髓母细胞瘤复发过程中星形胶质细胞的来源、功能和分化机制研究

髓母细胞瘤(MB)是最常见儿童恶性脑肿瘤，预后差且近30%的患者会复发。我们曾阐述星形胶质细胞(AS)构成了原发MB的肿瘤微环境。预实验通过谱系追踪和单细胞测序发现：与原发MB不同，复发MB中AS来源于肿瘤细胞；复发MB高表达Hif1α及其靶基因、Notch1通路靶基因和Sox9；而低氧培养及过表达Hif1α、Notch1或Sox9均促进MB细胞向AS转分化，过表达Hif1α可激活Notch1通路。因此提出假说：复发MB通过Hif1α-Notch1-Sox9信号轴启动肿瘤细胞向AS转分化，促进肿瘤复发。项目拟采用条件性转基因鼠验证Notch1通路和Sox9对肿瘤复发和AS转分化的作用，通过基因干预验证Hif1α对Notch1和Sox9的调控，利用ChIP-seq分析Hif1α-Notch1-Sox9信号轴的具体作用机制。项目从肿瘤微环境角度探究MB复发机制，为复发MB防治提供多个分子靶点。

Medulloblastoma (MB) is the most common malignant brain tumor in children, 20 to 30 percent will relapse following their initial treatment. Cellular and molecular basis for MB recurrence still remains elusive, which precludes development of effective strategies to prevent and treat relapsed MB. We previously demonstrated that astrocytes, the major glial cell type in MB microenvironment, play an indispensible role in MB progression. Using established primary and relapsed MB mouse models and by lineage-tracing and single cell RNA sequencing, we recently found that astrocytes and tumor cells were lineage-independent in primary MB; However, during MB relapse, astrocytes predominately derived from tumor cells that are generally believed to be neuronal-lineage committed. Further studies revealed that Notch1 pathway was up-regulated in tumor cells from relapsed MB compared with those from primary MB. Enforced expression of the intracellular domain of Notch1 effectively converted tumor cells into astrocytes. These data suggest that Notch1 signaling may reprogram tumor cells into astrocytes in relapsed MB. Expression of hypoxia inducible factor 1a (Hif1α) and its target genes was dramatically elevated during MB relapse, indicative of hypoxia present in relapsed tumor. Notch1 pathway was activated in tumor cells after overexpression of Hif1α, implying that hypoxia may be responsible for Notch1 signaling as well as astrocytogeneis in relapsed MB cells. Also, overexpression of Hif1α as well as cultured under hypoxic condition promoted MB cells trans-differentiate into astrocytes. Moreover, Sox9 is highly expressed in astrocytes derived from relapsed MB, and overexpression of it in MB cells gave rise to astrocytes generation. Based on these observations, we proposed that hypoxia-driven reprogramming of tumor cells generates astrocytes to facilitate MB relapse through Hif1α−Notch1−Sox9 axis. In this proposal, we will further determine the characteristics and functions of tumor cell-derived astrocytes and investigate the mechanisms underlying astrocytogenesis in relapsed MB. We will confirm the function of Notch1 pathway and Sox9 in astrocytogenesis during MB relapse by using conditional knockout mice, then determine the Hif1α’s role in regulation of Notch1 pathway/Sox9 as well as astrocytogenesis by genetic manipulation. Finally we will Examine whether Notch1 signaling induces Sox9 transcription in tumor cells during MB relapse by ChIP. We will demonstrate the important functions of astrocytes in MB relapse, highlighting the critical role of tumor microenvironment in tumor recurrence. Our studies will provide solid evidence for the intrinsic plasticity of tumor cells during MB relapse, which will deepen our understandings of tumor cells characteristics during MB recurrence and metastasis, and provide new prevention and therapeutic targets for relapsed MB.