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Abstract Background The plasma concentrations of acyl coenzyme A binding protein (ACBP, also known as diazepam-binding inhibitor, DBI, or ‘endozepine’) increase with age and obesity, two parameters that are also amongst the most important risk factors for cancer. Methods We measured ACBP/DBI in the plasma from cancer-free individuals, high-risk patients like the carriers of TP53 or BRCA1/2 mutations, and non-syndromic healthy subjects who later developed cancer. In mice, the neutralization of ACBP/DBI was used in models of non-small cell lung cancer (NSCLC) and breast cancer development and as a combination treatment with chemoimmunotherapy (chemotherapy + PD-1 blockade) in the context of NSCLC and sarcomas. The anticancer T cell response upon ACBP/DBI neutralization was characterized by flow cytometry and single-cell RNA sequencing. Results Circulating levels of ACBP/DBI were higher in patients with genetic cancer predisposition (BRCA1/2 or TP53 germline mutations) than in matched controls. In non-syndromic cases, high ACBP/DBI levels were predictive of future cancer development, and especially elevated in patients who later developed lung cancer. In preclinical models, ACBP/DBI neutralization slowed down breast cancer and NSCLC development and enhanced the efficacy of chemoimmunotherapy in NSCLC and sarcoma models. When combined with chemoimmunotherapy, the neutralizing monoclonal antibody against ACBP/DBI reduced the frequency of regulatory T cells in the tumor bed, modulated the immune checkpoint profile, and increased activation markers. Conclusion These findings suggest that ACBP/DBI acts as an endogenous immune suppressor. We conclude that elevation of ACBP/DBI constitutes a risk factor for the development of cancer and that ACBP/DBI is an actionable target for improving cancer immunosurveillance.

ABSTRACTExtra-cellular galectins 1, 3 and 9 (gal-1, −3 and −9) are known to act as soluble immunosuppressive agents in various malignancies. Previous publications have suggested that their expression is dependent on the metabolic status of producing cells and reciprocally that they can influence metabolic pathways in their target cells. Very little is known about the status of gal-1, −3 and −9 in patients bearing head and neck squamous cell carcinomas (HNSCC) and about their relationships with the systemic metabolic condition. This study was conducted in plasma samples from a prospective cohort of 83 HNSCC patients with advanced disease. These samples were used to explore the distribution of gal-1, −3 and −9 and simultaneously to profile a series of 87 metabolites assessed by mass spectrometry. We identified galectin and metabolic patterns within five disease categories defined according to the primary site and human papillomavirus (HPV) status (HPV-positive and -negative oropharyngeal carcinomas, carcinomas of the oral cavity, hypopharynx and larynx carcinomas). Remarkably, samples related to hypopharyngeal carcinomas displayed the highest average concentration of gal-9 (p = .017) and a trend toward higher concentrations of kynurenine, a potential factor of tumor growth and immune suppression. In contrast, there was a tendency toward higher concentrations of fatty acids in samples related to oral cavity. These observations emphasize the diversity of HPV-negative HNSCCs. Depending on their primary site, they evolve into distinct types of immune and metabolic landscapes that seem to be congruent with specific oncogenic mechanisms.

Tumor necrosis is a recurrent characteristic of head and neck squamous cell carcinomas (HNSCCs). There is a need for more investigations on the influence of biomolecules released by these necrotic foci in the HNSCC tumor microenvironment. It is suspected that a fraction of the biomolecules released by necrotic cells are damage-associated molecular patterns (DAMPs), which are known to be natural endogenous ligands of Toll-like receptors (TLRs), including, among others, proteins and nucleic acids. However, there has been no direct demonstration that biomolecules released by HNSCC necrotic cells can activate TLRs. Our aim was to investigate whether some of these molecules could behave as agonists of the TLR3, either in vitro or in vivo. We chose a functional approach based on reporter cell exhibiting artificial TLR3 expression and downstream release of secreted alkaline phosphatase. The production of biomolecules activating TLR3 was first investigated in vitro using three HNSCC cell lines subjected to various pronecrotic stimuli (external irradiation, serum starvation, hypoxia and oxidative stress). TLR3 agonists were also investigated in necrotic tumor fluids from five oral cancer patients and three mouse tumor grafts. The release of biomolecules activating TLR3 was demonstrated for all three HNSCC cell lines. External irradiation was the most consistently efficient stimulus, and corresponding TLR3 agonists were conveyed in extracellular vesicles. TLR3-stimulating activity was detected in the fluids from all five patients and three mouse tumor grafts. In most cases, this activity was greatly reduced by RNAse pretreatment or TLR3 blocking antibodies. Our data indicate that TLR3 agonists are consistently present in necrotic fluids from HNSCC cells and mainly made of dsRNA fragments. These endogenous agonists may induce TLR3, which might lead to a protumorigenic effect. Regarding methodological aspects, our study demonstrates that direct investigations—including functional testing—can be performed on necrotic fluids from patient tumors.

