The NPC microenvironment is characterized by the intense filtration of tumor-infiltrating immune cells (TIICs), which constitute 40 – 50% of the tumor mass in NPC, with EBV-negative CD3⁺ T-lymphocytes as the commonest infiltrate (28–30). Despite its abundance, lack of effective immune response, and the presence of immunosuppressive infiltrates such as regulatory T-cells (Tregs), M2 macrophages, and myeloid-derived suppressor cells (MDSCs) leads to immunotolerance, promoting tumor progression. TIICs can be further classified based on lymphoid and myeloid lineage. Tumor-infiltrating lymphocytes (TILs), including T-cells and B-cells, are abundant in the NPC microenvironment as a result of an immune response against EBV. On the other hand, tumor-infiltrating myeloid cells (TIMs) consist of various cell types, including mature mast cells, monocytes and macrophages, dendritic cells (DCs), and pathologically activated immature MDSCs (17, 31, 32).

CAFs, also termed tumor-associated fibroblasts, are found abundantly in the cancer stroma. CAFs have elongated spindle morphology, displayed mesenchymal biomarkers, such as alpha-smooth muscle actin (α-SMA), platelet-derived growth factor receptors (PDGFRs), vimentin and fibroblast activation protein (FAP), and lack of genetic mutations (60, 61). The CAFs predecessors are recruited from diverse origins, which include resident tissue fibroblasts, peri-tumoral adipocytes, endothelial cells through endothelial-mesenchymal transition (EndMT), epithelial cells through EMT, bone-marrow-derived mesenchymal stem cells and haematopoietic stem cells (25, 60, 62). In NPC, extracellular vesicle-packaged EBV-encoded LMP1 can promote the activation of normal fibroblasts into CAFs via the nuclear factor-kappa B (NF-κB) p65 pathway (63). α-SMA expression in CAFs predicted an adverse prognosis in patients with NPC. A high density of CAFs is correlated with shorter OS and lower 5-year survival rates, suggesting CAF density as an independent prognostic factor for NPC patients, suggesting their role in tumorigenesis (64). Consistent with this finding, several studies reported that CAFs promote neoangiogenesis, metastasis, and therapeutic resistance in NPC (65–67).

CAFs stimulate neoangiogenesis in NPC. Genes correlated with endothelial cells abundance were highly expressed by fibroblasts, implying their potential roles in endothelial cell recruitment and angiogenesis (18). The immunohistochemical method revealed high expression of α-SMA fibroblasts in NPC stroma, together with increased intensities of the chemokine stroma-derived factor-1 (SDF-1, also known as CXCL12), a mediator of the recruitment of endothelial progenitor cells, and its receptor CXCR4 in NPC cells. Vascular endothelial growth factor (VEGF) was detected in both cancer and stromal cells, indicating secretion of a significant amount of VEGF in these cells. Prominin 1 (PROM 1, or CD133) and VEGF receptor (VEGFR)-2 double-positive endothelial progenitor cells or CD34 positive cells were observed in the stroma, suggesting tumor-associated neoangiogenesis. Statistical analyses revealed a positive correlation between α-SMA and endothelial antigens CD34, suggesting that CAFs and NPC tumor cells may enhance neoangiogenesis in a VEGF- and SDF-1-dependent manner by the recruitment endothelial progenitor cells from the bone marrow into the tumor stroma (65).

CAFs support tumor metastasis in NPC. Increased density of α-SMA-expressing CAFs at metastatic sites of NPC compared with primary sites, along with upregulation of COX-2 or prostaglandin-endoperoxide synthase (PTGS2) in CAFs, indicated the involvement of fibroblasts and COX-2 in NPC metastasis. High expression of COX-2 catalysed CAF-secreted prostaglandin E2 (PGE2), which induces EMT, thereby promoting NPC cell migration and invasiveness in vitro. Furthermore, COX-2 in host fibroblasts promotes lung metastasis and correlated with the expression of TNF-α expression in mouse models, suggesting that high expression of COX-2 in fibroblasts promotes NPC metastasis through the COX-2-PGE2-TNF-α axis. Consistent with this finding, the expression of COX-2 in CAF was positively correlated with N stage, relapse, and poor survival in patients with NPC (66).

