMC no significant, the immune changes cell inprofile the percentage in tumor ofwas CD3 analyzed+ T cells after (Figure ICB6A). treatment However,. From the ourCD4 results,/CD8 ratio there in were the NPC-PDX no significant was decreased changes in (Figure the perc6B–D).entage Interestingly, of CD3+ T cells paired (Figure tumor 6A). biopsies However, from thethe patientCD4/CD8 donor ratio showed in the a NPCsimilar-PDX reduction was decreased in the CD4 (Figure/CD8 ratio 6B– afterD). Interestingly dual PD-1/CTLA4, paired blockade tumor biopsies(Figure6 E).from Similar the patient trend of donor the CD4 showed/CD8 a ratio similar was reduction also seen inin subsequentthe CD4/CD8 NPC-PDX ratio after derived dual fromPD- 1/CTLA4other patient blockade donor (Figure treated 6E). with Similar dual ICB trend (Figure of the S6), CD4/CD8 which further ratio w supportsas also seen that in the subsequent humanized NPC mice- PDXcan simulate derived from the immune other patient responses donor to treated ICB. Last with but dual not ICB least, (Figure tumor-infiltrating S6), which further CD3 +suppT cellsorts were that theisolated humanized for ex vivo mice activation. can simulate It was the found immune that responses the T cells to from ICB. the Last ICB-treated but not least humanized, tumor-infiltrating mice were CD3+ T cells were isolated for ex vivo activation. It was found that the T cells from the ICB-treated humanized mice were capable of secreting more IFN-γ after stimulation (Figure 6F), which suggests CancersCancers 20202020, 12,, x12 , 1025 9 of 1910 of 20 that capablethe tumor of secreting-infiltrating more IFN-T cellsγ after were stimulation re-activated (Figure and6F), the which ICB suggests might restore, that the tumor-infiltratingat least partially, the functionT cells of were the re-activatedT cells. and the ICB might restore, at least partially, the function of the T cells.

A B C CD3+ CD4+ CD8+

100 100 100

4 4

4 *

D D

D 80 80 80

60 60 60

e e

e 40 40 40

D D

D 20 20 20

% % 0 0 0 e i e i e i n Ip n Ip n Ip li + li + li + a o a o a o S iv S iv S iv N N N D E F CD4/CD8

5 0.0 150 g

n **

* i

l n.s.

i f

4 ) **

l o

r -0.5

g 100



p (

D -1.0

4 2



- D

8 50

C D

-1.5 F

I C

1 /

D C 0 -2.0 0 e i t t n Ip n n li + e e TransAct - + - + a o m m S iv t t N a a re re t t Saline Nivo+Ipi e r r te fo f e A B