ABSTRACT The immune regulator galectin-9 (Gal-9) is commonly involved in the regulation of cell proliferation, but with various impacts depending on the cell type. Here, we revealed that Gal-9 expression was persistently increased in Epstein-Barr virus (EBV)-infected primary B cells from the stage of early infection to the stage of mature lymphoblastoid cell lines (LCLs). This sustained upregulation paralleled that of gene sets related to cell proliferation, such as oxidative phosphorylation, cell cycle activation, and DNA replication. Knocking down or blocking Gal-9 expression obstructed the establishment of latent infection and outgrowth of EBV-infected B cells, while exogenous Gal-9 protein promoted EBV acute and latent infection and outgrowth of EBV-infected B cells at the early infection stage. Mechanically, stimulator of interferon gene (STING) activation or signal transducer and activator of transcription 3 (STAT3) inhibition impeded the outgrowth of EBV-infected B cells and promotion of Gal-9-induced lymphoblastoid cell line (LCL) transformation. Accordingly, Gal-9 expression was upregulated by forced EBV nuclear antigen 1 (EBNA1) expression in 293T cells in vitro. Clinical data showed that Gal-9 expression in B-cell lymphomas (BCLs) correlated positively with EBNA1 and disease stage. Targeting Gal-9 slowed LCL tumor growth and metastasis in xenografted immunodeficient mice. These findings highlight an oncogenic role of Gal-9 in EBV-associated BCLs, indicating that Gal-9 boosts the transformation of EBV-infected B cells. IMPORTANCE The cross talk between Epstein-Barr virus (EBV) and the host cell transcriptome assumes important roles in the oncogenesis of EBV-associated malignancies. Here, we first observed that endogenous Gal-9 expression was persistently increased along with an overturned V-type change in antivirus signaling during the immortalization of EBV-transformed B cells. Upregulation of Gal-9 promoted the outgrowth and latent infection of EBV-infected B cells, which was linked to B-cell-origin tumors by suppressing STING signaling and subsequently promoting STAT3 phosphorylation. EBV nuclear antigen EBNA1 induced Gal-9 expression and formed a positive feedback loop with Gal-9 in EBV-infected B cells. Tumor Gal-9 levels were positively correlated with disease stage and EBNA1 expression in patients with B-cell lymphomas (BCLs). Targeting Gal-9 slowed the growth and metastases of LCL tumors in immunodeficient mice. Altogether, our findings indicate that Gal-9 is involved in the lymphomagenesis of EBV-positive BCLs through cross talk with EBNA1 and STING signals.

Galectins are galactoside-binding proteins, exerting numerous functions inside and outside the cell, particularly conferring adaptation to stress factors. For most of them, aberrant expression profiles have been reported in the context of cancer. Albeit not being oncogenic drivers, galectins can be harnessed to exacerbate the malignant phenotype. Their impact on disease establishment and progression is not limited to making cancer cells resistant to apoptosis, but is prominent in the context of the tumor microenvironment, where it fosters angiogenesis, immune escape and exclusion. This review focuses mainly on Gal-1, Gal-3 and Gal-9 for which the involvement in cancer biology is best known. It presents the types of galectin dysregulations, attempts to explain the mechanisms behind them and analyzes the different ways in which they favor tumour growth. In an era where tumour resistance to immunotherapy appears as a major challenge, we highlight the crucial immunosuppressive roles of galectins and the potential therapeutic benefits of combinatorial approaches including galectin inhibition.