CAFs promote therapeutic resistance and immune evasion. CAFs induced the formation of radioresistance and promoted NPC cell survival following irradiation via the IL-8/NF-κB pathway to reduce irradiation-induced DNA damage. Moreover, CAFs express immunosuppressive factor IDO1, which encodes the enzyme IDO. IDO catalyses the production of L-kynurenine (Kyn), which subsequently promote the generation or differentiation of tolerogenic immune cells by interacting with aryl hydrocarbon receptors on immune cells. High expression of IDO is inversely correlated with the density of CD3⁺ T-cells and predicted poor survival outcomes in NPC patients. Hence, CAFs may promote immune suppression in NPC by the expression of IDO (18, 68).

CAFs exhibit a supportive role in tumor progression through the remodeling of the ECM (25). NPC-derived fibroblasts express genes that encode ECM components, including COL1A1, COL1A2, LUM, FN1. This suggests the complexity of ECM in the NPC microenvironment and the possible interaction with tumors and stromal cells via integrin signaling, indicating integrin receptors on tumor and immune cells as potential therapeutic targets for disruption of ECM-dependent tumor progression and suppressive immunomodulation (17). Increased production and crosslinking of collagen, such as COL1A1, increase the stiffness of ECM, leading to the promotion of tumor progression through increased integrin signaling (25, 27). On the other hand, fibronectin 1 (FN1) is shown to increase migration and invasion of NPC cells by upregulation of matrix metalloproteinase 9 (MMP9) and MMP2, which are ECM-digesting enzymes mainly produced by CAFs. FN1 also suppresses NPC cell apoptosis via the NF-κB pathway by upregulation of the expression of BCL2 and P65 (25, 69). CAFs mediated directional migration of cancer cells by assembling a fibronectin-rich ECM with anisotropic fiber orientation through increased non-muscle myosin II- and PDGFRα-mediated contractility and traction forces (62, 70).

Angiogenesis, the formation of blood vessels from existing vasculature in response to a hypoxic condition, is one of the hallmarks of cancer that contributes to tumor growth, development and metastases (71). The vascular niche is associated with stem cell-like NPC at the invasive front (72). The tumor vasculature is lined internally by TECs and surrounded externally by perivascular cells. TECs are characterized by their genetic instability, which contributes to the development of distinct phenotypes that promote therapeutic resistance, tumor progression and metastasis. TECs also interact with tumor cells by secretion of angiocrine factors, contributing to tumor migration and metastasis (73, 74). Drug-resistant human microvasculature endothelial cells (HMECs) are reported to promote progression, EMT and chemoresistance in NPC through secretion of exosomes (75). In NPC, TECs demonstrated enhanced capacity to recruit CTLs and Tregs via CXCL9–CXCR3 and CXCL10–CXCR3 while simultaneously inhibiting CTLs through PDL2–PD1 interaction, suggesting their role in the mediation of an immunosuppressive niche in the NPC microenvironment (18).

Hypoxia was detected in over 80% of NPC tumors as a result of the high requirement of oxygen due to increased proliferation and metabolism. It was reported to play an important role in NPC by regulating its apoptotic activity, metastasis, angiogenesis, metabolic adaptation, and therapeutic resistance, thereby representing a potential target for the treatment of NPC. Hypoxic stress prevents cell survival by inducing the upregulation of anti-apoptotic proteins such as growth arrest and DNA-damage-inducible beta (Gadd45β) and immediate early response 3 (IER3) (76). Hypoxia promotes tumor cell migration by EMT stimulation and ECM remodeling through Notch signaling (27). Lysyl oxidase (LOX), an extracellular matrix-remodeling enzyme, was significantly upregulated in hypoxic conditions and predicted poor prognosis in patients with NPC (76, 77). In addition, hypoxia induces the upregulation of VEGF, which is responsible for neoangiogenesis. Furthermore, hypoxia mediates metabolic adaptation and acidosis in NPC (76). A metabolic shift in NPC infiltrating lymphocytes was reported to induce the exhausted phenotype of T-cells through overexpression of miR-24. miR-24 overexpression suppresses the expression of MYC and FG11 in TILs and disrupts MFN1-mediated mitochondrial fusion, thereby inducing T-cell exhaustion (78).

