91 Impact of ultra-fast ‘FLASH’ radiotherapy on single cell immunogenomics in diffuse intrinsic pontine glioma (DIPG)

Background Diffuse intrinsic pontine gliomas (DIPG’s) are immunologically inert tumors with a median survival of 9–15 months. Radiation therapy (RT) is the mainstay treatment for DIPG but associated immunodepletion tumor microenvironment (TME) at high dose ranges. FLASH, or ultra-fast rate RT, represents novel ablative technique that may spare TME immune responses while decreasing burden. Here, we present single-cell profiling treated conventional RT (CONV) no (SHAM). Methods Murine H3.3K27M mutant cells were stereotactically injected and induction confirmed by magnetic resonance imaging (MRI) 15 days later. DIPG-bearing mice randomly assigned to one three groups (n=4/group), CONV SHAM. A fourth group (NML) was included as negative biological control. modified linear accelerator used deliver Gy electron brainstem rates 90 Gy/second 2 Gy/minute, FLASH groups, respectively. Four post-RT, brainstems harvested, homogenized, stained CD45 tagged hashtag antibody specific each group. CD45+ isolated sequenced using 10X Genomics chromium 3’ platform. After processing alignment reads CellRanger default parameters, data quality checked filtered before demultiplexing, unsupervised clustering downstream analysis implemented following Seurat R package. Differential expression evaluated based on non-parametric Wilcoxon rank sum test. Key genes determine an adjusted p value < 0.05 bonferroni correction |avg log2FC| > 0.8. Results Preliminary identifies clusters distinct phenotypes (figure 1). gene (treatment group) reveals 14 differentially expressing key genes, including 3 upregulated in compared NML, irradiated SHAM NML 2). Notably, demonstrates individual cluster versus all other (p = 3.07E-171). Further deconvolution represented ongoing. Abstract 91 Figure 1 tSNE plot RNA signatures, grouped Conclusions Our preliminary shows differential among NML. We also find several cell subsets unique unirradiated samples. Most notably, identify single subset exclusive alone, indicating elicits response murine DIPG.