TMIC-21. PYK2/MAPK/ERK SIGNALING PATHWAY REGULATES EXTRACELLULAR VESICLE RELEASE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM), the deadliest brain cancer, typically proves fatal within a year despite treatment. Tumor-associated myeloid cells (TAMs) support GBM growth, treatment resistance, and immune surveillance. Extracellular vesicles (EVs) facilitate communication between tumor and TAMs, exchanging proteins, lipids, RNA, and genetic material. As EVs can travel through the bloodstream to distant tissues, they trigger signaling pathways and metabolic changes in remote organs while also recruiting bone marrow-derived cells and modulating immune responses. Therefore, EVs serve as a crucial mechanism for the distant regulation of the tumor’s immune microenvironment. We hypothesize that Pyk2/MAPK/Erk signaling regulates EV release by modulating the actin cytoskeleton, impacting TAM infiltration and activation in tumors. This study aims to investigate the role of Pyk2/MAPK/Erk in specific EV release in GBM cells, potentially offering insights into therapeutic targets. Using confocal imaging and flow cytometric analysis of EVs, purified from medium conditioned from human primary GBM cell lines with and without Pyk2 CRISPR/Cas9 knock-out (Pyk2KO), the study identified that Pyk2KO cells do not release the population of EVs with diameter bigger then 600nm and up-regulate release of smaller EVs. Additionally, flow cytometric analysis of EVs, released from cells, fluorescently labeled with membrane dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), detected 14% of CFSE+ events in EV’s population, purified from Pyk2WT, vs. to 2.5% from Pyk2KO cells. Membrane labeling of cells enables the tracking of microvesicles (MV) shedding and was used to differentiate between MV and ES based on the process of their biogenesis. Considering that larger size and the transition of fluorescence from the plasma membrane are indicative of MVs, our data indicate that Pyk2 is involved in the regulation of MV release. FUNDING: PRSTRT2022 and NIH Grant 1R15CA287203