Various molecular signals and tumor-derived factors are involved in angiogenesis. VEGF is a pro-angiogenic factor that activates and stimulates proliferation and migration in VEGFR expressing endothelial cells. VEGF also alters vascular permeability by loosening the junctions between endothelial cells, favouring tumor intravasation (79). Besides hypoxia, increased VEGF production can be induced by EBV-encoded LMP1 through COX-2 expression in NPC cells (80). High expression of tissue VEGF is associated with reduced OS and DFS in NPC patients (81).

EBV-infected nasopharyngeal epithelial cells construct the NPC microenvironment, which is characterized by heavy infiltration of immune cells with suppressed immune activities, by secretion of EBV-encoded products that activate several inflammatory-associated signalling pathways. A variety of cytokines, such as IL-6, IL-8, interferon-inducible protein 10 (IP-10), TNF-α, VEGF, and macrophage inflammatory protein (MIP)-3α are elevated in NPC patients (84). LMP1 recruit immune cells into the tumor site by upregulating several cytokines through NF-κB and STAT3 signaling pathways (82, 85). These include regulatory cells with immunosuppressive influence in the TME. NPC cells induce polarization of macrophage into immunosuppressive M2-like phenotype through secretion of interferon-stimulated gene 15 (ISG15) (18). EBV nuclear antigen 1 (EBNA1) induce the production of CCL20, or MIP-3α to promote chemotaxis of Tregs to the tumor site (86, 87). In addition, EBV-encoded small RNAs (EBERs) activates the toll-like receptor 3 (TLR3) pathway to produce inflammatory cytokines such as CXCL8 which recruit and activate TAMs. The inflammatory responses in NPC cells are amplified by a positive feedback loop consisting of EBER, LMP1 and NF-κB (88). Metabolic reprogramming by LMP1 leads to alteration in Nod-like receptor family protein 3 (NLRP3) inflammasome, COX-2 and P-p65 signaling pathways, which results in the release of cytokines, including IL-1β, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF), contributing to the expansion of MDSCs (50). Stromal cells, including TECs, can attract CTLs and Tregs via chemokine-receptor interactions such as CXCL9/CXCR3 and CXCL10/CXCR3, however, cytotoxic activities of CTLs are inhibited by PD-L2/PD1 interaction (18).

Chemokines and cytokines facilitate tumor elusion from immune surveillance. EBER, LMP1 and EBV lytic transactivator, Zta, are positively correlated with the production of IL-10. BCRF1, or viral IL-10, share immunosuppressive properties with human IL-10. IL-10 induces immunosuppressive activities by inhibition of antigen-specific T-cell proliferation, induction of T-cell apoptosis and inhibition of IFN-γ secretion from NK cells. LMP1 upregulates IL-18 and IP-10 (CXCL10), which take part in the recruitment and immunosuppression of CXCR3⁺ NK cells and T-cells (45, 82, 85, 89). IL-6 and TNF-α mediate the expression of IDO, which demonstrates an immunosuppressive role by suppression of T-cell proliferation and impairment of CD8⁺ T-cells cytotoxic activities. Pre-treatment serum levels of IL-6 and TNF-α are negatively correlated with the 2-year survival rate in NPC patients (90, 91). Increased levels of IL-18 in NPC induces NK cells exhaustion via upregulation of expression of PD-1 on NK cells (45).

Furthermore, chemokines induce metastasis and promote tumor progression. IL-8 induce EMT by activating AKT signalling, enhancing migration and invasion of NPC cells. Enhanced level of IL-8 predicts adverse OS, DFS, and DMFS of NPC patients (92).