Abstract Mechanisms of tumor immune escape are quite diverse and require specific approaches for their exploration in syngeneic tumor models. In several human malignancies, galectin-9 (gal-9) is suspected to contribute to the immune escape. However, in contrast with what has been done for the infiltrating cells, the contribution of gal-9 produced by malignant cells has never been demonstrated in an animal model. Therefore, we derived isogenic clones—either positive or negative for gal-9—from the MB49 murine bladder carcinoma cell line. A progressive and consistent reduction of tumor growth was observed when gal-9-KO cells were subjected to serial transplantations into syngeneic mice. In contrast, tumor growth was unaffected during parallel serial transplantations into nude mice, thus linking tumor inhibition to the enhancement of the immune response against gal-9-KO tumors. This stronger immune response was at least in part explained by changing patterns of response to interferon-γ. One consistent change was a more abundant production of CXCL10, a major inflammatory factor whose production is often induced by interferon-γ. Overall, these observations demonstrate for the first time that serial transplantation into syngeneic mice can be a valuable experimental approach for the exploration of novel mechanisms of tumor immune escape.

Nasopharyngeal carcinoma (NPC) lacks effective diagnostic and therapeutic strategies, in particular at advanced stages. Here, the authors show that expression of the somatostatin receptor 2 is induced by Epstein-Barr virus in NPC and has a key role in the diagnosis, imaging, targeted therapies and prognosis of NPC.

Damage-associated molecular patterns (DAMPs) are endogenous molecules released from the necrotic cells dying after exposure to various stressors. After binding to their receptors, they can stimulate various signaling pathways in target cells. DAMPs are especially abundant in the microenvironment of malignant tumors and are suspected to influence the behavior of malignant and stromal cells in multiple ways often resulting in promotion of cell proliferation, migration, invasion, and metastasis, as well as increased immune evasion. This review will start with a reminder of the main features of cell necrosis, which will be compared to other forms of cell death. Then we will summarize the various methods used to assess tumor necrosis in clinical practice including medical imaging, histopathological examination, and/or biological assays. We will also consider the importance of necrosis as a prognostic factor. Then the focus will be on the DAMPs and their role in the tumor microenvironment (TME). We will address not only their interactions with the malignant cells, frequently leading to cancer progression, but also with the immune cells and their contribution to immunosuppression. Finally, we will emphasize the role of DAMPs released by necrotic cells in the activation of Toll-like receptors (TLRs) and the possible contributions of TLRs to tumor development. This last point is very important for the future of cancer therapeutics since there are attempts to use TLR artificial ligands for cancer therapeutics.

Background: Fibroblast-like synoviocytes (FLSs) are essential mediators in the expansive growth and invasiveness of rheumatoid synovitis, and patients with a fibroblastic-rich pauci-immune pathotype respond poorly to currently approved antirheumatic drugs. Galectin-9 (Gal-9) has been reported to directly modulate rheumatoid arthritis (RA) FLSs and to hold both pro- and anti-inflammatory properties. The objective of this study was to evaluate clinical and pathogenic aspects of Gal-9 in RA, combining national patient cohorts and cellular models. Methods: Soluble Gal-9 was measured in plasma from patients with newly diagnosed, treatment-naïve RA (n = 98). The disease activity score 28-joint count C-reactive protein (DAS28CRP) and total Sharp score were used to evaluate the disease course serially over a two-year period. Plasma and synovial fluid samples were examined for soluble Gal-9 in patients with established RA (n = 18). A protein array was established to identify Gal-9 binding partners in the extracellular matrix (ECM). Synovial fluid mononuclear cells (SFMCs), harvested from RA patients, were used to obtain synovial-fluid derived FLSs (SF-FLSs) (n = 7). FLSs from patients suffering from knee Osteoarthritis (OA) were collected from patients when undergoing joint replacement surgery (n = 5). Monocultures of SF-FLSs (n = 6) and autologous co-cultures of SF-FLSs and peripheral blood mononuclear cells (PBMCs) were cultured with and without a neutralizing anti-Gal-9 antibody (n = 7). The mono- and co-cultures were subsequently analyzed by flow cytometry, MTT assay, and ELISA. Results: Patients with early and established RA had persistently increased plasma levels of Gal-9 compared with healthy controls (HC). The plasma levels of Gal-9 were associated with disease activity and remained unaffected when adding a TNF-inhibitor to their standard treatment. Gal-9 levels were elevated in the synovial fluid of established RA patients with advanced disease, compared with corresponding plasma samples. Gal-9 adhered to fibronectin, laminin and thrombospondin, while not to interstitial collagens in the ECM protein array. In vitro, a neutralizing Gal-9 antibody decreased MCP-1 and IL-6 production from both RA FLSs and OA FLSs. In co-cultures of autologous RA FLSs and PBMCs, the neutralization of Gal-9 also decreased MCP-1 and IL-6 production, without affecting the proportion of inflammatory FLSs. Conclusions: In RA, pretreatment plasma Gal-9 levels in early RA were increased and correlated with clinical disease activity. Gal-9 levels remained increased despite a significant reduction in the disease activity score in patients with early RA. The in vitro neutralization of Gal-9 decreased both MCP-1 and IL-6 production in an inflammatory subset of RA FLSs. Collectively these findings indicate that the persistent overexpression of Gal-9 in RA may modulate synovial FLS activities and could be involved in the maintenance of subclinical disease activity in RA.