Figure 6. Modulation of immune cell profile in tumor after combination immunotherapy. Tumors Figure 6. Modulation of immune cell profile in tumor after combination immunotherapy. Tumors were harvested from saline-treated humanized mice (n = 5) and nivolumab plus ipilimumab-treated werehumanized harvested mice from (n =saline10; combined-treated fromhumanized 2 experiments) mice (n at = the 5) experimentaland nivolumab endpoint. plus Theipilimumab percentages-treated humanizedof CD3+ T mice cells (An ),= CD4 10;+ T combined cells (B), CD8 from+ cells 2 experiments) T cells (C) and at CD4 the/CD8 experimental ratio (D) were endpoint. analyzed. The percentage(E) Thes CD4 of /CD3CD8+cell T cells type ( profilingA), CD4 from+ T cells corresponding (B), CD8+ cells donor T was cells revealed (C) and by CD4/CD8 Nanostring ratio analysis. (D) were analyzed.(F) Tumor-infiltrating (E) The CD4/CD8 CD3 +cellT cellstype were profiling isolated from from corresponding the saline-treated donor group was (n revealed= 4) and nivolumab by Nanostring analysisplus. ipilimumab-treated (F) Tumor-infiltrating group (CD3n = 4).+ T The cells release were of isolated IFN-γ after from TransAct the saline stimulation-treated was group determined (n = 4) and by ELISA. Data are expressed as means SEM. n.s. means non-significant; * p < 0.05; ** p < 0.01. nivolumab plus ipilimumab-treated group± (n = 4). The release of IFN-γ after TransAct stimulation 3.was Discussion determined by ELISA. Data are expressed as means ± SEM. n.s. means non-significant; * p < 0.05; ** p < 0.01. EBV-associated NPC exhibits a distinct geographical distribution and most cases occur in Southern 3. DiscussionChina and South-East Asia. Nevertheless, due to limited in vitro and in vivo models, the progress of basic and translational research on NPC has been limited. Reliable EBV-positive patient-derived NPCEBV models-associated for both NPC oncology exhibits and a immunology distinct geographical studies, would distribution accelerate the and evaluation most cases of novel occur in Southernimmunotherapy China and regimens South-East and furtherAsia. Nevertheless, development of due effective to limited treatments. in vitro and in vivo models, the progressPrevious of basic attemptsand translational to establish research NPC-PDX on inNPC vivo hashave been been limited successful. Reliable in nude EBV mice-posi andtive NOD patient- derivedscid mice. NPC Inmodels the present for both study, oncology fresh NPC and biopsies immunology obtained studies, from stage would IV metastatic accelerate and the/or recurrentevaluation of novelpatients immunotherapy were transplanted regimens in NSG and micefurther and development the tumor take of rateeffective for PDX treatments. engraftment was 18.9%, Previous attempts to establish NPC-PDX in vivo have been successful in nude mice and NOD scid mice. In the present study, fresh NPC biopsies obtained from stage IV metastatic and/or recurrent patients were transplanted in NSG mice and the tumor take rate for PDX engraftment was 18.9%, which is comparable to those aforementioned studies [18–20,34]. Yet, the lack of comprehensive immune system in immunocompromised mice hinders the understanding of immunological responses during cancer progression, as well as the development and evaluation of the efficacy of Cancers 2020, 12, 1025 10 of 19 which is comparable to those aforementioned studies [18–20,34]. Yet, the lack of comprehensive immune system in immunocompromised mice hinders the understanding of immunological responses during cancer progression, as well as the development and evaluation of the efficacy of cancer immunotherapies [21,35]. In the presence of humanized immune system, the growth of the tumor was significantly inhibited. Notably, it has been reported that the NPC-PDX may lose their EBV episomes after prolonged passages [17], potentially altering the tumor-immune milieu and limiting the interpretation of experiments done with these models. To confirm the presence of EBV in the tumor from our PDX, EBER ISH and IHC staining for LMP1 and LMP2A were performed prior to subsequent experiments. Our results showed that the EBV were retained in the tumors in both NSG and humanized mice upon serial passages, and the NPC-PDX was EBV-positive at the time of experiments. The role of the human immune system in shaping immunogenicity of tumor has been extensively reviewed, where cancer immunoediting can be categorized into elimination, equilibrium and escape phases through activation of innate and adaptive immune responses [36,37]. From our results, there was an increase in human CD8+HLA-DR+ T cells in the NPC-bearing humanized mice, compared to their non-engrafted counterparts, suggesting that the immune system was activated. In addition, the transplantation of NPC led to an elevation of numerous cytokines, including IFN-γ, IL-6 and IL-8, as well as MCP-1, a chemokine that regulates migration and infiltration of monocytes/macrophages [38]. IFN-γ was shown to protect the host against the growth of transplanted tumors, chemically-induced tumors and spontaneous tumors, which forms the basis of tumor immunosurveillance and promotes elimination of tumor cells at early stage of engraftment [39,40]. The increase in circulating IFN-γ level in humanized mice may account for the reduction in tumor burden when compared to the immunocompromised NSG mice. Remarkably, more IL-6 and IL-8 were secreted in the NPC-PDX mice at later time point. These cytokines are believed to promote tumor growth by supporting angiogenesis, although the pro-tumorigenic role of IL-6 is still inconclusive as reported elsewhere [41,42]. TGF-β plays a dual role in modulating cancer development [43]. At the experimental endpoint, it was found that the plasma concentration of total TGF-β1 was higher in the NPC-bearing humanized mice, which is in line with the data in clinical settings [44]. Previous clinical studies have demonstrated that NPC is densely infiltrated by T cells [45], where the presence of more TIL, particularly CD3+ T cells, was associated with favorable outcomes [46,47]. In our model, TIL was detected in the humanized mice, and the percentage of intratumoral T cells was lower than those in circulation and spleen. More importantly, the expressions of several inhibitory receptors on the intratumoral CD8+ T cells, including PD-1, CTLA-4, LAG3 and TIM3 were upregulated, resulting in exhausted phenotypes that are frequently observed in cancer patients [48,49]. PD-1 is one of the major inhibitory receptors that regulates T-cell exhaustion and the T cells with high PD-1 expression fail to eradicate cancer [48], thus making PD-1 an attractive target in cancer immunotherapy using anti-PD-1 monoclonal antibody, although the reported objective response rates were around 20% to 30% in different single-arm trials [4]. Similarly, CTLA-4 plays a critical role in attenuating the early activation of naïve and memory T cells [50] and NPC patients with higher tumor CTLA-4 expression had poorer prognosis [51]. To evaluate the anti-tumor efficacy of combination therapy and align with our clinical trial, the NPC-PDX humanized mice were administrated with nivolumab plus ipilimumab. The tumors in humanized mice did not respond to the combination therapy in terms of the tumor burden, which is consistent with the lack of response in the donor patient in clinic. In addition, they displayed a similar trend in the modulation of CD4/CD8 ratio after treatment, suggesting that humanized mice could be used to mimic some of the responses in patients after treatment. Interestingly, ex vivo T-cell activation assay revealed that tumor-infiltrating CD3+ T cells isolated from the ICB-treated group were more responsive to the stimulation and secreted more IFN-γ. Yet, the re-activation of T cells may remain insufficient to exert significant anti-tumor effect in vivo, suggesting alternate pathways of resistance including other inhibitory checkpoints, such as LAG3 and TIM3. Of note, LAG3 was upregulated in our NPC-PDX model, and LAG3 upregulation was also seen in a recent trial of spartalizumab in NPC [52]. Indeed, relatlimab, an anti-LAG3 antibody, is Cancers 2020, 12, 1025 11 of 19 currently under clinical development in several tumors [53]. Apart from the inhibitory ligands, it is also anticipated that other immunosuppressive signals in tumor microenvironment, such as soluble mediators and metabolic milieu may contribute to the dysfunction states of the intratumoral T cells [30], which may be further investigated in the humanized mouse NPC-PDX model. To the best of our knowledge, this is the first report to describe the establishment and characterization of a humanized mouse NPC-PDX model (Figure7). However, several limitations remain. Firstly, the take rate can be improved. Hsu et al. reported that PDX engraftment-positive patients had shorter survival than PDX engraftment-negative patients, regardless of their EBV DNA load and previous treatments that the patients received [34]. Hence, it would be of interest to unravel the underlying factors that govern the tumor take rate, which can plausibly increase the bioavailability of NPC-PDX for EBV and NPC research. While most human hematopoietic lineages are engrafted, not all are fully developed in the humanized mice. Due to the absence of full HLA matching and human primary lymphoid organs, human pre-T cells are educated in mice thymus and rely on major histocompatibility complex (MHC) for antigen presentation. This results in limited education of T cells in the mice [54], while some publications demonstrated that human T cells in humanized mice could generate human HLA-restricted responses [55,56]. Bone marrow (BM) microenvironment is capable of supporting T-cell development in the absence of thymus, however, the difference in human and mouse BM microenvironment may further hinder T-cell education and function [57]. Notably, the lack of species-specific growth factors, cytokines and chemokines also hampers the maturation of certain immune cell subsets, including myeloid cells and NK cells [58]. To overcome these hurdles would involve manipulating the human-mouse immune interface. HLA class I and II transgenes have been introduced into NSG mice, which possibly allow the generation of functional HLA-restricted T cells [59]. In addition, efficient methods to improve mouse environment to support better human cell functions have been developed, including hydrodynamic delivery of constructs expressing human-specific cytokines [60,61], and multiple strains of transgenic mice bearing knock-in genes, which constitutively increase the levels of human cytokines in the mice and facilitate the growth of specific immune cell subsets are being developed [62]. These improved models may better recapitulate the human immune responses during cancer progression [63,64]. HLA mismatch could be overcome with PBMC or induced pluripotent stem cells (iPSC) that can be obtained or derived autologously to generate the humanized mouse NPC-PDX model with fully matched HLA. Nevertheless, injection of PBMC results in graft-versus-host-disease [54], whilst the repopulating capability of iPSC in mice is limited [65]. These barriers have to be overcome before a fully HLA-matched humanized mouse cancer model can be established. Cancers 2020, 12, 1025 12 of 19 Cancers 2020, 12, x 13 of 20

