Proteomic and phospholipidomic characterization of extracellular vesicles inducing tumor microenvironment in Epstein‐Barr virus‐associated lymphomas

Abstract

Epstein-Barr virus (EBV) causes malignant carcinomas including B cell lymphomas accompanied by the systemic inflammation. Previously, we observed that phosphatidylserine (PS)-exposing subset of extracellular vesicles (EVs) secreted from an EBV strain Akata-transformed lymphoma (Akata EVs) convert surrounding phagocytes into tumor-associated macrophages (TAMs) via induction of inflammatory response, which is in part mediated by EBV-derived micro RNAs. However, it is still unclear about EV-carried other potential inflammatory factors associated with TAM formation in EBV lymphomas. To this end, we sought to explore proteomic and phospholipidomic profiles of PS-exposing EVs derived from EBV-transformed lymphomas. Mass spectrometric analysis revealed that several immunomodulatory proteins including integrin αLβ2 and fibroblast growth factor 2 (FGF2) were highly expressed in PS-exposing Akata EVs compared with another EBV strain B95-8-transformed lymphoma-derived counterparts which significantly lack TAM-inducing ability. Pharmacological inhibition of either integrin αLβ2 or FGF2 hampered cytokine induction in monocytic cultured cells elicited by PS-exposing Akata EVs, suggesting the involvement of these proteins in EV-mediated TAM induction in EBV lymphomas. In addition, phospholipids containing precursors of immunomodulatory lipid mediators were also enriched in PS-exposing Akata EVs compared with B95-8 counterparts. Phospholipidomic analysis of fractionated Akata EVs by density gradient centrifugation further demonstrated that PS-exposing Akata EVs might be identical to certain Akata EVs in low density fractions containing exosomes. Therefore, we 2 of 15 | ITO eT al. Technology (CREST); Research Program on Hepatitis from Japan Agency for Medical Research and Development, Grant/ Award Number: 16fk0210114h0001 concluded that a variety of immunomodulatory cargo molecules in a certain EV subtype are presumably conducive to the development of EBV lymphomas.

New insights into Epstein‑Barr virus‑associated tumors: Exosomes (Review)

“…An immunosuppressive TME exists in most EBV-associated tumors, which impedes the efficacy of immunotherapies ( 119 ). The majority of previous studies have confirmed that exosomes derived from EBV + tumor cells serve a role in inhibiting body immunity and promoting tumor cell immune evasion in the process of tumor progression ( 63 , 95 , 104 ). Therefore, targeting EBV + exosomes is considered as a promising new treatment for EBV + tumors.…”

“…The proteins or nucleic acids encoded by EBV may serve pivotal roles in tumorigenesis and tumor development as tumor regulators. In addition, exosomes serve essential roles in the transfer of oncogenic molecules, as well as in the tumorigenesis and tumor metastasis of EBV-associated tumors ( 62 , 63 ).…”

“…In this process, the expression levels of TNF-α, IL-10 and ARG1 are partially regulated by EBV- Bam HI A rightward transcripts (BART)-miRNAs. Ito et al ( 63 ) observed that a phosphatidylserine-exposing subset of EVs secreted from lymphoma cells transformed with the EBV strain Akata converted surrounding phagocytes into tumor-associated macrophages (TAMs) by inducing an inflammatory response partially mediated by EBV-miRNAs. Using mass spectrometric analysis, the study indicated that several immunomodulatory proteins, especially integrin αLβ2 and fibroblast growth factor 2 (FGF2), are key factors in the TAM-inducing ability of EVs.…”

“…Although there are a number of different kinds of EBV-associated tumors, they primarily include B cell lymphoma, NPC and EBVaGC. These three different tumors have different TMEs but share an analogous immunosuppressive characteristic, which might be induced by EBV + exosomes ( 63 , 95 , 104 ). The known mechanisms of different EBV + tumor cell exosomes acting on various target cells are summarized in Fig.…”

Significance of trogocytosis and exosome-mediated transport in establishing and maintaining the tumor microenvironment in lymphoid malignancies

“…Mass spectrometric and phospholipidomic analysis revealed that several immunomodulatory proteins and lipid mediators containing EVs are important for inducing the tumor microenvironment in EBV-positive lymphoma. 80 Exosome-mediated establishment of a tumor-supporting environment has been reported. Waldenström macroglobulinemia, which is characterized by a mutation in the innate immune-signaling adaptor myeloid differentiation response 88 (MyD88), was reported to secrete a mutant of MyD88 in EVs.…”