The Epstein-Barr Virus (EBV) is involved in the etiology of multiple hematologic and epithelial human cancers. EBV+ tumors employ multiple immune escape mechanisms, including the recruitment of immunosuppressive regulatory T cells (Treg). Here, we show some EBV+ tumor cells express high levels of the chemokines CCL17 and CCL22 both in vitro and in vivo and that this expression mirrors the expression levels of expression of the EBV LMP1 gene in vitro. Patient samples from lymphoblastic (Hodgkin lymphoma) and epithelial (nasopharyngeal carcinoma; NPC) EBV+ tumors revealed CCL17 and CCL22 expression of both tumor cell-intrinsic and -extrinsic origin, depending on tumor type. NPCs grown as mouse xenografts likewise showed both mechanisms of chemokine production. Single cell RNA-sequencing revealed in vivo tumor cell-intrinsic CCL17 and CCL22 expression combined with expression from infiltrating classical resident and migratory dendritic cells in a CT26 colon cancer mouse tumor engineered to express LMP1. These data suggest that EBV-driven tumors employ dual mechanisms for CCL17 and CCL22 production. Importantly, both in vitro and in vivo Treg migration was effectively blocked by a novel, small molecule antagonist of CCR4, CCR4-351. Antagonism of the CCR4 receptor may thus be an effective means of activating the immune response against a wide spectrum of EBV+ tumors.

Nasopharyngeal carcinoma (NPC) is a highly malignant epithelial cancer linked to EBV infection. Addition of interferon-β (IFNβ) to chemo- and radiochemotherapy has led to survival rates >90% in children and adolescents. As NPC cells are sensitive to apoptosis via tumor necrosis factor-related apoptosis inducing ligand (TRAIL), we explored the role of TRAIL and IFNβ in the killing of NPC cells by natural killer (NK) cells. NPC cells, including cells of a patient-derived xenograft were exposed to NK cells in the presence or absence of IFNβ. NK cells killed NPC- but not nasoepithelial cells and killing was predominately mediated via TRAIL. Incubation of NK cells with IFNβ increased cytotoxicity against NPC cells. Concomitant incubation of NK- and NPC cells with IFNβ before coculture reduced cytotoxicity and could be overcome by blocking the PD-1/PD-L1 axis leading to the release of intracellular TRAIL from NK cells. In conclusion, combination of IFNβ and anti-PD-1, augmenting cytotoxicity of NK cells against NPC cells, could be a strategy to improve NPC-directed therapy and warrants further evaluation in vivo.

The lack of appropriate models restricts pre-clinical research for nasopharyngeal carcinoma (NPC). Here the authors report the development and characterization of NPC patient-derived xenografts (PDXs), and EBV positive NPC cell line from patient tumor, and suggest their potential use in future NPC research.