FigureFigure 7. 7. SchematicSchematic representation representation of of the the immunomodulation immunomodulation in in humanized humanized mice mice after after NPC NPC transplantationtransplantation and and immune immune checkpoint checkpoint blockade blockade (ICB) (ICB) treatment. treatment. Ten Ten to to twelve twelve weeks weeks after after HSC HSC injection,injection, major major human human immune immune cell cell subsets subsets were were repopulated repopulated in in the the circulation circulation of of humanized humanized mice. mice. Subsequently,Subsequently, NPC NPC-PDX-PDX was was transplanted transplanted in in one one flank ﬂank of the humanized mice. The The humanized humanized + immuneimmune system system was activatedactivated asas revealed revealed by by an an increase increase in in plasma plasma IFN- IFNγ.- Inγ. addition,In addition, more more CD4 CD4and+ + + andCD8 CD8T cells+ T cells were were present present in the in circulation,the circulation, accompanied accompanied by a decreaseby a decrease in the in percentage the percentage of CD19 of + + CD19B cells+ B after cells the after transplant. the transplant At experimental. At experimental endpoint, endpoint, it was it found was found that less that CD4 less CD4and+CD8 and CD8T cells+ T cellswere were present present in the in tumor the tumor (Blue (Blue in color) in color) than thosethan those in the in circulation. the circulation. Combination Combination therapy therapy using usingnivolumab nivolumab and ipilimumab and ipilimumab further further activated activated the immune the immune system system in NPC-bearing in NPC-bearing humanized humanized mice, γ mice,with awith higher a higher level of level plasma of plasma IFN- . IFN Moreover,-γ. Moreover, a reduction a reduction of CD4/ CD8of CD4/CD8 ratio was ratio observed was observed in the tumor in the(Red tumor in color) (Red after in ICB color) treatment. after In ICB additional treatment. to the current In additional combination to the immunotherapy, current combination other ICB, such as anti-LAG3 and TIM3 antibodies, or cell-mediated therapy involving chimeric antigen receptor immunotherapy, other ICB, such as anti-LAG3 and TIM3 antibodies, or cell-mediated therapy (CAR)-engineered T cells and NK cells can be evaluated using the humanized mouse NPC-PDX model. involving chimeric antigen receptor (CAR)-engineered T cells and NK cells can be evaluated using the humanized mouse NPC-PDX model. Cancers 2020, 12, 1025 13 of 19

4. Materials and Methods

4.1. Cord Blood Processing and Isolation of Human CD34+ HSC Human umbilical cord blood was collected from KK Women’s and Children’s Hospital (KKH), Singapore, with written consent obtained from guardians of donors and in strict accordance with the institutional ethical guidelines of KKH. SingHealth and National Health Care Group Research Ethics Committees Singapore specifically approved this study (IRB no: 2013/778/D). The cord blood was purified by density centrifugation with Lymphoprep (Stemcell Technologies, Vancouver, BC, Canada) and red blood cells (RBC) were lysed by EasySep RBC lysis buffer (Stemcell Technologies). Thereafter, human CD34+ HSCs were enriched using EasySep Human Cord Blood CD34 Positive Selection kit (Stemcell Technologies) according to manufacturer’s instructions.

4.2. Mice and Transplantation of CD34+ Cells NSG mice were purchased from The Jackson Laboratory and bred in a specific pathogen-free condition at Biological Resource Centre (BRC) in Agency for Science, Technology and Research (A*STAR), Singapore. To generate humanized mice, one- to three-days-old NSG pups were sub-lethally irradiated at 1 Gy and human CD34+ HSC (2 105/mouse) were inoculated into the pups by intra-hepatic × injection [66]. Human immune cells engraftment (chimerism) in the mice was determined ten to twelve weeks after injection. In essence, blood samples were collected and composition of immune cells was analyzed by flow cytometry. The chimerism was calculated by (% human CD45+/(% human CD45+ + % mouse CD45+)), and humanized mice with the chimerism between 20% and 50% were used for subsequent experiments. All animal experiments were conducted in strict accordance to the guidelines on Care and Use of Animals for Scientific Purposes, which are released by National Advisory Committee on Laboratory Animal Research, Agri-Food and Veterinary Authority of Singapore and International Animal Care and Use Committee (IACUC) at A*STAR. The IACUC specifically approved this study (IACUC no: 181367 and 191440).

4.3. Transplantation of NPC-PDX and Drug Treatment NPC samples were collected from National Cancer Center Singapore (NCCS, Singapore), with written consent obtained from patients and in strict accordance with the institutional ethical guidelines of NCCS. SingHealth and National Health Care Group Research Ethics Committees Singapore specifically approved this study (IRB no: 2016/2887). Fresh NPC sample was cut into fragments (3 3 mm) and × transplanted in one flank of NSG mouse subcutaneously using aseptic technique. Size and volume of the tumor were recorded weekly using a caliper and passaged in the flank of recipient NSG mice once the tumor reached 10 10 mm in size. Tumor volume was calculated by using the formula: × Tumor volume = (length width2)/2, where length represents the longest tumor diameter and width × represents the perpendicular tumor diameter [67]. To evaluate the anti-tumor efficacy of immune checkpoint blockades, NPC was transplanted in one flank of humanized mice subcutaneously. Once the tumor reached 3 3 mm in size, nivolumab × (anti-PD-1 antibody; 3 mg/kg) and ipilimumab (anti-CTLA-4 antibody; 1 mg/kg) were administered into the mice intravenously weekly for four consecutive weeks.

