Background The progression of nasopharyngeal carcinoma (NPC) is profoundly affected by Epstein-Barr virus (EBV) infection. However, the role of EBV in the intercommunication between NPC and surrounding stromal cells has yet to be explored. Methods NPC biopsies were obtained for immunohistochemical (IHC) analyses. Clinical correlations between the expression of active YAP1/FAPα and the fibrotic response and between YAP1/FAPα and the density of cytotoxic CD8a+ T lymphocytes were determined. Survival times based on IHC scores were compared between groups using Kaplan-Meier survival and log-rank tests. Independent prognostic factors for metastasis/recurrence-free survival and overall survival were identified using univariate and multivariate Cox regression models. Fibroblasts were isolated from human nasopharyngeal biopsies. Exosomes were purified from culture supernatants of EBV+-positive NPC cells. The effects of EBV product-containing exosomes on fibroblast activation, fibrotic response, tumor growth, immune response, and correlations between the expression of featured genes were investigated using gel contraction assays, ELISAs, EdU incorporation assays, real-time impedance assays, RNA sequencing, immunostaining, 3D cancer spheroid coculture systems, and an NPC xenograft model. Results NPC patients who developed metastasis had significantly higher levels of active YAP1 and FAPα in their tumor stroma, which was further correlated with tumor fibrosis and poorer metastasis-free survival. Exosomes released from EBV+-NPC cells contained abundant FAPα protein and EBV-encoded latent membrane protein 1. Viral product-containing exosomes markedly enhanced the fibrotic response and tumor growth in a mouse xenograft model. IHC analyses of human NPC and NPC xenografts revealed positive correlations between levels of active YAP1 and FAPα, YAP1 and the fibrotic response, and FAPα and the fibrotic response. Mechanistic studies showed that treatment of fibroblasts with viral product-containing exosomes promoted the characteristics of cancer-associated fibroblasts by stimulating YAP1 signaling and the production of the immunosuppressive cytokines IL8, CCL2, and IL6. Inhibition of YAP1 activation markedly reversed these exosome-mediated protumoral effects, resulting in reduced contractility, inactivation of YAP1 signaling, and decreased production of immunosuppressive cytokines in fibroblasts. Furthermore, fibroblasts stimulated with these viral product-containing exosomes promoted NPC resistance to T cell-mediated cytotoxicity within tumor spheroids. In NPC tissues, a significant negative correlation was found between YAP1/FAPα and the density of CD8a+ T lymphocytes with a granzyme B signature. Conclusion EBV orchestrates interactions with the host and surrounding stroma by stimulating the functions of YAP1 and FAPα in fibroblasts through exosome cargos to create a more immunosuppressive, proinvasive microenvironment.
Epstein-Barr virus (EBV) infection is intimately associated with type III nasopharyngeal carcinoma (NPC). Most NPCs respond well to chemoradiation. Nevertheless, approximately 20% of patients experience recurrence and distant metastasis. Tumor desmoplastic responses and high densities of cancer-associated fibroblasts (CAFs) within tumors have been correlated with poorer prognosis. In this study, we demonstrate that EBV activates fibroblasts via exosomes to remodel a pro-tumoral microenvironment. Exosomes derived from NPC cells contained a high level of fibroblast activation protein alpha (FAPα) and EBV-encoded latent membrane protein 1 (LMP1). Treating primary fibroblasts with these exosomes induced functional changes resembling CAFs. Transcriptomic analysis on exosome-treated fibroblasts revealed genes involved in fibrosis-associated pathways were enriched. Noticeably, treating with EBV products-containing exosomes in fibroblasts enhanced the production of several immune-suppressive cytokines, including IL6, IL8, and MCP-1. Moreover, stimulation with these exosomes enhanced activation of YAP1 signaling, a key pathway linking to fibrosis. The in vivo mouse xenografted NPC studies showed that EBV products-containing exosomes markedly enhanced the severity of tissue fibrosis and tumor growth. IHC analysis on mouse NPC xenografts and human NPC biopsies showed a positive correlation between levels of nuclear YAP1 and FAPα, which were further correlated with an enhanced fibrotic response within tumors. Pharmaceutical intervention with YAP1 inhibitors blocked the exosome-mediated effects on fibroblasts. Collectively, our data suggest that EBV products-containing exosome is a causal factor in CAF formation, which constitutes a pro-tumoral microenvironment to benefit the survival of both tumor and virus itself. Citation Format: Po-Ju Lee, Yun-Hua Sui, Chen-Han Huang, Tzu-Tung Liu, Ngan-Ming Tsang, Shu-Chen Liu. EBV-products containing exosomes remodel NPC tumor microenvironment by activating fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3192.
