Single-cell transcriptomics reveals antitumor immune responses of proton treatment in glioblastoma

Abstract Proton therapy is an innovative radiation therapy modality that has been shown to induce higher cytotoxicity to tumor cells compared to standard x-ray therapy while sparing normal tissue. However, the impact of proton radiotherapy on the growth and progression of glioblastoma (GBM) has not been defined. We hypothesize that understanding tumor evolution and micro environmental responses to radiotherapy is critical for developing and targeting interventions. In this study, we investigate the effect of proton versus standard photon radiotherapy on tumor growth and the microenvironment in an orthotopic syngeneic GBM model over the course of treatment. We characterized tumor tissues at early, progressing, and terminal phases post irradiation by single cell transcriptomics, bulk RNA sequencing, and histology. We find that proton irradiation led to a drastic reduction in tumor volume at early stages before eventual recurrence. Proton treatment exhibits equivalent if not superior, tumor control when compared to normal photon radiation. Single cell transcriptomics analyses show a dramatic increase in immune cell populations including T cell and macrophage populations which peak shortly before tumor recurrence. In addition, we identify an increase in exhausted T cell markers such as PD1, Tim-3, and Lag-3 in T cells post irradiation. Treatment with therapeutic agents that reprogram microglia/macrophages to an anti-tumor phenotype and activate T cell effector functions effectively eradicate GBM recurrence post irradiation. Thus, our study demonstrates a powerful combined treatment strategy with radiation against GBM and provides valuable information about how tumor growth and microenvironment respond to proton therapies, as well as potential mechanisms of tumor response and recurrence in GBM.