Tumor cell population in nasopharyngeal carcinomas (NPC) is highly heterogeneous. In addition of being heavily infiltrated by nonmalignant leucocytes, malignant NPC cells can display various phenotypes in terms of epithelial maturation and viral gene expression. These various cell sub-populations communicate through membrane contacts, secretion of cytokines and exosomes. Understanding their interactions is crucial for the elucidation of tumor growth and immune escape as well as for designing better therapeutic approaches. This chapter deals with 3 major questions: what are the local factors responsible for leucocyte attraction and retention in NPC tumors? What are the suspected autocrine and paracrine mechanisms of tumor growth? What are the mechanisms of tumor immune evasion which could explain the growth of malignant epithelial cells containing viral antigenic proteins in a context of local inflammation?

Introduction

One of the most striking and consistent characteristic of NPC is the presence of a very abundant leucocyte infiltrate containing mainly T-lymphocytes. This infiltrate often accounts for a large fraction of the tumor mass. Therefore, among the pathologists who originally described NPC, several authors called this tumor lympho-epithelioma (see the chapter by J. Nicholls and G. Niedobitek in this book).^1,2 This designation suggested a dual phenotype of NPC cells, in other words, the combination in the same tumor of malignant lymphoid and epithelial cells. This hypothesis has been ruled out in the late years 1970.³ It is now obvious that only epithelial cells are malignant and latently infected by EBV. When NPC are successfully xenografted on nude mice, the leucocyte infiltrate is rapidly eliminated.^3,4 Moreover, although the leucocyte infiltrate is very consistent in the primary tumor, it is generally absent in visceral metastatic lesions (see chapter by J. Nichols and G. Niedobitek in this book).^1,5 However, there are clinical and experimental observations suggesting that the lymphoid infiltrate plays a role in tumor growth at least at the initial stage of tumor development. This chapter will address several issues related to the formation and function of this infiltrate: what are the inflammatory cytokines produced by malignant NPC cells? Which vascular factor could favor leucocyte entry in the tumor? Conversely, which factor from the leucocyte infiltrate are likely to influence NPC cell growth and survival? We will also comment the role of other potential players involved in NPC tumor growth; for example stromal fibroblasts or rare malignant epithelial cells entering the lytic cycle. Finally this chapter will address a major paradox of NPC physiopathology which is the failure of the immune system to prevent tumor growth despite the presence of antigenic viral products in malignant cells and the presence of multiple immune effectors in the tumor tissue.

Our Limited Knowledge of NPC Histogenesis. The Hypothesis of a Tubal Tonsil Epithelial Origin

Primary lympho-epithelial carcinomas with an histological appearance almost identical to NPC have been reported in various anatomic sites outside the nasopharynx, for example in the stomach and salivary glands (consistently associated to EBV), in the lung (consistently associated with EBV in Asian population) and thymus (rarely associated with EBV).^6-10 However one should acknowledge that these “ectopic” primary NPCs are extremely rare. A contrario, it should be recognized that the overwhelming majority of EBV-associated lymphoepithelial carcinomas do occur in the nasopharynx. This observation suggests that NPC oncogenesis is strongly dependent on local factors related to anatomical, histological and physiological characteristics of the nasopharyngeal cavity. Such local characteristics are probably important not only for EBV-infection of epithelial cells but also for the formation of the leucocyte infiltrate. On the basis of direct or endoscopic observation of small tumors, it is well established that NPC consistently arise in the fossae of Rosenmuller which are lateral extensions of the nasopharyngeal cavity also called “pharyngeal recesses”.¹¹ These recesses are situated just above the Eustachian tube openings. They are also in close proximity to the “tubal tonsils” which is a part of the Waldeyer ring. This ring of lymphoid structures comprises the nasopharyngeal tonsil (NT) or adenoid, attached at the roof of the pharynx; the paired tubal tonsils (TT) as mentioned previously; the paired palatine tonsils (PT) positioned in the oropharynx; and the lingual tonsil (LT) on the posterior third of the tongue.¹² At least some of these human lymphoepithelial elements are homologue to a unique rodent lymphoid structure called nasopharynx-associated lymphoid tissue (NALT). In rodents, NALT is located on both sides of the nasopharyngeal duct which is homologous to the human nasopharyngeal cavity.¹³