Type III Nasopharyngeal carcinoma (NPC) is characterized by Epstein-Barr virus (EBV) infection and immune infiltration. M2 macrophages in the NPC microenvironment are considered to associate with poorer prognosis and promote disease progression. How the tumor-derived EBV or viral products regulate macrophage polarization within NPC microenvironment remains to be explored. We demonstrated that EBV products can be transferred to uninfected cancer cells or stromal cells via exosomes. The exosome-mediated intercellular communications between EBV+-NPC cells and macrophages were further investigated. Results of mass spectrometry and western blotting showed that NPC cells deposited EBV-encoded latent protein 1 (LMP1) into exosomes. The uptake of these exosomes by human monocyte-derived macrophages led to a M2-dominant polarization. Specifically, multiple properties of M2 macrophages, including higher levels of CD206, CD163, CD209, and VEGF, as well as a reduced expression of iNOS were found in macrophages stimulated with LMP1-containing exosomes (LMP1_exosomes), compared to those treated with exosomes derived from EBV-negative NPC cells. The LMP1_exosome-treated macrophages also exhibited a reduced cytotoxicity towards primary cancer cells. RNA sequencing data revealed that LMP1_exosomes suppressed the function of antigen presentation while activating the macrophage stimulating 1 receptor (MST1R)-STAT3 signaling in macrophages. Results of mechanistic studies showed that LMP1-CTAR2 domain contributed to the activation of MST1R. Additionally, treating macrophages with LMP1_exosomes activated yes-associated protein 1 (YAP1), the LMP1-mediated M2 phenotype and MST1R expression were partially suppressed by treating cells with the YAP1 inhibitor (verteporfin). It appears that YAP1 acts as a key regulator of MST1R. Clinically, higher expressions of MST1R and nuclear YAP1 were correlated with disease progression of NPC (p < 0.001). The role of LMP1_exosomes in regulating macrophage immune responses was further investigated using single-cell RNA sequencing (scRNA-seq) technique. Results showed that treating primary macrophages isolated from human head and neck tumors with LMP1_exosomes resulted in an increase in M2 proportion. Results of gene set variation analysis (GSVA) demonstrated enhanced signatures in IL10 and VEGF signalings. Collectively, our data suggest that LMP1-containing exosomes facilitate M2 macrophage polarization, which may assist cancer immune evasion in the NPC microenvironment. Citation Format: Tzu-Tung Liu, Yun-Hua Sui, Fang-Yu Tsai, Shih-Sheng Jiang, Kai-Ping Chang, Chen-Han Huang, Shu-Chen Liu. EBV positive NPC-derived exosomes promote macrophage M2 polarization via activating MST1R and YAP1 signalings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2500.
Purpose Nasopharyngeal carcinoma is highly metastatic but difficult to detect in its early stages. It is critical to develop a simple and highly efficient molecular diagnostic method for early detection of NPC in clinical biopsies. Methods The transcriptomic data of primary NPC cell strains were used as a discovery tool. Linear regression approach was used to define signatures distinctive between early and late stage of NPC. Expressions of candidates were validated with an independent set of biopsies (n = 39). Leave-one-out cross-validation technique was employed to estimate the prediction accuracy on stage classification. The clinical relevance of marker genes was verified using NPC bulk RNA sequencing data and IHC analysis. Results Three genes comprising CDH4, STAT4, and CYLD were found to have a significant differentiating power to separate NPC from normal nasopharyngeal samples and predicting disease malignancy. IHC analyses showed stronger CDH4, STAT4, and CYLD immunoreactivity in adjacent basal epithelium compared with that in tumor cells (p < 0.001). EBV-encoded LMP1 was exclusively expressed in NPC tumors. Using an independent set of biopsies, we showed that a model combining CDH4, STAT4, and LMP1 had a 92.86% of diagnostic accuracy, whereas a combination of STAT4 and LMP1 had a 70.59% accuracy for predicting advanced disease. Mechanistic studies suggested that promoter methylation, loss of DNA allele, and LMP1 contributed to the suppressive expression of CDH4, CYLD, and STAT4, respectively. Conclusion A model combining CDH4 and STAT4 and LMP1 was proposed to be a feasible model for diagnosing NPC and predicting late stage of NPC.
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