Background: Nasopharyngeal carcinoma (NPC) in endemic regions and younger patients is characterized by a prominent lymphomononuclear infiltration. Radiation is the principal therapeutic modality for patients with NPC. Recent data suggest that the efficacy of radiotherapy in various cancers can be augmented when combined with immune checkpoint blockade. Here, we investigate the effect of radiotherapy on the killing of NPC cells by Natural Killer (NK) cells. Methods: NPC cell lines and a patient-derived xenograft were exposed to NK cells in the context of radiotherapy. Cytotoxicity was measured using the calcein-release assay. The contribution of the PD-L1/PD-1 checkpoint and signaling pathways to killing were analyzed using specific inhibitors. Results: Radiotherapy sensitized NPC cells to NK cell killing and upregulated expression of PD-1 ligand (PD-L1) in NPC cells and PD-1 receptor (PD-1) in NK cells. Blocking of the PD-L1/PD-1 checkpoint further increased the killing of NPC cells by NK cells in the context of radiotherapy. Conclusion: Radiation boosts the killing of NPC cells by NK cells. Killing can be further augmented by blockade of the PD-L1/PD-1 checkpoint. The combination of radiotherapy with PD-L1/PD-1 checkpoint blockade could therefore increase the efficacy of radiotherapy in NPC tumors.

Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor, most commonly located in the pharyngeal recess and endemic to parts of Asia. It is often detected at a late stage which is associated with poor prognosis (5-year survival rate of 63%). Treatment for this malignancy relies predominantly on radiotherapy and/or systemic chemotherapy, which can be associated with significant morbidity and impaired quality of life. In endemic regions NPC is associated with infection by Epstein–Barr virus (EBV) which was shown to upregulate the somatostatin receptor 2 (SSTR2) cell surface receptor. With recent advances in molecular techniques allowing for an improved understanding of the molecular aetiology of this disease and its relation to SSTR2 expression, we provide a comprehensive and up-to-date overview of this disease and highlight the emergence of SSTR2 as a key tumor biomarker and promising target for imaging and therapy.

Extra-cellular galectin-9 (gal-9) is an immuno-modulatory protein with predominant immunosuppressive effects. Inappropriate production of gal-9 has been reported in several human malignancies and viral diseases like nasopharyngeal, pancreatic and renal carcinomas, metastatic melanomas and chronic active viral hepatitis. Therefore therapeutic antibodies neutralizing extra-cellular gal-9 are expected to contribute to immune restoration in these pathological conditions. Two novel monoclonal antibodies targeting gal-9 -Gal-Nab 1 and 2-have been produced and characterized in this study. We report a protective effect of Gal-Nab1 and Gal-Nab2 on the apoptotic cell death induced by gal-9 in primary T cells. In addition, they inhibit late phenotypic changes observed in peripheral T cells that survive gal-9-induced apoptosis. Gal-Nab1 and Gal-Nab2 bind nearly identical, overlapping linear epitopes contained in the 213-224 amino-acid segments of gal-9. Nevertheless, they have some distinct functional characteristics suggesting that their three-dimensional epitopes are distinct. These differences are best demonstrated when gal-9 is applied on Jurkat cells where Gal-Nab1 is less efficient than Gal-Nab2 in the prevention of apoptotic cell death. In addition, Gal-Nab1 stimulates non-lethal phosphatidylserine translocation at the plasma membrane and calcium mobilization triggered by gal-9 in these cells. Both Gal-Nab1 and 2 cross-react with murine gal-9. They bind its natural as well as its recombinant form. This cross-species recognition will be an advantage for their assessment in pre-clinical tumor models.

The presence of the Epstein-Barr virus (EBV)-encoded nuclear antigen-1 (EBNA1) protein in all EBV-carrying tumours constitutes a marker that distinguishes the virus-associated cancer cells from normal cells and thereby offers opportunities for targeted therapeutic intervention. EBNA1 is essential for viral genome maintenance and also for controlling viral gene expression and without EBNA1, the virus cannot persist. EBNA1 itself has been linked to cell transformation but the underlying mechanism of its oncogenic activity has been unclear. However, recent data are starting to shed light on its growth-promoting pathways, suggesting that targeting EBNA1 can have a direct growth suppressing effect. In order to carry out its tasks, EBNA1 interacts with cellular factors and these interactions are potential therapeutic targets, where the aim would be to cripple the virus and thereby rid the tumour cells of any oncogenic activity related to the virus. Another strategy to target EBNA1 is to interfere with its expression. Controlling the rate of EBNA1 synthesis is critical for the virus to maintain a sufficient level to support viral functions, while at the same time, restricting expression is equally important to prevent the immune system from detecting and destroying EBNA1-positive cells. To achieve this balance EBNA1 has evolved a unique repeat sequence of glycines and alanines that controls its own rate of mRNA translation. As the underlying molecular mechanisms for how this repeat suppresses its own rate of synthesis in cis are starting to be better understood, new therapeutic strategies are emerging that aim to modulate the translation of the EBNA1 mRNA. If translation is induced, it could increase the amount of EBNA1-derived antigenic peptides that are presented to the major histocompatibility (MHC) class I pathway and thus, make EBV-carrying cancers better targets for the immune system. If translation is further suppressed, this would provide another means to cripple the virus.