There are specific characteristics applying to each elements of the Waldeyer ring. PT and LT are directly exposed to ingested pathogens and antigens whereas NT and TT are strategic for interactions with airborne pathogens and antigens. The surface of the nasopharyngeal tonsils (NT and TT) are covered mainly with a ciliated respiratory epithelium whereas that of oropharyngeal tonsils (PT and LT) are protected by stratified squamous nonkeratinized or parakeratinized epithelium respectively. Unfortunately from the point of view of NPC biology, among the elements of the Waldeyer ring, TT have not been in the focus of most biological investigations. Very few things are known about tubal tonsils (TT). However we know that all human tonsils share common structural and functional properties. The lymphoid subepithelial compartment contains germinal centers surrounded by a mantle zone which are more or less similar to their lymph node homologs. Two major epithelial components are the surface epithelium and the epithelium lining the crypts which has the structure of a sponge with interstices containing infiltrating T- and B-cells, macrophages, interdigitating dendritic cells and Langherans cells.¹² This specialised reticulated crypt epithelium, often called lymphoepithelium, has the same embryonic origin as the thymic epithelium (third pharyngeal pouche) and expression of a distinct subset of cytokeratins (CK 8, 18 and 19).¹⁴ The tonsil crypt epithelium has been sometimes described as a model of “lymphoepithelial symbiosis”. In addition, tonsil lymphoepithelium has a network of intra-epithelial capillary vessels, some of them ending in high endothelial venules (HEVs) with specific expression of adhesion molecules favoring lymphocyte extravasation.¹²

Because tonsillar epithelium has specific features supporting lymphoepithelial interactions, there is a suggestion that NPC cells derive from tubal tonsillar epithelial cells. This hypothesis needs to be substantiated by more experimental evidence. We know that small numbers of epithelial cells from both tonsil surface and reticulated crypts can be infected by EBV in vitro but we do not know yet whether specific markers of the tonsil crypt epithelium are expressed by NPC cells.¹⁵ We also ignore whether NPC tumor vessels have some properties in common with tonsillar lymphoepithelium vessels that would favor lymphocytes infiltration. Even if such vessels are not present in the tumor, their presence in close proximity to the tumor in tonsil crypts and interfollicular spaces might facilitate leucocyte entry in the neighbouring tumor tissue. Answering these question will be essential to better understand the formation of the lymphoepithelial stroma and its possible influence on tumor growth.

Subpopulations of Stromal Cells in NPC Tumors

Infiltrating Leucocytes

As previously mentioned, the abundance of infiltrating leucocytes is a major characteristic of NPC tumor stroma (in many reports, the terms “tumor infiltrating lymphocytes” or TILs are synonymous of “tumor infiltrating leucocytes”). Infiltrating leucocytes are often located around malignant cell clusters but sometimes disseminated within epithelial cell nests (see the chapter by J. Nicholls and G. Niedobitek in this book).¹ As shown by immunohistochemistry using an anti-CD3 antibody, most of them are CD3-positive T-cells with a morphology of small resting lymphocytes.¹⁶ Among the CD3-positive cells, CD8 and CD4 T-cells are present in varying proportions depending on the tumor specimens.¹⁷ Collagenase dispersion of cells from tumor pieces allow quantitative assessment of subpopulations of tumor infiltrating leucocytes by flow cytometry. Using this technique, Ferradini et al have found a CD4 to CD8 ratio varying from 0.4 to 2.2.¹⁸ On average, 15% of these T-cells express the integrin αEβ7 (or CD103), a surface marker frequently expressed by intra-epithelial lymphocytes. NK-cells positive for CD56 or CD94 are also detected both by flow cytometry on TILs (about 5% TIL) or immunohistochemistry.^18,19 Small populations of B-cells stained with anti-CD19 or -CD20 are also consistently detected.^17,18 Several studies have reported the presence of monocytes and dendritic cells in NPC biopsies.^20-23 Dendritic cells are often found inside malignant cell nests whereas monocytes are more often interspersed at some distance of epithelial cell clusters.²¹ Some NPC dendritic cells occasionally display CD23 expression like follicular dendritic cells.²⁴ A fraction of dendritic cells contained in NPC tumors have features of Langherans cells (also called T-zone histiocytes) (see the chapter by J. Nicholls and G. Niedobitek in this book).^1,22 Eosinophils are also detected in the leucocyte infiltrate of NPC tumors.^25,26