4.4. H&E, EBER ISH and IHC Staining Tumors from NSG and humanized mice were harvested at experimental endpoint, which were formalin-fixed (Sigma, Saint Louis, MO, USA) at room temperature for 24 h. For H&E staining, the fixed tissues were paraffin-embedded (Leica, Germany), sliced into 5 µm sections (Leica) and stained with hematoxylin and eosin (Thermo Fisher Scientific, Waltham, MA, USA). The EBER ISH was conducted for the detection of EBV-positive cells, using Leica Bond-Max autostainer as previously described [68]. For the IHC staining, the tumor sections were stained for LMP1 and LMP2A using their respective Cancers 2020, 12, 1025 14 of 19 antibodies purchased from Abcam (Cambridge, MA, USA), followed by hematoxylin counterstain. All the stained images were captured using a ZEN fluorescence microscope (Zeiss, Germany) with ZEN 2 acquisition software (Zen Blue Version, Zeiss, Germany).

4.5. Isolation of Mononuclear Cells from Blood, Spleen and Tumor Humanized mice were cheek-bleed at specified time point. Blood samples were collected in EDTA tubes (Greiner Bio-One, Monroe, NC, USA) and centrifuged at 10, 000 g for 5 min. The plasma was × stored at 80 C for subsequent cytokines quantification. The cell pellet was resuspended in ACK lysis − ◦ buffer (Gibco, Grand Island, NY, USA) prior to DAPI and surface marker staining. At experimental endpoint, the spleen and tumor were harvested from the mice, and mononuclear cells (MNC) were isolated as reported elsewhere [69]. For the isolation of spleen MNC, the spleen was meshed with a syringe tip and the cell suspension was filtered through a 70 µm cell strainer (Thermo Fisher Scientific). To isolate TIL, the tumor was cut into 2 2 mm fragments and digested using Tumor × Dissociation Kit, human (Miltenyi Biotec, Germany) and gentleMACS Dissociator (Miltenyi Biotec). The cell suspension was filtered through a 70 µm cell strainer and the TIL was enriched by density centrifugation with Percoll (GE Healthcare, Piscataway, NJ, USA). All the MNC were subjected to RBC lysis using ACK lysis buffer prior to DAPI and surface marker staining.

4.6. Flow Cytometry Before surface marker staining, dead MNC were labeled with the dye provided in LIVE/DEAD Fixable Blue Dead Cell Stain kit (Sigma) at room temperature for 15 min. The MNC were then washed once with fluorescence-activated cell sorting (FACS) buffer and stained with antibodies at 4 ◦C for 15 min. The antibodies used for FACS are listed in Table S1. Flow cytometric analysis was performed on a LSRII flow cytometer using FACSDiva software (Becton Dickinson, Sparks, MD, USA). A total of 100,000 events were collected per sample and analyzed using Flowjo software version 10 (Treestar, Ashland, OR, USA).

4.7. Cytokines and Chemokines Quantification Cytokines and chemokines levels in the plasma from humanized mice were determined by LEGENDplex Human Inflammation Panel (13-plex) according to manufacturer’s instructions from BioLegend (San Diego, CA, USA). The profile was analyzed by flow cytometry and the LEGENDplex data analysis software (BioLegend).

4.8. Nanostring Analysis RNA was extracted from frozen tumor biopsy using AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. In brief, a total of 125 ng RNA (>300 bp) was assayed using nCounter PanCancer IO360 on the nCounter MAX Analysis System (NanoString Technologies, Inc., Seattle, WA, USA). The data was normalized and processed using Advance Analysis module in nSolver Software version 4.0 (NanoString Technologies, Inc.) [70]. Cell type proﬁling score was calculated as previously described [71].

4.9. ELISA for Human TGF-β1 and IFN-γ Total TGF-β1 level in the plasma from humanized mice was quantified by TGF beta 1 Human ELISA Kit (Abcam) according to manufacturer’s instructions. Tumor-infiltrating T cells in humanized mice were isolated using EasySep Human T Cell Isolation kit (Stemcell Technologies). Purified T cells (5 105 cells/mL) were seeded into a 96-well plate and × stimulated with T Cell TransAct (Miltenyi Biotec) for 24 h. After incubation, supernatant was collected and centrifuged once to remove cell debris. Human IFN-γ level in the cell-free supernatant was determined by LEGEND MAX Human IFN-γ ELISA Kit (BioLegend). Cancers 2020, 12, 1025 15 of 19

4.10. Statistical Analysis All statistical analysis was performed using GraphPad Prism version 8 (GraphPad, Inc., San Diego, CA, USA) and data are expressed as mean SEM. Unpaired Student’s t-test and one-way analysis of ± variance (ANOVA) with post hoc Tukey’s Multiple Comparison Test were used, whenever appropriate. The diﬀerences are considered as statistically signiﬁcant at p < 0.05.

