Single cell RNAseq of murine gliomas induced by PDGFB overexpression

Glioblastoma progression remains elusive, especially in its first stages. Here, by simultaneous transfer of PDGFB and genetic barcode in mouse brain, we triggered gliomagenesis in univocally labelled cells in vivo, hence enabling direct tracing of glioblastoma evolution from the earliest possible stage. We observed an unexpectedly high incidence of clonal extinction events and progressive clonal size divergence, even after the acquisition of a malignant phenotype. Computational modelling suggests these dynamics as the consequence of a clonal-based cell-cell competition. Bulk and single-cell transcriptome analyses, coupled to lineage tracing, revealed that the strongest correlation with clonal size unbalancing is shown by Myc transcriptional targets. Additionally, higher levels of Myc signalling discriminate "winner" from "loser" clones in early gliomagenesis. These results agree with recent understanding of the Myc-mediated induction of supercompetitive phenotype in mammalian embryo. Our findings shed light on aspects of glioblastoma evolution inaccessible by conventional retrospective approaches and highlight the potential of the combined use of clonal tracing and transcriptomic analyses in this field. Overall design: Gliomas induced by in-utero somatic gene transfer of PDGFB in embryonic telencephalic ventricles were collected at about 40 days from the birth. Tumor masses were dissociated, tumor cells were enriched by FACSorting exploiting the GFP expression. The resulting samples were pooled two by two prior to 10X partitioning.

胶质母细胞瘤（Glioblastoma）的发生发展过程仍有待阐明，尤其是其早期阶段。本研究通过在小鼠脑内同时转导PDGFB与遗传条形码，在体内对唯一标记的细胞诱导胶质瘤发生，从而可从最早阶段直接示踪胶质母细胞瘤的演化进程。我们观察到意外高发的克隆消亡事件，以及即便在获得恶性表型后仍存在的进行性克隆大小分化。计算建模表明，此类动态变化源于基于克隆的细胞间竞争。批量转录组与单细胞转录组分析结合谱系示踪结果显示，与克隆大小失衡相关性最强的为Myc转录靶基因。此外，更高水平的Myc信号通路可区分早期胶质瘤发生中的获胜克隆与落败克隆。上述结果与近期关于Myc介导哺乳动物胚胎中超级竞争表型诱导的研究认知相符。本研究揭示了传统回顾性研究方法难以触及的胶质母细胞瘤演化特征，并凸显了联合应用克隆示踪与转录组分析在该领域的研究潜力。

实验整体设计：通过在胚胎端脑室开展PDGFB的宫内体细胞基因转移诱导胶质瘤，于小鼠出生后约40天收集样本。将肿瘤组织解离后，利用GFP表达通过荧光激活细胞分选（FACSorting）富集肿瘤细胞。所得样本每两份合并后，再进行10X分区处理。