Recently, several studies have refined the description of infiltrating T-lymphocytes contained in NPCs (Fig. 1). Lau et al have shown that about 12% of TILs recovered after collagenase cell dispersion have a phenoptype of regulatory T-cells (T-reg), CD4⁺CD25^high, most of them being Foxp3-positive and CCR7-negative.²⁷ By immunohistology on tissue sections, it has been shown that CD25 high and Foxp3 T-cells are more abundant in tumor tissue than in nonmalignant nasopharyngeal mucosa.²⁸ On the other hand, lymphocytes expressing CXCR3 (the CXCL10 or IP10 receptor) are consistently detected in NPC tissue sections; in contrast, they were apparently rare in squamous carcinomas of the tongue investigated in the same study.²⁹ CXCR3 expression is generally associated with Th1 differentiation. Detection of a subpopulation of CXCR3-positive TILs suggests that Th1 differentiation is taking place inside NPC tumors despite the presence of regulatory T-cells. CXCR3-postive cells are often detected within nests of malignant cells.²⁹ It will be interesting to know whether intra-tumoral T-reg have the same distribution or accumulate at some distance of the tumor cells.

Figure 1

Summary of lympho-epithelial interactions in NPC tumors. Malignant epithelial cells are admixed with several sub-populations of T-lymphocytes. CD4+ Th1 T-cells are characterized by surface expression of CXCR3 and production of interferon γ (IFNγ). (more...)

Heterogeneity of Malignant Cells in NPC Tumors

In addition to being heavily infiltrated by various categories of nonmalignant cells, the tumor cell population itself is heterogeneous in NPC tumors. This heterogeneity is obvious in terms of epithelial differentiation. Although most NPCs are nonkeratinizing undifferentiated carcinomas, one can consistently find areas of squamous cell maturation in this type of tumors (see the chapter by J. Nicholls and G. Niedobitek in this book).¹ One can assume that there is an inverse relationship between proliferation and differentiation, although this has not been formally proven for NPC cells. It is not yet known whether one can isolate tumor stem cells from NPC specimens. A recent publication by Wang et al has reported the presence of stem cell-like side population cells in the CNE2 cell line which was depicted as an NPC cell line.³⁴ This “side-population” or “SP” phenotype which is demonstrated by flow cytometry rely on a capacity of active extrusion of the DNA binding dye Hoechst 33342. It is characteristic of stem cells in several normal tissue lineages as well as in some malignant tumor lines. Wang et al have found that a fraction of 2.6% CNE2 cells display an SP phenotype which is associated with a series of characteristics suggestive of stem cell behavior. By comparison with nonSP cells, CNE2 SP cells have a much higher clonogenic potential in vitro. They are more tumorigenic in SCID mice and more resistant to some cytotoxic drugs. They also have a higher production of interleukin 19. One limitation of this study is that CNE2 cells are not representative of NPC cells. For example, they are not latently EBV-infected. Nevertheless, it is obvious that characterization of NPC tumor stem cells will become a major field of investigation in the next years. It will be important to determine which factors control NPC stem cell asymmetrical division and transient proliferation of tumor cells that leaves the stem cell compartment. It will be also important to confirm that morphological epithelial maturation is associated with both loss of stemness and decrease of proliferation.