Exosomes are small (30-100 nm) extracellular vesicles that originated from the plasma membrane. They are often involved in the intercellular interactions in the TME, influencing various cellular processes, including cell growth, angiogenesis, EMT, metastasis, immune tolerance, and therapeutic resistance (93). Exosomes may play a significant role in the formation of a premetastatic milieu by acting as a vehicle for tumor-derived factors that modulate pre-metastatic sites (15). Exosome contents include transcription factors, enzymes, ECM proteins, lipids, and nucleic acids, such as DNA, mRNA, and non-coding RNA. Based on their parental cells, exosomes in NPC can be classified into nasopharyngeal carcinoma-derived exosomes (NPC-Exo) and EBV-related exosomes (94–96).

NPC is a promising candidate for immunotherapy owing to its immunosuppressive property and expression of immunogenic EBV antigen. Several approaches which include immune checkpoint inhibitors and cellular-based immunotherapy have been employed to reinvigorate the exhausted immune cells in the NPC microenvironment (83) ( Table 2 ). PD-1/PD-L1 axis has been exploited as a potential therapeutic target for reversing T-cells exhaustion and restoration of their anti-tumor function (136, 137). PD-1 is an immune checkpoint encoded by the PDCD1 gene and expressed on the T-cell surface, whereas PD-L1 is expressed on tumor cells and immune cells. Studies reported expression of PD-L1 in approximately 70 - 90% of NPC tissues and its prognostic significance. The difference in interpretational methods leads to inconsistency of results, suggesting the need for further large-scale study (138–141).

PD-1/PD-L1 blockade therapies with anti-PD-1 antibodies, such as nivolumab, pembrolizumab, cemiplimab and anti-PD-L1 antibodies, including atezolizumab, avelumab and duravulumab have been implicated in immunotherapy against various neoplasms (136, 137). Anti-PD-1 and anti-PD-L1 therapies are generally safer than chemotherapy, with a lower incidence of treatment-related adverse events (142, 143). Single-agent studies involving PD-L1 antibodies showed lower overall response rate (ORR), OS and progression-free survival (PFS) when compared with studies with PD-1 antibodies, likely due to alternative binding of PD-1 to PD-L2 following blockade of PD-L1. Combination therapy of chemoradiation and PD-1/PD-L1 promote a synergistic anti-tumor immunity by enhancing host recognition, elimination of tumor cells and preventing T-cell apoptosis. For instance, a phase I trial investigating anti-PD1 antibody camrelizumab achieved an ORR of 34%, whereas a combination trial of camrelizumab with gemcitabine and cisplatin achieved an ORR of 91%. In spite of that, combination therapy exhibited a higher incidence of treatment-related adverse events when compared with anti-PD-1 monotherapy, suggesting synergistic toxicity (142, 143). There is also limited anti-tumor response rate towards PD-1/PD-L1 blockade therapy. Resistance to PD-1/PD-L1 therapy involves multiple mechanisms, including low tumor immunogenicity, T-cell dysfunction by other immune checkpoint receptors and modulation by noncoding RNAs.

Strategies to overcome the challenge include the incorporation of PD-L1/PD-1 inhibitors with other anti-tumor therapy. Ongoing clinical trials propose the combination of PD-1/PD-L1 blockade with other immune checkpoint inhibitors such as anti-CTLA-4 or anti-angiogenic agents such as anti-VEGFR inhibitors as potential therapeutic strategies for NPC (144, 145). Besides that, the identification of predictive biomarkers of PD-1 inhibitors will allow the selection of appropriate patients for PD-1/PD-L1 targeted treatment (136). Other immune regulatory checkpoints, for instance, the Gal-9/TIM-3 (HAVCR2) interaction represents a promising alternative as they are prominently expressed in recurrent NPC (20, 146).

