Single-cell lineage tracing of ispinesib-resistant glioma cells
To study the dynamics of GBM resistance and identify potential synergistic targets , we transfected PDGFR-amplified, patient-derived glioma neurospheres (TS543) with a barcoded lineage tracing library (CellTag), and treated the neurospheres with ispinesib. These genetically-modified, patient-derived neurospheres, which recapitulate key aspects of GBM heterogeneity, allow for surveillance of resistant phenotype on multiple timescales, and the barcode lineage tracing allows us to selectively analyze clones that are destined for resistance in the drug-naïve setting. We analyzed the phenotypes of the glioma cells during the long-term ispinesib treatment with single-cell RNAseq (scRNAseq), assess the stability and survival impact of drug-resistant phenotypes in the absence of drug and in orthotopic xenografts, and identified molecular markers of resistant clones in the drug-naïve setting to nominate effective drug combination. Overall design: TS543 were seeded at ~1000 cells and were transduced with CellTag virus-laden media or normal media. TS543 were propagated for three weeks before start of treatment. TS543 were treated with 75 nM ispinesib or with vehicle DMSO. Media with ispinesib or DMSO were replenished every two or three days. Viability of TS543 were monitored as guidepost for resistance. Dead cells were removed with Dead Cell Removal Kit from Miltenyi Biotec, and scRNA-seqs were initially performed at three-day interval, and later at two- and four-week intervals.  Orthopotic cell transplantations were performed on six-year-old CrTac:NCr-Foxn1nu female mice with latest time point of TS543 treated with DMSO or ispinesib, ten mice per cohort. Mice were clinically monitored daily and sacrificed once endstage criteria were met. One mouse from each cohort was used for scRNAseq