Qualitative and quantitative variations in EBV-gene expression is another major factor of heterogeneity among NPC tumor cells. Another chapter of this book by N. Raab-Traub is focused on EBV gene expression and their role in tumor development. In the scope of this chapter we will simply mention a few points in close connection with tumor heterogeneity and cellular interactions. There is a consensus that EBV-infection is mainly latent in NPC. However, the amount of EBV latent gene products is variable from one cell to another, especially the amount of the Latent Membrane Protein 1 (LMP1).³⁵ EBV viral particles have never been observed in fresh NPC biopsies although they can be produced by NPC cells used in short term culture in vitro and incubated with BUdR or other inducers of viral replication.^36-38 Nevertheless partial expression of EBV genes involved in the lytic-productive cycle can occur in NPC tumors in situ. Several EBV-proteins specifically expressed during the lytic cycle have been detected in NPC tissue sections in limited areas of the tumor; for instance, elements of the EA complex, the BZLF1 protein and the EBV-Dnase.^39-41 Consistently, linear replicative forms of the EBV genome are occasionally detected in a subset of NPC biopsies.⁴² The factors which control partial expression of the lytic EBV-genes in NPC cells are still poorly understood. There is some evidence that maturation from a phenotype of immature basal cell toward a phenotype of intermediate squamous tumor cells favors lytic gene expression.⁴³ Local production of TGF β is also suspected to increase lytic cycle gene expression in some malignant epithelial cells.⁴⁴ Recent findings made on EBV-associated SCID lymphoma models has spurred a renewed interested for lytic cycle gene expression in NPC. Hong et al have shown that a minority of lymphoma cells entering the lytic cycle play a critical role in the emergence of the disease through production of cytokines and angiogenic factors, especially interleukin-6.⁴⁵ Similar mechanisms might be important for NPC tumor growth although interleukin-6 does not seem to be very abundant in NPC tissue sections.⁴⁶

Dynamic Cellular Interactions: Possible Contribution to Tumor Growth

Role of Cytokines in Leucocyte Attraction and Retention

The leucocyte infiltrate consistently account for about 50% of the tumor mass. Obviously, it cannot be accounted for by a remnant of tonsilar leucocytes pre-existing to tumor development. For this reason, we hypothesized a long time ago that malignant epithelial cells were playing an active role in the formation of the infiltrate (Fig. 1, Table 1). Initially, we could demonstrate that malignant NPC cells constitutively produce interleukine-1 alpha (IL-1α), a cytokine with various inflammatory effects, including T-cell proliferation.⁴⁷ This observation was later confirmed by Huang et al (1999) who detected both IL-1 α and β in malignant NPC cells by antibody staining of tissue sections. Simultaneously, investigations made by RT-PCR demonstrated IL-1 α and β transcripts in most NPC primary tumors and a fraction of metastatic lesions and its absence in control fragments of nonmalignant nasopharyngeal mucosa.⁴⁸ Production of other inflammatory cytokines was investigated in NPC specimens in the subsequent years. IL-18 which has structural similarities with IL-1 is known to stimulate the proliferation of activated T-cells, to enhance their production of γ interferon and to promote their Th1 differentiation. By immunohistochemistry, IL18 was shown to be consistently produced by malignant NPC cells but not by epithelial cells of the non malignant mucosa.¹⁹ Data regarding IL-10 in NPCs remain controversial. According to three publications based on immunohistochemistry, IL-10 is detected in malignant cells of about 60% primary tumor biopsies of NPCs.^49-51 In contrast, Beck et al (2001) has failed to detect IL-10 transcripts by in situ hybridization in malignant NPC cells whereas in some cases it was detected in the leucocyte infiltrate. The same group has also found a very rare expression of IL-6 and IL-8 transcripts by malignant NPC cells contrasting with occasional expression by infiltrating leucocytes.⁴⁶

Table 1

Main cytokines involved in NPC cell interactions.