Two approaches of cellular-based immunotherapy include adoptive immunotherapy, which is the infusion of ex vivo generated activated effector cells, such as EBV-specific CTLs, chimeric antigen receptor (CAR) engineered T-cells and cytokine-induced killer cells (CIKs) (147–150). An increase in the frequency of MDSCs, and immunosuppressive cytokines, such as CCL2 and CXCL10, underlie the development of resistance towards adoptive immunotherapy. Chemotherapeutic agents, such as gemcitabine and carboplatin may alleviate the resistance by limiting the expansion of MDSCs, secretion of pro-inflammatory cytokines and depletion of immune checkpoint molecules (151). A phase II study combining chemotherapy with adoptive immunotherapy using engineered EBV-specific CTLs as first-line treatment for metastatic or recurrent NPC patients yield a satisfactory result, with a 2-year OS of 62.9% (118). On the other hand, active immunotherapy or tumor vaccines aim to enhance recognition by the immune system by the delivery of tumor-specific antigens through antigen-presenting cells or viral vectors. These include LMP2 expressing DCs and recombinant modified vaccinia Ankara vaccine (MVA-EL), which has been proved to be safe and well-tolerated (152, 153). Besides targeting EBV-encoded oncoproteins, a peptide vaccine targeting non-EBV associated tumor specific antigen, four-jointed box 1 (FJX1), was also designed in a preclinical setting (154).

Further research should consider [1] large-scale studies for the evaluation of clinical efficacy and the optimization of dosage for the above therapy; [2] combination of therapies to augment the clinical responses, for example, combining immune checkpoint blockade with adoptive T-cell therapy (155–157); [3] development of personalized immunotherapy targeting neoantigens (158); and [4] localization of NPC-specific CTLs at the malignant site, for example, via chemokine-receptor interactions (159). Other alternatives, for example, CAR-NK, an emerging cellular immunotherapy strategy for cancer, should be evaluated in NPC, given its promising clinical efficacy in other settings (160).

Immune score, established based upon the density of lymphocyte populations, is predictive of disease progression and distant metastases. High immune scores predict better OS, DFS and distant-metastasis free survival (DMFS) of NPC patients (37). Among TILs, CD3⁺, CD4⁺, and CD8⁺ T-cells as well as CD56⁺ NK cells are good prognostic factors for NPC patients (161–163). Despite its immunosuppressive role, high Foxp3⁺ T-cells to CD8⁺ T-cells ratio was associated with favorable PFS in early-stage NPC patients (164). Similarly, Ooft et al. reported Foxp3 as an independent predictor for better OS in NPC patients (165). On the other hand, CD68+ TAM density predicted favorable DFS, whereas stromal CD163⁺ M2-like TAMs correlated with poor OS and PFS in NPC patients (20, 166, 167).

Nonetheless, there are discrepancies among studies investigating the prognostic significance for different TIICs, due to different methodologies used. Thus, further studies should consider the development of a standardized method for the evaluation of TIICs as an indicator or predictor for the progression and therapeutic response of NPC patients.

Angiogenesis is important to meet the huge demand for oxygen and nutrients by tumor cells, thus, anti-angiogenic therapy using angiogenic inhibitors is a promising anti-cancer therapeutic strategy. Conventional chemotherapeutic agents, for instance, epirubicin, elicits inhibitory activity on angiogenesis by inducing non-specific vascular toxicity (168). On the other hand, hypericin-mediated photodynamic therapy exerts its antitumor activity by targeting the tumor vasculature (169). Several small-molecule inhibitors of VEGFR such as apatinib are employed in angiogenesis-targeting therapy for NPC patients (170, 171). The inhibition of angiogenesis could enhance the effectiveness of standard chemoradiotherapy against NPC. Combination of VEGF inhibitor bevacizumab with chemotherapy promoted microvasculature maturation, enhanced immune infiltration and exhibited promising tumor response (172). Combination therapy of apatinib with cisplatin showed a synergistic effect in inhibition of tumor growth, repression of VEGFR-2 expression and reduction in microvascular density in preclinical models (173). On the other hand, combining apatinib and radiation enhances the anti-angiogenic effect and increases hypoxia in tumor tissues, leading to an anti-tumor effect (174). Endostatin, an endogenous angiogenesis inhibitor is another anti-tumor agent tested in clinical trials. Endostar, a recombinant endostatin, sensitized NPC towards radiation therapy by induction of endothelial cell and tumor cell apoptosis and repression of radiation-induced pro-angiogenic factors (175). Phase II multicentre randomized controlled combining Endostar therapy with standard chemoradiation reported an improved remission rate of cervical lymph node metastasis, with a slight improvement in objective response rate (121). To sum, anti-angiogenic agents targeting VEGF and VEGFR can enhance the effectiveness of chemoradiotherapy through inhibition of blood vessels formation, normalization of blood vessels, which restore vascular function, improve tumor perfusion, and drug delivery, and activation of immune response (176).

