Epco-50. Tert Promoter Mutation-Based Cell Sorting for Single Cell Transcriptomic and Epigenetic Profiling of Glioblastoma Cells

TERT promoter mutations are the most frequent single nucleotide variants (SNVs) in glioblastoma and many other cancers. These hotspot mutations provide novel sites for transcription factor binding, activating expression of TERT gene and leading to telomere elongation and cancer cell immortalization. Moreover, these SNVs disrupt the highly structured G-quadruplex region of TERT promoter, impacting chromatin organization more broadly. Since epigenetic cell state is highly dynamic and regulated by cues from the tumor microenvironment, studying these changes in freshly resected tumor tissue is highly relevant. To enable such studies in glioblastoma, we developed STAR-FACS, a method allowing for cell sorting based on an SNV, that can be applied to freshly dissociated human tumor tissue. The single cell suspension is fixed, and a mutation-specific PCR is performed in the intact cells. Incubation of the cells with a labeled probe that hybridizes to the PCR product renders the cells fluorescent and amenable for cell sorting. Using TERTp WT and MUT glioblastoma cell lines and neurospheres, we demonstrated that STAR-FACS protocol is compatible with downstream profiling of the sorted cells by bulk and single cell transcriptomics and epigenetic profiling with CUT&Tag. We also show that TERT promoter mutation-based cancer cell enrichment can be performed on freshly dissociated glioblastoma tissue. STAR-FACS allowed us to compare expression profiles of TERT promoter mutant glioblastoma cells accumulating 5-ALA with 5-ALA- cancer cells from the same tumor, which would be challenging with standard methods. Our mutation-based cell sorting method provides a novel, inexpensive tool for studies linking the effect of subclonal non-coding SNVs on transcriptomic and epigenetic heterogeneity.