More recently, several studies have been focused on CC chemokines. Using in situ hybridisation, Teichman et al demonstrated a consistent and intense expression of the CXCL10 cytokine messenger by malignant NPC cells (CXCL10 is also called IP10 for γ-interferon inducible protein 10).²⁹ CXCL10 induces chemotaxis of activated T-cells and inhibits angiogenesis. It is the agonist of the CXCR3 receptor.

As mentioned previously, CXCR3 which is often associated with Th1-differentiation is consistently detected in a fraction of T-cells infiltrating NPC. However, there is no precise relationships between CXCL10 and CXCR3 expression in terms of abundance or spatial distribution in the tissue sections.²⁹ To our knowledge CXCL10 production has not yet been confirmed at the protein level in NPC tissue sections. Its status in the nonmalignant nasopharyngeal mucosa is not known. CCL20 or MIP-3α (Macrophage inflammatory protein-3 α), is another CC-chemokine that induces leukocyte migration into inflammation sites and regulates leukocyte trafficking through lymphoid tissues. It is a chemoattractant for memory regulatory T-cells.⁵² Chang et al have reported a high expression of CCL20 in NPC tumor cells. Interestingly, CCL20 is detected at a high concentration in serum samples from NPC patients. Its concentration is correlated with tumor mass and has prognostic value.⁵³

In summary, on the basis of currently published data, the main inflammatory cytokines produced by malignant NPC cells are IL-1 α and β, CCL20, IL-18 and probably CXCL10 (Fig. 1, Table 2). We do not know yet which factors up-regulate their production by NPC cells. So far there is no evidence of a direct role of an EBV product in their induction. Except for CCL20 whose expression is induced by EBNA1 in the background of malignant Hodgkin cells.⁵² Whether or not the same applies to epithelial cells remain to be investigated. All these cytokines are produced by NPC cells not only in situ but also by several NPC tumor lines used in the laboratory, suggesting that their production is constitutive and do not require the presence of the leucocyte infiltrate.^19,29,47 However some leucocytes might be involved in positive regulatory loops contributing to additional infiltration. For example CD3-positive T-lymphocytes and CD94 NK cells abundantly produce γ-interferon when they are located in primary NPC tumors whereas they do not or at a very low level when they are in nonmalignant NP mucosa.^23,54 Concentration of γ-interferon is increased in the plasma of NPC patients.⁵⁵ Production of γ-interferon by infiltrating lymphocytes is thought to be induced by local IL-18 whereas γ-interferon will in turn enhance CXCL10 production by malignant epithelial cells.^19,29 Similarly, CD68-positive monocytes have been shown to abundantly produce two chemokines, CCL2 (also called MCP1 or monocyte chemoattractant protein 1) and CCL3 (also called MIP-1α), when they are located in the tumor infiltrate but not—or at a low level—when they are observed in nonmalignant nasopharyngeal mucosa or sub-mucosa.²³ CCL2, like other monocyte chemoattractant proteins, recruits and/or activates monocytes, activated T-cells, NK cells and immature dendritic cells.²³ CCL3 is an attractant for CD8+ T-cells, B-cells and dendritic cells.

Table 2

Peripheral blood modifications related to cell interactions in NPC tumors.

Tumor infiltrating leucocytes which are so abundant in the primary tumor often disappear in the metastatic lesions.⁵ This might be explained by the decrease or loss of the production of some cytokines during the metastatic process. Alternatively, a positive balance between leucocyte entry and exit in the primary tumor might be dependent on specific anatomic factors in its local environment. As mentioned earlier, the proximity of the tubal tonsils with their network of specialised vessels—especially “high endothelial veinules”—probably facilitates leucocyte extravasation and tissue penetration.¹²