Currently, anti-angiogenic therapy faces the challenge of the development of resistance, which leads to limited survival benefits. The resistance is likely due to hypoxic events as a consequence of vascular depletion, resulting in the promotion of cancer invasion and metastasis, activation of alternative pro-angiogenic signalling pathways, VEGF-independent vascular mimicry, and increased recruitment of pro-angiogenic cells (176, 177). Studies unveiled upregulation of several proangiogenic factors, including VEGF, TNF-α, IFN-α, and basic fibroblast growth factor (bFGF), following hypericin-mediated photodynamic therapy. This indicates the need of combination therapies such as photodynamic therapies with angiogenesis inhibitors to enhance the efficacy of treatment (169, 178). Other challenges include toxicity effects, which include haemorrhage, hypertension, and thrombosis (79). Several strategies direct targeting the tumor vessels are introduced to increase the effectiveness of the therapy and minimise the side effects. Nanoparticles might serve as vectors for the delivery of anti-angiogenic drugs to the target site (79). Given the high effectivity for DNA enzyme (DNAzyme) to cleave targeted sequence with high specificity, DNAzyme also serves as a potential therapeutic agent for anti-angiogenic therapy. DNAzyme targeting VEGFR-1 mRNA significantly downregulated VEGFR-1, inhibited angiogenesis and altered the vascular permeability. No toxicity effect was observed in vivo, indicating anti-VEGFR-1 DNAzyme as potential drug candidates for further clinical evaluation (179).

The increased oxygen demand by the highly proliferative and metabolically active tumor cells with inadequate oxygen supply from the impaired vasculature leads to hypoxia, a condition that mediates tumor progression, metastasis, and therapeutic resistance. This tumor-specific event has been studied extensively for the development of anti-tumor therapy. Several strategies targeting hypoxia are employed, which include a cytotoxic approach with hypoxic selectivity. In the hypoxic TME, hypoxia-activated prodrugs such as tirapazamine are preferentially activated into cytotoxic drugs and eliminate hypoxic tumor cells with high selectivity. The second approach direct inhibits hypoxia-inducible proteins. HIF-1α siRNA and PX-478 directly target HIF-1α and elicit anti-tumor activity. Hypoxia-modifying therapy improves the hypoxic condition in tumors by increasing the blood oxygen level through blood transfusion, carbogen, and nicotinamide treatment (76).

The combination of hypoxia-targeting therapy and radiotherapy exhibited a synergistic effect. Topotecan is reported to enhance the radiosensitivity of tumors by inhibiting topoisomerase I and subsequently downregulating the expression of HIF-1α target genes (180, 181). Nanotechnology is also employed to enhance the therapeutic efficacy of hypoxia-targeting therapy. In a preclinical study, nanoparticle delivering siRNA targeting HIF-1α significantly suppressed tumor growth in in vivo animal models of NPC (182). In recent studies, hypoxia-responsive nanoparticles have been designed for the selective release of drugs in the hypoxic tumor environment, thereby prolonging the bioavailability and improving the effectiveness of the drug (183). These nanomedicines are still in the early phase, their mechanism, delivery efficacy and safety in human bodies remain to be studied.

Chemokines and cytokines are potential targets for the development of diagnostic biomarkers or therapeutic strategies. CCL5, also known as RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), is a pro-angiogenic chemokine that can be detected in NPC patients’ plasma with 90.07% sensitivity and 56.34% specificity. Increased screening efficacy was observed when combining CCL5 with EBV viral capsid antigen (VCA-IgA) or EBV DNA assay. Inhibition of the CCL5 receptor, CCR5 with its antagonist maraviroc could suppress CCL5-associated migration of NPC cells (184, 185). Leukaemia inhibitory factor (LIF), a member of the IL-6 type cytokine family, is remarkably increased in the TME and NPC patients’ serum. Secreted by tumor cells and inflammatory infiltrates, LIF mediates the mTOR pathway in NPC, which leads to tumor growth and enhanced radioresistance. Elevated serum LIF in NPC patients is predictive of local recurrence and radiosensitivity. The blockade of the LIF signalling pathway through a LIF antagonist, soluble LIF receptor, or mTOR inhibitor, rapamycin, could sensitize NPC towards irradiation (186, 187).

