PROJECT SUMMARY Diffuse midline gliomas with H3K27M alteration (DMG-H3K27M) are the leading cause of pediatric brain tumor-associated deaths. All DMG-H3K27M becomes resistant to radiation, the standard of care, and most children succumb to their disease within two years of diagnosis. New treatment options are urgently needed. The applicant's long-term goal is to combine engineering and cancer cell biology to lead a research group that advances extracellular vesicle (EV)-based drug delivery for central nervous system diseases like DMG-H3K27M. EVs are potent signaling vehicles in the tumor microenvironment and are understudied in DMG-H3K27M. The overall objectives are to define how DMG-H3K27M derived EVs contribute to radiation resistance within a heterogenous tumor and to develop EVs as a delivery vehicle for brain-penetrant radiosensitizers while equipping the applicant with the skills to become a strong, independent cancer researcher. Preliminary data show that some subclones within DMG-H3K27M are inherently radiation resistant and can release EVs readily taken up by radiosensitive cells in a receptor-mediated manner. These EVs protect the recipient tumor cells from radiation-induced cell death. Therefore, the central hypothesis is that the cargo of DMG-H3K27M extracellular vesicles, particularly microRNAs, drives radiation resistance within these tumors and that targeting these factors can sensitize DMG-H3K27M cells to radiotherapy. Additionally, the selective uptake of EVs by DMG-H3K27M cells suggests that EVs can be exploited as a drug delivery tool. The rationale is that this research will provide novel insights into mechanisms of radioresistance in DMG-H3K27M and may result in new approaches to target this cancer. The hypothesis will be tested by pursuing two specific aims: 1) Determine the mechanism of EV-mediated radioresistance in DMG-H3K27M and 2) Engineer extracellular vesicles to selectively target brain tumor cells and deliver radiosensitizers. The first aim will be carried out as part of the dissertation research and will use patient-derived DMG-H3K27M cells, proteomics approaches, and chemical inhibitors to define the mechanism of EV uptake. miRNA mimics and antagomirs will also be used to overexpress and silence miRNAs found in EV cargo released from radioresistant cells to identify miRNAs that confer enhanced radioresistance in DMG-H3K27M. Targets will be validated using zebrafish xenograft models. The second aim will encompass the post-doctoral research and focus on using single-cell microfluidic methods to engineer non-tumor extracellular vesicles for specificity towards brain tumor cells and assessing their capability to cross the blood-brain barrier to deliver radiosensitizers. The research proposed in this application is significant because it will provide new insights into the role of tumor heterogeneity in driving therapy resistance. This project will also identify new mechanisms of DMG-H3K27M radioresistance and develop ...