5. Conclusions We have successfully established and characterized a novel humanized mouse NPC-PDX model where the anti-tumor eﬃcacy of combination cancer therapy can be robustly examined. It is anticipated that other therapeutic options, including but not limiting to cell-mediated therapy or other immune checkpoint blockades can be evaluated in our model.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/4/1025/s1, Figure S1: Gating strategy for NK cell, macrophage and T cell subsets, Figure S2: Modulation of immune cell subsets in blood in the presence of NPC in humanized mice, Figure S3: Modulation of immune cell subsets in spleen in the presence of NPC in humanized mice, Figure S4: Modulation of tumor-infiltrating immune cells in another NPC-PDX, Figure S5: Evaluation of the anti-tumor efficacy of nivolumab and ipilimumab in humanized mice, Figure S6: Modulation of immune cell profile in tumor after combination immunotherapy using another NPC-PDX, Table S1: FACS antibodies used in the present study. Author Contributions: Conceptualization, Q.C.; data curation, W.N.L.; formal analysis, W.N.L.; funding acquisition, D.W.-T.L., and Q.C.; investigation, W.N.L., S.Y.F., W.W.S.T., S.Y.T., M.L., J.Y.C., S.L. and L.S.; resources, E.K.H., J.K.Y.C., N.G.I., J.P.S.Y., D.W.-T.L. and Q.C.; supervision, D.W.-T.L. and Q.C.; writing—original draft, W.N.L.; writing—review and editing, D.W.-T.L., and Q.C. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the National Medical Research Council Singapore, CS-IRG (CIRG19may-0006) and NMRC/CSA-INV/0025/2017 to D.W.-T.L.; the VICTORY Programme (NMRC/OFLCG/003/2018), the Senior Clinician Scientist Award (CSA, NMRC/CSASI16nov006) and NMRC/TCR/015-NCC/2016, and by the National Research Foundation Singapore Fellowship (NRF-NRFF2017-03) and NRF-ISF joint grant (NRF2019-NRF-ISF003-3127) to Q.C., and also by A*STAR IAF-PP (H18/01/a0/022). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Young, L.S.; Yap, L.F.; Murray, P.G. Epstein–Barr virus: More than 50 years old and still providing surprises. Nat. Rev. Cancer 2016, 16, 789–802. [CrossRef][PubMed] 2. Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018, 68, 394–424. [CrossRef][PubMed] 3. Chen, W.; Zheng, R.; Baade, P.D.; Zhang, S.; Zeng, H.-M.; Bray, F.; Jemal, A.; Yu, X.Q.; He, J. Cancer statistics in China, 2015. CA Cancer J. Clin. 2016, 66, 115–132. [CrossRef][PubMed] 4. Chen, Y.P.; Chan, A.T.C.; Le, Q.T.; Blanchard, P.; Sun, Y.; Ma, J. Nasopharyngeal Carcinoma. Lancet 2019, 394, 64–80. [CrossRef] 5. Ia, W.-H.; Qin, H.-D. Non-viral environmental risk factors for nasopharyngeal carcinoma: A systematic review. Semin. Cancer Boil. 2012, 22, 117–126. 6. Jain, A.; Chia, W.K.; Toh, H.C. Immunotherapy for nasopharyngeal cancer—A review. Chin. Clin. Oncol. 2016, 5, 22. [CrossRef] 7. Tsao, S.W.; Tsang, C.M.; Lo, K.W. Epstein–Barr virus infection and nasopharyngeal carcinoma. Philos. Trans. R. Soc. B Boil. Sci. 2017, 372, 20160270. [CrossRef] 8. Chin, Y.-M.; Mushiroda, T.; Takahashi, A.; Kubo, M.; Krishnan, G.; Yap, L.F.; Teo, S.-H.; Lim, P.V.-H.; Yap, Y.-Y.; Pua, K.-C.; et al. HLA-ASNPs and amino acid variants are associated with nasopharyngeal carcinoma in Malaysian Chinese. Int. J. Cancer 2014, 136, 678–687. [CrossRef] 9. Jin, Y.; Shi, Y.X.; Cai, X.Y.; Xia, X.Y.; Cai, Y.C.; Cao, Y.; Zhang, W.D.; Hu, W.H.; Jiang, W.Q. Erratum To: Comparison of Five Cisplatin-Based Regimens Frequently Used as the First-Line Protocols in Metastatic Nasopharyngeal Carcinoma. J. Cancer Res. Clin. Oncol. 2015, 141, 767. [CrossRef] Cancers 2020, 12, 1025 16 of 19

