Glioblastoma (GBM) is the most aggressive brain cancer and usually fatal within a year after diagnosis. Current treatment approaches provide only a modest, few months, life extension. Tumor infiltrating myeloid cells (TIM) provide strong impact on tumor growth, dispersal and treatment resistance. The current unmet need in GBM treatment is to address the TIM contribution on tumor growth and treatment resistance in treatment protocols. The goal of the study is to identify molecular targets for the GBM treatment approaches aimed at modulation of tumor immune microenvironment. Our recent studies identified significant up-regulation of Proline-Rich Tyrosine Kinase 2 (Pyk2) in GBM tumors, compared with healthy brain tissue, as well as, a positive correlation between Pyk2 activation in tumor cells and cytokines expression profile of TIM. Additionally, both tumor resection and temozolomide treatment up-regulate Pyk2 in GBM tumors and affect activation state of TIM. This identified Pyk2 as a candidate prognostic marker for the immune state in GBM tumor microenvironment. Preliminary studies in GL261 glioma cells identified that cells, knocked out for Pyk2, do not release the population of EVs with diameter bigger them 600nm and reduce expression of monocyte chemoattractant protein (CCL2), CCL12 and vascular endothelium growth factor (VEGF) in EV fraction, compared with Pyk2WT cells. Correspondingly, increase of CD86+/CD206+ inflammatory myeloid cell and CD8+ lymphocytes populations were found in TIMs, purified from Pyk2KO tumors, compared with Pyk2WT tumors, in GL261/C57Bl/6 mouse glioma implantation model. Based on these findings we hypothesize that Pyk2 is involved in signaling regulation of release of inflammatory cytokines CCL2, CCL12 and CCL5 through the EVs mechanism, leading to modulation of TIM cells polarization. In this study we will utilize mouse glioma implantation model and primary human GBM cell cultures, with high and low levels of Pyk2 expression, coupled with cell biology approaches, to dissect the role of Pyk2 in regulation of biogenesis and cytokines content of glioma cell derived EVs. We will also examine, in mouse and human in vitro models, the modulation of activation state of microglial cells through the glioma-derived EV mechanism. The purpose of the project is to identify mechanisms of interaction between glioma and TIM cells and the role of Pyk2 signaling in this communication. To test our hypothesis, we propose the following specific aims: Specific Aim 1. To investigate the role of Pyk2 in release of cytokines through the EV mechanism in GBM cells. Specific Aim2. To investigate the role of Pyk2 in regulation of actin-related release of EVs populations. Specific Aim3. To assess the role of vesicular Pyk2 in EV internalization by microglial cells.

This project is aimed at improvement of glioblastoma (GBM) therapeutic strategies, taking into consideration the supporting role of tumor infiltrating myeloid cells on tumor progression and treatment resistance. The studies will elucidate the Pyk2-dependent machinery of cytokines release through the extracellular vesicles (EVs) mechanism in GBM cells, Pyk2 dependent populations of tumor-derived EVs and their effect on tumor infiltrating myeloid cells activation. The results of the study will help to identify molecular targets for managing tumor immune microenvironment and to enhance current GBM treatment protocols.