MODL-29. EXPLORING CELL STATE TRANSITIONS IN GLIOBLASTOMA WITH LIVE REPORTING OF CELL STATES USING CRISPR/CAS9

Abstract

Abstract Glioblastoma (GBM) is the most common malignant primary brain tumor, with a median survival time of 12-15 months. The heterogeneity of GBM, ranging from inter-patient to intra-tumoral, poses a significant challenge in identifying effective treatments. At the single-cell level, GBMs comprise a multiplicity of transcriptional cell states that can change over time and in response to therapy. To explore how cell state plasticity shapes tumor progression, new experimental tools to monitor cell state dynamics in real-time will be important, if not essential. Here, we describe a live reporting of GBM cell states that utilize CRISPR/Cas9 and fluorescent proteins for tracking individual glioblastoma patient-derived cells’ (GBM-PDC) physiological gene expression, and cell state transitions over time and during treatment. The method aims to provide real-time reporting of accurate transcriptional phenotypes (e.g. OPC/NPC-like, Mesenchymal, Astrocyte states) over extended periods or in relevant in vitro or in vivo contexts. Our protocols achieved successful endogenous tagging of a common stem cell marker with full-length mCherry in up to 15 % of GBM-PDC (an order of magnitude more effective than previous reports). Real-time imaging (Incucyte S3 and IMX-C) allows us to trace individual cells’ states over time and during treatment, supported by ICC, qPCR, and Western blot, providing a reliable approach. Our next objective is to employ this method to tag several cell-state marker genes with fluorescent proteins. We can directly follow gene expression and cellular state transitions when several state-selective genes are fused with different fluorescent proteins. We anticipate establishing a robust framework to investigate the temporal plasticity and invasion patterns within GBM cells across different patients which may open for new therapeutic opportunities.