Other Potential Mechanisms of Growth Based on Cellular Interactions

In addition to lympho-epithelial interactions, other cellular interactions are suspected to contribute to tumor growth. We have previously mentioned an hypothetical role of stromal fibroblasts possibly mediated by the SDF1/CXCR4 ligand-receptor pair. Autrocrine growth mechanisms should also be taken in account (Fig. 2). Sheu et al have suggested a role for an autocrine loop involving the c-kit tyrosine kinase receptor and its ligand SCF (stem cell factor). Both molecules are co-expressed by malignant cells from primary tumors and metastases in about 70% NPC patients. SCF/c-kit co-expression is also observed in epithelial cells of nonmalignant nasopharyngeal mucosa but at a lower frequency.⁶² On the other hand NPC patients with c-kit expression in their tumors do not have a more severe prognosis.⁶³ In our hands, there is no significant constitutive phosphorylation of c-kit in an EBV-positive, c-kit-positive NPC xenograft (P. Busson, unpublished data). As mentioned in the chapter on systemic therapy (E.P. Hui and A.T.C. Chan in this book), a therapeutic trial based on SU 11248 (sunitinib malate) is in progress for NPC patients.⁶⁴ This drug is active on several tyrosine kinases including c-kit. Positive results would encourage further investigations about the role of the c-kit-SCF pair. In addition to promoting leucocyte infiltration, CCL20 or MIP-3α is suspected to have an autocrine enhancing effect on NPC cell migration and invasion.⁵³

Figure 2

Candidate autocrine growth factors for NPC cells. In a fraction of NPC tumors, there is co-expression of SCF (stem cell factor) and its receptor c-kit with a potential growth-promoting effect.^, CCL20 is a chemokine abundantly produced by NPC cells which (more...)

Several viral products are suspected to participate in autocrine growth mechanisms of NPC cells. A 33 Kd EBV protein called BARF1—according to the name of the corresponding viral ORF—is frequently produced by malignant cells in a large fraction if not all NPC tumors.⁶⁵ This protein which is secreted in the extracellular medium has homology with the human CSF gene. According to Houali et al, it is detected in the plasma of NPC patients.⁶⁶ A publication from the same group provides evidence that extra-cellular BARF1 has growth-promoting activity and suggest that it might be an autocrine growth factor for malignant NPC cells.⁶⁷ The small viral untranslated RNAs called EBERs have been previously mentioned for their role in cell resistance to interferon. These viral RNAs which are partially double-stranded are mainly concentrated in the nucleus. However, a small fraction of them leaks in the cytoplasm and even further in the extra-cellular space. These extra-nuclear EBERs have the power to activate some cellular receptors of double-strand RNAs, such as RIG1 and TLR-3.⁶⁸ Activation of these receptors can result in growth-promoting signals for example an increase in IGF-1 secretion.⁶⁹ Finally, a possible contribution to tumor growth of a small number of cells entering the lytic cycle has already been mentioned in this chapter.