The development of anti-tumor therapeutics targeting chemokines or their receptors need to consider [1] the optimal dosage of drugs with concern on their safety and efficacy; [2] evaluation of synergism effect when combined with conventional therapy and immunotherapy; [3] development of drugs with high affinity and high specificity; and [4] identification of tumor-specific targets, with minimal effects on non-tumor-associated cells.

Tumor-derived exosomes are involved in a wide range of biological and pathological processes as well as intercellular crosstalk and thus are promising targets for the development of biomarkers and anti-cancer therapies. Exosomes are highly stable and can be detected in patients’ body fluids, such as urine, saliva, cerebrospinal fluid, and serum, and hence can be used in liquid biopsies as non-invasive biomarkers (96). Sera exosome concentrations have clinical significance and prognostic value in NPC patients, whereby a positive correlation between tumor lymph node metastasis and shorter disease-free survival was observed in NPC patients.

Nasopharyngeal brush or swab is a less invasive alternative for the collection of tumor tissue samples when compared to the conventional biopsy method. Elevation of EBV miRNAs, including mir-BART1-5p, mir-BART5, mir-BART6-5p, mir-BART17-5p, were reported in nasopharyngeal brush samples from NPC patients. miR-BART1-5p shows potential as a diagnostic indicator of NPC, with 93.5% sensitivity and 100% specificity, its expression level positively correlated with tumor progression (188).

In addition, miR-BART7 and miR-BART13 are significantly elevated in plasma specimens from NPC patients, the combination of both markers offers a 90% prediction of NPC. Furthermore, the level of miR-BART7 is associated with metastatic progression. Diminished levels of miR-BART7 and miR-BART13 were reported after radiotherapy, suggesting their potential as biomarkers for the monitoring of therapeutic efficacy (189). Post-treatment detection of circulating miR-BART17-5p is a potential biomarker of a poor prognosis (190). Plasma EBV-miR-BART7-3p showed 96.1% sensitivity and 96.7% specificity, whereas miR-BART13-3p has a sensitivity of 97.9% and specificity of 96.7% for the detection of NPC. miR-BART7-3p is a potential prognostic biomarker, whereby prominent levels of pre-treatment miR-BART7-3p in plasma indicated a higher risk of distant metastasis whereas post-treatment miR-BART7-3p is associated with short DMFS and OS (191, 192). Significant upregulation of miR-1301-3p was detected in the exosomes from early-stage NPC patients’ plasma, indicating their potential application as diagnostic biomarkers in early-stage NPC (193).

Upregulation of circulating exosomal circRNA circMYC is correlated with increase cell proliferation and resistance to radiotherapy in NPC patients. Circulating circMYC is a potential biomarker for the differentiation of radiosensitive and radioresistant patients with 90.24% sensitivity and 94.51% specificity (103). The detection of exosomal cyclophilin A (CYPA) combined with EBV VCA-IgA could increase the accuracy of diagnosis, indicating the utility of exosomal CYPA as a potential non-invasive biomarker for the diagnosis of NPC (194).

Besides serving as biomarkers, exosomes are potential vehicles for the delivery of cancer drugs to the target molecules. iRGD-tagged exosomes targeting αvβ3 integrin-positive NPC cells containing antagomir-BART10-5p and antagomir-18a showed remarkable anti-angiogenic efficacy in in vitro and in vivo NPC models (110). Exosomes are also employed as vehicles for tumor antigen in cancer vaccines, however, to our knowledge, exosome vaccines have not been tested specifically in NPC yet (195).