10. Zhang, Y.; Chen, L.; Hu, G.Q.; Zhang, N.; Zhu, X.D.; Yang, K.Y.; Jin, F.; Shi, M.; Chen, Y.P.; Hu, W.H.; et al. Gemcitabine and Cisplatin Induction Chemotherapy in Nasopharyngeal Carcinoma. N. Engl. J. Med. 2019, 381, 1124–1135. [CrossRef] 11. Le, Q.-T.; Colevas, A.D.; O’Sullivan, B.; Lee, A.W.M.; Lee, N.; Ma, B.; Siu, L.L.; Waldron, J.; Lim, C.-M.; Riaz, N.; et al. Current Treatment Landscape of Nasopharyngeal Carcinoma and Potential Trials Evaluating the Value of Immunotherapy. J. Natl. Cancer Inst. 2019, 111, 655–663. [CrossRef][PubMed] 12. Lam, W.K.J.; Chan, J.Y.K. Recent advances in the management of nasopharyngeal carcinoma. F1000Research 2018, 7, 1829. [CrossRef][PubMed] 13. Chang, Y.-S.; Lin, S.-Y.; Lee, P.-F.; Chung, H.-C.; Tsai, M.-S. Establishment and Characterization of a Tumor Cell Line from Human Nasopharyngeal Carcinoma Tissue. Cancer Res. 1989, 49, 6752–6757. [PubMed] 14. Huang, D.P.; Ho, J.H.C.; Poon, Y.F.; Chew, E.C.; Saw, D.; Lui, M.; Li, C.L.; Mak, L.S.; Lai, S.H.; Lau, W.H. Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx. Int. J. Cancer 1980, 26, 127–132. [CrossRef] 15. Cheung, S.T.; Huang, D.P.; Hui, A.B.; Lo, K.W.; Ko, C.W.; Tsang, Y.S.; Wong, N.; Whitney, B.M.; Lee, J.C. Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int. J. Cancer 1999, 83, 121–126. [CrossRef] 16. Sacks, P.G.; Parnes, S.M.; Gallick, G.E.; Mansouri, Z.; Lichtner, R.; Satya-Prakash, K.L.; Pathak, S.; Parsons, D.F. Establishment and characterization of two new squamous cell carcinoma cell lines derived from tumors of the head and neck. Cancer Res. 1988, 48, 2858–2866. 17. Dittmer, D.P.; Hilscher, C.J.; Gulley, M.L.; Yang, E.V.; Chen, M.; Glaser, R. Multiple pathways for Epstein-Barr virus episome loss from nasopharyngeal carcinoma. Int. J. Cancer 2008, 123, 2105–2112. [CrossRef] 18. Busson, P.; Ganem, G.; Flores, P.; Mugneret, F.; Clausse, B.; Caillou, B.; Braham, K.; Wakasugi, H.; Lipiński,M.; Tursz, T. Establishment and characterization of three transplantable EBV-containing nasopharyngeal carcinomas. Int. J. Cancer 1988, 42, 599–606. [CrossRef] 19. Huang, D.P.; Ho, J.H.C.; Chan, W.K.; Lau, W.H.; Lui, M. Cytogenetics of undifferentiated nasopharyngeal carcinoma xenografts from southern chinese. Int. J. Cancer 1989, 43, 936–939. [CrossRef] 20. Lin, W.; Yip, Y.L.; Jia, L.; Deng, W.; Zheng, H.; Dai, W.; Ko, J.M.Y.; Lo, K.W.; Chung, G.T.Y.; Yip, K.Y.; et al. Establishment and Characterization of New Tumor Xenografts and Cancer Cell Lines from Ebv-Positive Nasopharyngeal Carcinoma. Nat. Commun. 2018, 9, 4663. [CrossRef] 21. Decker, W.K.; da Silva, R.F.; Sanabria, M.H.; Angelo, L.S.; Guimaraes, F.; Burt, B.M.; Kheradmand, F.; Paust, S. Cancer Immunotherapy: Historical Perspective of a Clinical Revolution and Emerging Preclinical Animal Models. Front Immunol. 2017, 8, 829. [CrossRef] 22. Zitvogel, L.; Pitt, J.M.; Daillère, R.; Smyth, M.J.; Kroemer, G. Mouse models in oncoimmunology. Nat. Rev. Cancer 2016, 16, 759–773. [CrossRef] 23. Sanmamed, M.F.; Rodriguez, I.; Schalper, K.A.; Onate, C.; Azpilikueta, A.; Rodriguez-Ruiz, M.E.; Morales-Kastresana, A.; Labiano, S.; Perez-Gracia, J.L.; Martin-Algarra, S.; et al. Nivolumab and Urelumab Enhance Antitumor Activity of Human T Lymphocytes Engrafted in Rag2-/-Il2rgammanull Immunodeficient Mice. Cancer Res. 2015, 75, 3466–3478. [CrossRef][PubMed] 24. Zhao, Y.; Shuen, T.W.H.; Toh, T.B.; Chan, X.Y.; Liu, M.; Tan, S.Y.; Fan, Y.; Yang, H.; Lyer, S.G.; Bonney, G.K.; et al. Development of a new patient-derived xenograft humanised mouse model to study human-specific tumour microenvironment and immunotherapy. Gut 2018, 67, 1845–1854. [CrossRef][PubMed] 25. Aspord, C.; Tramcourt, L.; Leloup, C.; Molens, J.-P.; Leccia, M.; Charles, J.; Plumas, J. Imiquimod Inhibits Melanoma Development by Promoting pDC Cytotoxic Functions and Impeding Tumor Vascularization. J. Investig. Dermatol. 2014, 134, 2551–2561. [CrossRef][PubMed] 26. Roth, M.D.; Harui, A. Human tumor infiltrating lymphocytes cooperatively regulate prostate tumor growth in a humanized mouse model. J. Immunother. Cancer 2015, 3, 12. [CrossRef] 27. Chang, D.-K.; Moniz, R.J.; Xu, Z.; Sun, J.; Signoretti, S.; Zhu, Q.; Marasco, W.A. Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo. Mol. Cancer 2015, 14, 119. [CrossRef] 28. Zhang, Y.-L.; Li, J.; Mo, H.-Y.; Qiu, F.; Zheng, L.; Qian, C.-N.; Zeng, Y.-X. Different subsets of tumor infiltrating lymphocytes correlate with NPC progression in different ways. Mol. Cancer 2010, 9, 4. [CrossRef] Cancers 2020, 12, 1025 17 of 19

