Tmic-32. parallel single cell nucleosome occupancy and rna sequencing on recurrent gbm

Nearly all patients diagnosed with Glioblastoma (GBM) will experience a fatal recurrence of the tumor. Thought to be in part driven by immense tumor heterogeneity it is important to explore the small subpopulations of tumor cells with in depth single cell sequencing pre and post therapy in order to understand recurrence. To this end, we created an experimental in vivo pipeline that allows us profile not only gene expression changes during therapy, but also epigenetic shifts due to methylation across the genome at the single cell level from a single tumor. Single Cell NomE-Seq libraries were created and correctly identified characteristic CTCF binding regions within DNase1 hypersensitivity regions when compared back to representative encode data sets. Primary tumors analyzed by single cell RNA sequencing showed enrichment for on average 5 significant principle components (St. Dev >2 verified by Elbow Plot). Comparatively, recurrent temozolomide (TMZ) exposed tumors averaged 10 significant principle components (St. Dev >2 verified by Elbow Plot) suggesting TMZ therapy causes tumors to diversify their gene expression profiles. For direct comparison samples were merged into a continuous data set and dimensional analysis with TSNE reduction (15 dimensions used) identified 6 unique clusters, with one cluster being over-represented by cells from recurrent, TMZ exposed mice. Gene Ontology analysis on expression values contained within in this cluster demonstrated significant enrichment for ribosome biogenesis/ribosomal structure, (p=2.87e-17) and mRNA catabolic processes (p=2.47e-30). Further, comparison to a similar data set gathered using cells in vitro culture conditions demonstrates superior intraturmoral heterogeneity from cells isolated freshly from the brains of mice as opposed to tissue culture, indicating the effectiveness of our model for assaying heterogeneity. Ultimately, these data illuminate possible new mechanisms for TMZ induced ribosomal modifications underlying intraturmoral heterogeneity in GBM and provide an expandable technique for further experimentation.