The oncogenic process leading to nasopharyngeal carcinoma (NPC) requires the combination of genetic and epigenetic alterations, latent infection by the Epstein-Barr virus and local inflammation. A transcriptome analysis of NPC xenografts identified the gene encoding the cellular inhibitor of apoptosis protein 2 (c-IAP2) among the top five most intensely expressed. Consistently, the very high levels of the c-IAP2 protein were detected in 11 of 13 NPC biopsies. RMT 5265, a structural analog of second mitochondria-derived activator of caspase (SMAC), induced the rapid degradation of c-IAP2 in nasopharyngeal epithelial cells, whether malignant or not, but blocked clonal cell growth in NPC cells only. In short-term experiments, RMT 5265 induced apoptosis in a fraction of NPC cells, and this apoptosis was dramatically enhanced when RMT 5265 was combined with Toll-like receptor 3 (TLR3) stimulation. By contrast, the cooperative effect with tumor necrosis factor α was only marginal. The apoptosis induced by the combination of RMT 5265 and TLR3 stimulation was mediated by caspase-8 and associated with a decrease in the cellular content of the long isoform of FLICE-like inhibitory protein. Similar caspase-8 activation was obtained when siRNA knockdown of c-IAP2 was combined with TLR3 stimulation. In conclusion, c-IAP2 has a specific protective function in NPC cells challenged by TLR3 agonists. This protective function is probably important to make NPC cells tolerant to their own production of small viral RNAs, which are potential agonists of TLR3. Our data will help to design a rational use of IAP inhibitors in NPC patients.

EBV-related nasopharyngeal carcinomas (NPCs) still raise serious therapeutic problems. The therapeutic potential of the histone-deacetylase (HDAC) inhibitor Abexinostat was investigated using 5 preclinical NPC models including 2 patient-derived xenografts (C15 and C17). The cytotoxicity of Abexinostat used either alone or in combination with cis-platin or irradiation was assessed in vitro by MTT and clonogenic assays using 2 EBV-negative (CNE1 and HONE1) and 3 EBV-positive NPC models (C15, C17 and C666-1). Subsequently, the 3 EBV-positive models were used under the form of xenografts to assess the impact of systemic treatments by Abexinostat or combinations of Abexinostat with cis-platin or irradiation. Several cell proteins known to be affected by HDAC inhibitors and the small viral non-coding RNA EBER1 were investigated in the treated tumors. Synergistic cytotoxic effects of Abexinostat combined with cis-platin or irradiation were demonstrated in vitro for each NPC model. When using xenografts, Abexinostat by itself (12.5 mg/kg, BID, 4 days a week for 3 weeks) had significant anti-tumor effects against C17. Cooperative effects with cis-platin (2 mg/kg, IP, at days 3, 10 and 17) and irradiation (1 Gy) were observed for the C15 and C17 xenografts. Simultaneously two types of biological alterations were induced in the tumor tissue, especially in the C17 model: a depletion of the DNA-repair protein RAD51 and a stronger in situ detection of the small viral RNA EBER1. Overall, these results support implementation of phase I/II clinical trials of Abexinostat for the treatment of NPC. A depletion of RAD51 is likely to contribute to the cooperation of Abexinostat with DNA damaging agents. Reduction of RAD51 combined to enhanced detection of EBER 1 might be helpful for early assessment of tumor response.

Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by in vitro and in vivo assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. FOXN4, GLI1), immune response (CCR7, IL8) and transmembrane transport (e.g. ABCC3, ABCC11) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 in vitro. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients.

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (approximately 70-80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-gamma, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4(+)CD25(+)FoxP3(+)CD127(low) regulatory T cells, contraction of CD4(+) effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.