29. Lakhdar, M.; Ben Aribia, M.H.; Maalej, M.; Ladgham, A. Selective homing of phenotypically lytic cells within nasopharyngeal carcinoma biopsies: Numerous CD8- and CD16-positive cells in the tumor. Int. J. Cancer 1991, 48, 57–61. [CrossRef] 30. Thommen, D.S.; Schumacher, T.N. T Cell Dysfunction in Cancer. Cancer Cell 2018, 33, 547–562. [CrossRef] 31. Hsu, C.; Lee, S.-H.; Ejadi, S.; Even, C.; Cohen, R.B.; Le Tourneau, C.; Mehnert, J.M.; Algazi, A.; Van Brummelen, E.M.; Saraf, S.; et al. Safety and Antitumor Activity of Pembrolizumab in Patients With Programmed Death-Ligand 1–Positive Nasopharyngeal Carcinoma: Results of the KEYNOTE-028 Study. J. Clin. Oncol. 2017, 35, 4050–4056. [CrossRef][PubMed] 32. Ma, B.B.Y.; Lim, W.T.; Goh, B.C.; Hui, E.P.; Lo, K.W.; Pettinger, A.; Foster, N.R.; Riess, J.W.; Agulnik, M.; Chang, A.Y.C.; et al. Antitumor Activity of Nivolumab in Recurrent and Metastatic Nasopharyngeal Carcinoma: An International, Multicenter Study of the Mayo Clinic Phase 2 Consortium (Nci-9742). J. Clin. Oncol. 2018, 36, 1412–1418. [CrossRef][PubMed] 33. Fang, W.; Yang, Y.; Ma, Y.; Hong, S.; Lin, L.; He, X.; Xiong, J.; Li, P.; Zhao, H.; Huang, Y.; et al. Camrelizumab (SHR-1210) alone or in combination with gemcitabine plus cisplatin for nasopharyngeal carcinoma: Results from two single-arm, phase 1 trials. Lancet Oncol. 2018, 19, 1338–1350. [CrossRef] 34. Hsu, C.-L.; Lui, K.-W.; Chi, L.-M.; Kuo, Y.-C.; Chao, Y.-K.; Yeh, C.-N.; Lee, L.-Y.; Huang, Y.; Lin, T.-L.; Huang, M.-Y.; et al. Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma. J. Exp. Clin. Cancer Res. 2018, 37, 233. [CrossRef] 35. Cho, S.-Y.; Kang, W.; Han, J.Y.; Min, S.; Kang, J.; Lee, A.; Kwon, J.Y.; Lee, C.; Park, H. An Integrative Approach to Precision Cancer Medicine Using Patient-Derived Xenografts. Mol. Cells 2016, 39, 77–86. 36. Dunn, G.P.; Old, L.J.; Schreiber, R.D. The Three Es of Cancer Immunoediting. Annu. Rev. Immunol. 2004, 22, 329–360. [CrossRef] 37. Mittal, D.; Gubin, M.M.; Schreiber, R.D.; Smyth, M.J. New insights into cancer immunoediting and its three component phases–elimination, equilibrium and escape. Curr. Opin. Immunol. 2014, 27, 16–25. [CrossRef] 38. Deshmane, S.L.; Kremlev, S.; Amini, S.; Sawaya, B.E. Monocyte Chemoattractant Protein-1 (MCP-1): An Overview. J. Interf. Cytokine Res. 2009, 29, 313–326. [CrossRef] 39. Dighe, A.S.; Richards, E.; Old, L.J.; Schreiber, R.D. Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFN gamma receptors. Immunity 1994, 1, 447–456. [CrossRef] 40. Kaplan, D.H.; Shankaran, V.; Dighe, A.S.; Stockert, E.; Aguet, M.; Old, L.J.; Schreiber, R.D. Demonstration of an interferon γ-dependent tumor surveillance system in immunocompetent mice. Proc. Natl. Acad. Sci. USA 1998, 95, 7556–7561. [CrossRef] 41. Fisher, D.T.; Appenheimer, M.M.; Evans, S.S. The two faces of IL-6 in the tumor microenvironment. Semin. Immunol. 2014, 26, 38–47. [CrossRef][PubMed] 42. David, J.M.; Dominguez, C.; Hamilton, D.H.; Palena, C. The Il-8/Il-8r Axis: A Double Agent in Tumor Immune Resistance. Vaccines (Basel) 2016, 4, 22. [CrossRef][PubMed] 43. Massague, J. Tgfbeta in Cancer. Cell 2008, 134, 215–230. [CrossRef][PubMed] 44. Xu, J.; Menezes, J.; Prasad, U.; Ahmad, A. Elevated serum levels of transforming growth factor beta1 in Epstein-Barr virus-associated nasopharyngeal carcinoma patients. Int. J. Cancer 1999, 84, 396–399. [CrossRef] 45. Larbcharoensub, N.; Mahaprom, K.; Jiarpinitnun, C.; Trachu, N.; Tubthong, N.; Pattaranutaporn, P.; Sirachainan, E.; Ngaimphaiboon, N. Characterization of PD-L1 and PD-1 Expression and CD8+ Tumor-infiltrating Lymphocyte in Epstein-Barr Virus-associated Nasopharyngeal Carcinoma. Am. J. Clin. Oncol. 2018, 41, 1204–1210. [CrossRef] 46. Wang, Y.-Q.; Chen, Y.-P.; Zhang, Y.; Jiang, W.; Liu, N.; Yun, J.-P.; Sun, Y.; He, Q.-M.; Tang, X.-R.; Wen, X.; et al. Prognostic significance of tumor-infiltrating lymphocytes in nondisseminated nasopharyngeal carcinoma: A large-scale cohort study. Int. J. Cancer 2018, 142, 2558–2566. [CrossRef] 47. Zhu, Q.; Cai, M.-Y.; Chen, C.-L.; Hu, H.; Lin, H.-X.; Li, M.; Weng, D.-S.; Zhao, J.-J.; Guo, L.; Xia, J. Tumor cells PD-L1 expression as a favorable prognosis factor in nasopharyngeal carcinoma patients with pre-existing intratumor-infiltrating lymphocytes. OncoImmunology 2017, 6, e1312240. [CrossRef] 48. Jiang, Y.; Li, Y.; Zhu, B. T-cell exhaustion in the tumor microenvironment. Cell Death Dis. 2015, 6, e1792. [CrossRef] 49. He, Q.-F.; Xu, Y.; Li, J.; Huang, Z.-M.; Li, X.-H.; Wang, X. CD8+ T-cell exhaustion in cancer: Mechanisms and new area for cancer immunotherapy. Briefings Funct. Genom. 2018, 18, 99–106. [CrossRef] Cancers 2020, 12, 1025 18 of 19

