Single cell transcriptome dynamics and subpopulation heterogeneity in radiation treated recurrent glioma. Mus musculus

Radiation is the frontline treatment for malignant gliomas. Intra-tumoral heterogeneity has been proposed to grant cancer cells a superior trajectory and survival advantage to avoid therapeutic interventions including radiation. However, direct evidence to support the hypothesis via the transcriptome dynamics of glioma during radiation therapy is limited. The current study aim to measure the functional subpopulation dynamics before and after radiation treatment that assist the radiation resistance at single cell resolution. We investigate the single cell transcriptome and biological pathways of primary glioma mouse model and post-radiation early/late time point. Specifically, we used the RCAS mouse model for gliomas, which overexpress PDGFRA as the model. Using single cell transcriptome, for the first time, we confirmed the proneural classification of PDGFRA RCAS glioma mouse model and its heterogeneity. We found that recurrent dominant subpopulations are featured with elevated proliferation rate and hypoxia. In addition, we identified a subpopulation of radiation resistant cells in at early time points with elevated stemness. Lastly, the subpopulations composition undergoes large changes at late time point when the tumor recurred. Single cell transcriptome profiling of radiation treated mouse glioma mouse model identified tumor cell subpopulations dynamics. It provides novel insights into the molecular phenotype and biological functions of radiation resistant tumor cell population. Overall design: To study mechanism of glioma relapse posterior to therapeutic dose irradiation, we have used PDGF-driven murine RCAS-tva glioma model as described in Methods. These mice develop high-grade glioma at approximately week 6. The tumors mimic pathological radiological and characteristics of glioblastoma. Since tumors in the RCAS-tva mice were driven by enhanced PDGF signaling and tumor cells were potentially derived from oligodendritic progenitor cells, majority of transformed cells were are abundantly expressing PDGFRa. Once we detected the radiographic primary high-grade tumor in RCAS-tva mice, we started to irradiate mice’s brain at 2 Gy every other day for five times. During week one post-radiation, we observed complete regression of primary tumors after radiation, confirmed by both MRI and histology. Thereafter, we had followed up the recurrence of tumors by MRI every week by MRI. We demonstrated that the mice had developed secondary high-grade tumors from week 4 to week 8 post-irradiation. We collected tumor samples from three time points: primary glioma (T1, primary stage), early phase of relapse post-radiation (T2, early stage), and late phase of relapse post-radiation (T3, late stage). Tumor samples were dissociated and subjected for FCAS. The stromal cell including immune cells and endothelial cells were removed and PDGFRa positive cells were isolated for subsequent analysis. To examine the transcriptome level heterogeneity of tumor cells of these three stages, we performed bulk RNA-seq and single cell RNA-seq experiments for the FCAS sorted tumor cells using Fluidgm C1 system.