EPCO-57. DISSECTING GLIOBLASTOMA RADIATION RESPONSE ACROSS DOSE AND TIME: A SINGLE-CELL MULTI-OMICS ANALYSIS OF SINGLE AND FRACTIONATED EXPOSURE

Abstract Glioblastoma is a heterogeneous brain tumor with limited responsiveness to radiation therapy. To uncover the molecular basis of radiation response in tumor subpopulations, we performed single-cell RNA-seq and single-cell ATAC-seq on glioblastoma stem cells (GSC20) treated with 2Gy, 6Gy, or fractionated 2 + 2Gy radiation. Single-cell gene-expression patterns revealed 7 distinct subpopulations, or clusters, several of which had ubiquitous enrichment for DNA damage response, immune signaling, and cell cycle regulation across all doses. Unexpectedly, certain clusters had dose-specific gene expression changes in response to different radiation doses and schedules. For example, the largest cluster showed increased extracellular matrix (ECM) activity and lipid metabolism when treated with 2Gy, but was instead enriched in immune signaling pathways when challenged with 6Gy. In other clusters, enriched gene pathways were involved in amino acid transport after 2Gy, cell adhesion and FOXO suppression after 6Gy, and neuronal downregulation after fractionated radiation (2 + 2Gy). Differences such as these may reflect a dose-dependent stress adaptation in each cluster. These transcriptional trends were mirrored by chromatin accessibility changes. Key transcription factors (TFs) involved in apoptosis, survival, and cell cycle control, such as IRF1, IRF2, E2F1, and TFDP1, showed dose- and cluster-specific accessibility. For example, suppression of immune and DNA repair pathways after 2Gy corresponded with reduced accessibility at interferon-related TF motifs, while mitotic and transcriptional activation after 2 + 2Gy aligned with increased chromatin accessibility at E2F1/TFDP1 binding sites. Pathway analysis of distinct TF-linked chromatin states revealed that 2Gy repressed immune and DNA repair pathways, while 6Gy and 2 + 2Gy silenced TP53 and interferon signaling. Meanwhile, 2 + 2Gy radiation induced divergence in chromatin accessibility: certain clusters favored differentiation, while other clusters promoted mitosis and transcription. Our findings suggest that radiation doses reshape gene expression and chromatin accessibility to direct divergent cell fates toward differentiation or proliferation.