50. Callahan, M.K.; Wolchok, J.D. At the bedside: CTLA-4- and PD-1-blocking antibodies in cancer immunotherapy. J. Leukoc. Boil. 2013, 94, 41–53. [CrossRef] 51. Huang, P.-Y.; Guo, S.-S.; Zhang, Y.; Lu, J.-B.; Chen, Q.-Y.; Tang, L.-Q.; Zhang, L.; Liu, L.-T.; Zhang, L.; Mai, H.-Q. Tumor CTLA-4 overexpression predicts poor survival in patients with nasopharyngeal carcinoma. Oncotarget 2016, 7, 13060–13068. [CrossRef][PubMed] 52. Lim, D.T.W.; Wang, H.M.; Li, S.H.; Ngan, R.; Dechaphunkul, A.; Zhang, L.; Yen, C.J.; Chan, P.C.; Chakrabandhu, S.; Ma, B.; et al. Phase Ii Study of Spartalizumab (Pdr001) Vs Chemotherapy (Ct) in Patients with Recurrent/Metastatic Nasopharyngeal Cancer (Npc). Cancer Res. 2019, 79, 615. 53. Andersson, P.; Ostheimer, C. Editorial: Combinatorial Approaches to Enhance Anti-tumor Immunity: Focus on Immune Checkpoint Blockade Therapy. Front. Immunol. 2019, 10, 2083. [CrossRef][PubMed] 54. Yong, K.S.M.; Her, Z.; Chen, Q. Humanized Mice as Unique Tools for Human-Specific Studies. Arch. Immunol. Ther. Exp. 2018, 66, 245–266. [CrossRef][PubMed] 55. Traggiai, E.; Chicha, L.; Mazzucchelli, L.; Bronz, L.; Piffaretti, J.-C.; Lanzavecchia, A.; Manz, M.G. Development of a Human Adaptive Immune System in Cord Blood Cell-Transplanted Mice. Science 2004, 304, 104–107. [CrossRef][PubMed] 56. Ishikawa, F.; Yasukawa, M.; Lyons, B.; Yoshida, S.; Miyamoto, T.; Yoshimoto, G.; Watanabe, T.; Akashi, K.; Shultz, L.D.; Harada, M. Development of Functional Human Blood and Immune Systems in Nod/Scid/Il2 Receptor {Gamma} Chain(Null) Mice. Blood 2005, 106, 1565–1573. [CrossRef][PubMed] 57. Zhao, E.; Xu, H.; Wang, L.; Kryczek, I.; Wu, K.; Hu, Y.; Wang, G.; Zou, W. Bone marrow and the control of immunity. Cell. Mol. Immunol. 2011, 9, 11–19. [CrossRef][PubMed] 58. Shultz, L.D.; Ishikawa, F.; Greiner, D.L. Humanized mice in translational biomedical research. Nat. Rev. Immunol. 2007, 7, 118–130. [CrossRef] 59. Shultz, L.D.; Saito, Y.; Najima, Y.; Tanaka, S.; Ochi, T.; Tomizawa, M.; Doi, T.; Sone, A.; Suzuki, N.; Fujiwara, H.; et al. Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice. Proc. Natl. Acad. Sci. USA 2010, 107, 13022–13027. [CrossRef] 60. Chen, Q.; Khoury, M.; Chen, J. Expression of human cytokines dramatically improves reconstitution of specific human-blood lineage cells in humanized mice. Proc. Natl. Acad. Sci. USA 2009, 106, 21783–21788. [CrossRef] 61. Chen, Q.; He, F.; Kwang, J.; Chan, J.K.Y.; Chen, J. GM-CSF and IL-4 stimulate antibody responses in humanized mice by promoting T, B, and dendritic cell maturation. J. Immunol. 2012, 189, 5223–5229. [CrossRef][PubMed] 62. Shultz, L.D.; Keck, J.; Burzenski, L.; Jangalwe, S.; Vaidya, S.; Greiner, D.L.; Brehm, M.A. Humanized mouse models of immunological diseases and precision medicine. Mamm. Genome 2019, 30, 123–142. [CrossRef] [PubMed] 63. Malaney, P.; Nicosia, S.V.; Davé, V. One mouse, one patient paradigm: New avatars of personalized cancer therapy. Cancer Lett. 2013, 344, 1–12. [CrossRef][PubMed] 64. De La Rochère, P.; Guil-Luna, S.; Decaudin, D.; Azar, G.; Sidhu, S.S.; Piaggio, E. Humanized Mice for the Study of Immuno-Oncology. Trends Immunol. 2018, 39, 748–763. [CrossRef] 65. Taoka, K.; Arai, S.; Kataoka, K.; Hosoi, M.; Miyauchi, M.; Yamazaki, S.; Honda, A.; Aixinjueluo, W.; Kobayashi, T.; Kumano, K.; et al. Using patient-derived iPSCs to develop humanized mouse models for chronic myelomonocytic leukemia and therapeutic drug identification, including liposomal clodronate. Sci. Rep. 2018, 8, 15855. [CrossRef] 66. Keng, C.T.; Sze, C.W.; Zheng, D.; Zheng, Z.; Yong, K.S.; Tan, S.Q.; Ong, J.J.; Tan, S.Y.; Loh, E.; Upadya, M.H.; et al. Characterisation of Liver Pathogenesis, Human Immune Responses and Drug Testing in a Humanised Mouse Model of Hcv Infection. Gut 2016, 65, 1744–1753. [CrossRef] 67. Tomayko, M.M.; Reynolds, C.P. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother. Pharmacol. 1989, 24, 148–154. [CrossRef] 68. Wee, Y.T.F.; Alkaff, S.M.F.; Lim, J.C.T.; Loh, J.J.H.; Hilmy, M.H.; Ong, C.; Nei, W.L.; Jain, A.; Lim, A.; Takano, A.; et al. An integrated automated multispectral imaging technique that simultaneously detects and quantitates viral RNA and immune cell protein markers in fixed sections from Epstein-Barr virus-related tumours. Ann. Diagn. Pathol. 2018, 37, 12–19. [CrossRef] Cancers 2020, 12, 1025 19 of 19

69. Ke, Y.; Liu, W.N.; Her, Z.; Liu, M.; Tan, S.Y.; Tan, Y.W.; Chan, X.Y.; Fan, Y.; Huang, E.K.; Chen, H.; et al. Enterovirus A71 Infection Activates Human Immune Responses and Induces Pathological Changes in Humanized Mice. J. Virol. 2019, 93, e01066-18. [CrossRef] 70. Cesano, A. Ncounter((R)) Pancancer Immune Proﬁling Panel (Nanostring Technologies, Inc., Seattle, Wa). J. Immunother Cancer 2015, 3, 42. [CrossRef] 71. Danaher, P.; Warren, S.; Dennis, L.; D’Amico, L.; White, A.; Disis, M.L.; Geller, M.A.; Odunsi, K.; Beechem, J.; Fling, S.P. Gene expression markers of Tumor Inﬁltrating Leukocytes. J. Immunother. Cancer 2017, 5, 18. [CrossRef][PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).