Mechanisms of Tumor Immune Evasion

Role of Tumor Exosomes

Exosomes are bi-lamellar nanovesicles secreted by many cell types which are paradoxically derived from structures of the endosomal pathway called multivesicular bodies.⁸² Exosomes contains various types of cellular proteins which are either luminal or membrane-inserted. They also carry RNAs.⁸³ There is growing evidence that exosomes are major players in cell communications including developmental processes, neural communications and immune responses.^82,84 Exosomes also appear to play an important role in tumor growth and host-tumor relationships.⁸⁵ Initial evidence that exosomes could play a role in immune evasion of EBV-infected cells came from a study by J. Middeldorp's group in Amsterdam dealing with the EBV-encoded LMP1 oncoprotein. They could show that this type III membrane protein contains an immunosuppressive motif in its first transmembrane segment and is secreted by EBV-transformed B-cells in association with exosomes.⁸⁶ These LMP1-positive exosomes have an inhibitory effect on T-cell proliferation. More recently, inspired by this study, our group could demonstrate that NPC cells also produce exosomes. These NPC exosomes contain LMP1 only when they are produced by NPC cells with strong LMP1 expression (for example cells from the C15 xenograft).⁸⁷ In contrast, regardless of the presence of LMP1, they have a high content of HLA class II molecules and galectin 9. Galectin-9 is a β-galactosyl binding lectin which is very abundant in NPC and has been identified as one specific ligand of the Tim-3 receptor.⁸⁸ Tim-3 is expressed by mature CD4+ Th1 lymphocytes. In the context of CD4+ Th1 lymphocytes, galectin 9 binding to Tim-3 triggers a very rapid process of apoptosis. We have shown that the galectin 9 CRDs (Carbohydrate Recognition Domains) are presented at the surface of NPC exosomes. Accordingly, these exosomes can induce massive apoptosis of CD4+ T-cells expressing Tim-3, including CD4+ T-cells directed to EBV-antigens.⁸⁹ In addition, we have demonstrated that HLA-class II-positive exosomes carrying galectin 9 are detected specifically in plasma samples from NPC patients but not from control patients, for example patients with nonNPC head and neck tumors.⁸⁹ We believe that this presence of galectin 9 positive exosomes in the plasma reflects passive diffusion from tumor interstitial fluids to the blood stream. In addition to cytotoxic effects against mature CD4+ Th1 lymphocytes, NPC exosomes are suspected to enhance T-reg expansion possibly in cooperation with CCL20 (C. Durieu and P. Busson, personal data).⁵² They probably also have some influence on dendritic cell maturation.⁹⁰ According to our current hypothesis depicted in Figure 3, NPC exosomes might subvert CD4+ Th1 functions by combining effects on dendritic cells favouring initial Th1 maturation with direct cytotoxic effects on fully mature Tim-3 positive Th1 cells. Development of novel in vitro and in vivo models will be required to validate this hypothesis.

Figure 3

Hypothesis of an abortive maturation of CD4+ Th1+ lymphocytes in NPC tumor microenvironment. Tumor exosomes containing galectin 9 are expected to stimulate maturation of monocyte-derived dendritic cells (Mo DC) in a way that will favor differentiation (more...)

Conclusion

In conclusion, it is interesting to address these two questions: what can we do to make further progress in this field? What will be the practical consequences of our knowledge of cellular interactions in NPC tumors?

Detection of cell population markers and cytokines on tumor tissue sections will have more and more importance in this field for the years to come. One important challenge is to achieve simultaneous detection of multiple targets on the same tissue section. One recent exciting trend in clinical investigations of NPC has been the discovery of a series of novel tumor markers in the peripheral blood of NPC patients.^53,92,93 Interestingly many of these markers are related to cellular interactions in NPC tumor microenvironment. ^53,92,93 Their detection in the blood is related to their production in the tumor and provide indirect informations on cellular interactions. Several of these markers which have relevance to cellular interactions inside NPC tumors are listed in Table 2. In the coming years one may expect that some of them will contribute to assessment of intra-tumoral cellular interactions in combination with data obtained by immunohistology.

A major challenge is to connect observations made on clinical samples with in vitro experimental systems allowing assessment of functional roles of cytokines, exosomes and other cell communication agents. For this aim, novel methods allowing 3D in vitro cultures of cells derived from NPC biopsies need to be explored more intensively. In the past, some attempts to culture NPC cells in vitro under the form of spheroids have resulted in some partial success.^94,95

In terms of therapeutic, there are several consequences of our knowledge of cellular interactions in NPC. Some therapeutic agents will be useful to block stimuli given by infiltrating cells to malignant cells. For example inhibitors of the met-receptor which are currently in phase I or II trials might be useful to block tumor growth promoting effects by HGF released by stromal lymphocytes or fibroblasts.^60,61 Other therapeutic approaches will aim to antagonize the immunosuppressive effects of cytokines or exosomes released by malignant epithelial cells. In this regard, it is noteworthy that a monoclonal antibody directed to the CRD (carbohydrate recognition domain) of galectin 9 can block the stimulation of Tim-3 by galectin 9 exosomes and protect CD4+ Th1 lymphocytes.⁸⁹ Finally based on our appreciation of cellular interactions in NPC tumor growth, it is useful to end with a word of caution about therapeutic strategies based on induction of the lytic cycle which might favour the release of a wide range of cytokines with potential enhancement of tumor growth and angiogenesis.

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