Csig-16. pten loss in glioma cell lines facilitates immune evasion by upregulating extracellular vesicle biogenesis and pd-l1 cargo

Abstract Phosphatase and tensin homologue (PTEN) is deleted or mutated in over 50% of all glioblastoma (GBM) cases. Recently, PTEN loss has been implicated as a prognostic biomarker for patients likely to be non-responders treated with immune checkpoint inhibitor (ICI) therapy. Indeed, a growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1) and the development of an immunosuppressive tumor immune microenvironment (TIME), likely the result of an altered secretome. This raised the question of whether the loss of PTEN impacts the biogenesis and function of extracellular vesicles (EVs), given their functional contributions to immunosuppression and tumor growth. Here we show that glioma cell lines with no or reduced PTEN expression produce elevated numbers of EVs, as measured through nanoparticle tracking analysis. Pharmacological inhibition of PI3K reversed this observation, indicating that this phenomenon occurs in a PI3K-dependent manner. Moreover, we show through flow cytometry and immunoblot analysis that the ability for the inflammatory cytokine interferon-γ (IFN-γ) to upregulate PD-L1 at the cell surface and as EV-associated cargo is markedly increased in cells lacking PTEN. Lastly, we show that in response to IFN-γ, EVs derived from glioma cells lacking PTEN exhibit an enhanced ability to inhibit TCR signaling in a Jurkat reporter system. Taken together, these findings shed new light on how the loss of a major tumor suppressor, PTEN, can alter EV biogenesis and function and contribute to an immunosuppressive extracellular milieu. Future studies will be directed toward exploring whether loss of PTEN function alters additional EV-associated immuno-modulatory proteins relevant to other immune cell populations, toward gaining a further understanding of how EVs shape the “cold” TIME of GBM and the potential therapeutic benefit of combining PI3K inhibitors with ICIs.