Adenosine leakage from perforin-burst extracellular vesicles inhibits perforin secretion by cytotoxic T-lymphocytes

Abstract

Extracellular vesicles (EVs) in the tumor microenvironment facilitate intercellular communication. Cancer cell-derived EVs act as an immunosuppressor by transporting cargos and presenting transmembrane proteins. By contrast, CD8+ cytotoxic T-lymphocytes (CTLs) exert anti-cancer cytotoxicity via the pore-forming protein perforin. Here, we hypothesize that although EVs are destroyed by perforin, cancer cell-derived EVs might possess mechanisms that enable them to avoid this destruction. We used a breast cancer cell line, MDA-MB-231luc-D3H2LN (D3H2LN), to generate EVs. Destruction of the EVs by perforin was demonstrated visually using atomic force microscopy. To investigate immunosuppressive metabolites within cancer cell-derived EVs, we performed metabolomic profiling of EVs from D3H2LN cells cultured for 48 h with or without IFN-γ, which induces metabolic changes in the cells. We found that both types of EV from IFN-γ treated D3H2LN cells and non-treated D3H2LN cells contained adenosine, which has immunosuppressive effects. When we exposed cancer cellderived EVs to CTLs, perforin secretion by CTLs fell significantly. In addition, the decreases in perforin secretion were ameliorated by treatment with adenosine deaminase, which degrades extracellular adenosine. Taken together, these results suggest that after perforin secreted by CTLs disrupts the membrane of EVs, adenosine released from the EVs acts as an immunosuppressive metabolite by binding to the adenosine receptor on the CTL membrane. This mechanism provides a novel survival strategy using cancer cell-derived EVs.

Metabolomic Analysis of Small Extracellular Vesicles Derived from Pancreatic Cancer Cells Cultured under Normoxia and Hypoxia

“…The existence of intermediate metabolites of purine and pyrimidine metabolism in sEVs has already been reported. For example, sEVs derived from a breast cancer cell line were reported to have high concentrations of uridine and guanosine [ 37 ]. In addition, it has been reported that sEVs derived from head and neck squamous cell carcinoma cell lines and plasma from patients also contain intermediates for purine metabolism, which exhibit an immunosuppressive effect [ 38 , 39 ].…”

“…Cationic metabolites were measured using LC-MS, as described previously [ 37 ]. Briefly, LC-MS analysis was performed using an Agilent 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Q Exactive Orbitrap MS system.…”

Adenine-Based Purines and Related Metabolizing Enzymes: Evidence for Their Impact on Tumor Extracellular Vesicle Activities

“…On this aspect, there is a wide consensus. Indeed, ADO, via the interactions with P1 receptors [ 75 ], exhibits an indirect “protective” role on tumors by affecting the function of almost all cells of the immune system, including MCs and B, CD8 + CT, NK [ 50 , 58 , 60 , 64 ], and also Treg lymphocytes, of which ADO promotes the immune-suppressive activity [ 47 ]. Since it is even more evident that chronic inflammation may contribute to about 25% of human cancers [ 76 ], the overall behavior of TME/EV purines and enzymes is similar to that of the same compounds in inflammatory conditions [ 77 ].…”

“…Other findings would also suggest ADO-induced immunosuppression via EV contribution. Thus, Tadokoro et al [ 58 ] showed that EVs secreted by cell lines of breast cancer are disrupted by perforin, a pore-forming protein liberated by CD8 + cytotoxic lymphocytes (CTLs), when activated by the known cytokine interferon-gamma (IFN-γ), whose release in vivo is generally stimulated by the tumor presence and aims at rejecting the tumor itself [ 59 ]. The EV destruction by perforin may liberate ADO in the TME, as detected by a sophisticated procedure coupling liquid chromatography to mass spectrometry analysis (LC-MS), which in turn hinders perforin secretion by CTLs.…”

EGFR‐rich extracellular vesicles derived from highly metastatic nasopharyngeal carcinoma cells accelerate tumour metastasis through PI3K/AKT pathway‐suppressed ROS

“…Extracellular vesicles (EVs) are cell‐derived membranous particles of sub‐micrometre sizes that play important roles in cancer biology by mediating intercellular communication through carrying proteins, nucleic acids, and lipids (Reategui et al., 2018; Tadokoro et al., 2020; Wan et al., 2018). EVs comprise many subtypes, including exosomes, microvesicles, and apoptotic bodies (Wang, Tan, & Guan, 2019).…”