While exploiting exosomes as therapeutic and diagnostic tools, there are some challenges that need to be addressed. These include [1] time and cost consumption; [2] unsatisfactory yield, purity, and quality, which could affect [3] the sensitivity and specificity of assays, [4] absence of endogenous control for miRNA quantification, and [5] limited sample size in current studies, which affect the reliability of these studies (196, 197). On the other hand, several problems need to be considered when using exosomes as therapeutic vehicles or targets: [1] enhancing the specificity of therapy to prevent undesirable adverse events; [2] prolonging the half-life of exosomes after administration into the body; [3] effective and safe therapeutic dose of exosomes; [4] effective drug loading into the exosomes; [5] cost and time effectiveness in the production of bioengineered exosomes (93, 196).

Overall, exosomes play a pivotal role in the interplay communication between EBV-infected NPC cells and stromal cells, which contribute to the construction of the TME that favours tumor progression, development, invasion, and metastasis. Further investigations should consider [1] understanding the mechanisms underlying cellular crosstalk via exosomes, including the biogenesis and internalization of exosomes; [2] development of standard methods for scalable isolation and purification of exosomes; [3] large-scale clinical studies for the evaluation of exosome as reliable cancer biomarkers.

The vast influence of CAFs on tumor development and progression made them promising therapeutic targets. Several strategies for CAF-directed anti-cancer therapies have been tested in preclinical settings, but only a few studies are focusing on CAFs in NPC (198). Disulfiram/copper exhibited anti-tumor activity by targeting both cancer cells and CAFs. It could induce apoptosis and inactivate CAFs by inhibiting the expression of α-SMA (199). Treatment with CAF inhibitor, for instance, Tranilast, inhibits CAFs from activating the NF-κB pathway, thus sensitizing NPC cells to irradiation (67). The targeting of CAFs is challenging as they demonstrate complexity in intercellular signaling, phenotype and source and lack of specific surface marker (198). Thus, further understanding of CAFs, with regards to their different phenotype and respective protumorigenic or tumor suppressive properties in TME is required for CAF-targeting therapies.

The NF-κB signaling pathway is constitutively activated in 90% of EBV-associated NPC by EBV oncoproteins and genetic mutations. This pathway plays a pivotal role in the intercellular communication and regulation of immune cells in the TME, which renders it a promising target for anti-cancer therapy against NPC (200–202). Strategies targeting the NF-κB signaling pathway include anti-inflammatory compounds such as aspirin, inhibition of IκB kinase (IKK) and proteasome inhibition (130, 203, 204). Acting as an NF-κB inhibitor, aspirin reverses LMP1-induced EMT by suppressing NF-κB-exosomal secretion of LMP1 and promoting miR-203 expression (130). Inhibition of lung metastasis by aspirin is also observed in mouse models. PS1145, an inhibitor of IKK, could abrogate the NF-κB signaling pathway and subsequently inhibit the production of pro-inflammatory cytokine and cell proliferation. PS1145 significantly inhibits the tumor growth of NPC in in vitro cell lines and in vivo xenograft models (203). Bortezomib, a proteasome inhibitor, targeting STAT1 and NF-κB, could relieve the immune tolerance in NPC (204). Vitexin, a natural flavonoid glycoside targeting NF-κB, displayed promising anti-tumor activity against NPC in preclinical studies (205). Restoration of Ras-like estrogen-regulated growth inhibitor (RERG), an NF-κB inhibitor, by 5-Aza-2’-deoxycytidine and trichostatin A attenuated ERK/NF-κB signaling pathway, resulting in the inhibition of tumor growth and angiogenesis in vivo. Therefore, RERG might be employed as a target molecule in cancer therapy (206).

Whilst translating these bench findings into bedside application, several issues need to be addressed. As the NF-κB signaling pathway regulates multiple physiological processes, the development of therapeutic tools should consider developing drug delivery strategies with high specificity to prevent undesirable adverse events. It is reported that the NF-κB complex p50/p50/Bcl3 is prevalent in NPC but seldom found in a normal cell. Thus, Bcl3 inhibitors may represent promising therapeutic agents against NPC (202, 207). Other than that, the route of administration and dosage of NF-κB inhibitors should consider their bioavailability and safety.