Identification of diverse tumor endothelial cell populations in malignant glioma

Background: Glioblastoma multiform is the most common and aggressive type of primary brain tumor, as most patients succumb to the disease less than two years after their diagnosis. Critically, studies demonstrate that glioma recruits surrounding blood vessels, while some work suggests that tumor stem cells themselves may directly differentiate into endothelial cells, yet the molecular and cellular dynamics of the endothelium in glioma are poorly characterized. The goal of this study was to establish clear molecular and morphological benchmarks for tumor associated vessels (TAVs) and tumor derived endothelial cells (TDECs) during GBM disease progression. Methods: Using In-Utero Electroporation and CRISPR/Cas9 genome engineering to generate a native, immunocompetent mouse model of glioma, we characterized vascular-tumor dynamics in three dimensions during tumor progression. We also employed bulk and single-cell RNA-Sequencing to elucidate the relationship between TAV and TDECs. We confirmed our findings in a patient derived orthotopic xenograft (PDOX) model. Results: Using a mouse model of glioma, we identified progressive alteration of vessel function and morphogenesis over time. We also showed that TDECs exist, and that these cells contribute to vessels within the tumor. Furthermore, transcriptional profiling demonstrates that both TAVs and TDECs are molecularly distinct, and that both populations feature extensive molecular heterogeneity. Finally, the distinct molecular signatures of these heterogenous populations are also present in human glioma. Conclusions: Our findings show extensive endothelial cell heterogeneity within the tumor and tumor microenvironment, and provide insights into the diverse cellular and molecular mechanisms that drive glioma vascularization and angiogenesis during tumorigenesis. Overall design: Using In-Utero Electroporation and CRISPR/Cas9 genome engineering to generate a native, immunocompetent mouse model of glioma, we characterized vascular-tumor dynamics in three dimensions during tumor progression. We also employed bulk and single-cell RNA-Sequencing to elucidate the relationship between TAV and TDECs. We confirmed our findings in a patient derived orthotopic xenograft (PDOX) model.

背景：多形性胶质母细胞瘤（Glioblastoma multiform）是最常见且侵袭性最强的原发性脑肿瘤，多数患者在确诊后生存期不足两年。尤为关键的是，已有研究证实胶质瘤可募集周围血管；另有研究提示肿瘤干细胞可直接分化为内皮细胞，但胶质瘤内皮组织的分子与细胞动态特征仍未得到充分表征。本研究旨在为胶质母细胞瘤（GBM）疾病进展过程中的肿瘤相关血管（tumor associated vessels, TAVs）与肿瘤源性内皮细胞（tumor derived endothelial cells, TDECs）建立明确的分子与形态学基准。
方法：本研究通过子宫内电穿孔（In-Utero Electroporation）与CRISPR/Cas9基因组编辑技术，构建原生免疫健全的胶质瘤小鼠模型，对肿瘤进展过程中血管-肿瘤的三维动态特征进行表征。同时采用批量RNA测序与单细胞RNA测序，阐明肿瘤相关血管与肿瘤源性内皮细胞之间的关联，并在患者来源原位异种移植模型（patient derived orthotopic xenograft, PDOX）中验证了上述研究发现。
结果：借助胶质瘤小鼠模型，我们发现随着病程进展，血管功能与形态发生进行性改变。研究证实了肿瘤源性内皮细胞的存在，且此类细胞可参与构成肿瘤内血管。转录组分析显示，肿瘤相关血管与肿瘤源性内皮细胞均具有独特的分子特征，且两类群体均存在广泛的分子异质性。最后，这些异质性群体的独特分子特征也可在人类胶质瘤中检测到。
结论：本研究揭示了肿瘤及肿瘤微环境内广泛存在的内皮细胞异质性，为肿瘤发生过程中驱动胶质瘤血管化与血管新生的多样细胞与分子机制提供了新的研究见解。
整体设计：本研究通过子宫内电穿孔与CRISPR/Cas9基因组编辑技术构建原生免疫健全的胶质瘤小鼠模型，对肿瘤进展过程中血管-肿瘤的三维动态特征进行表征；同时采用批量RNA测序与单细胞RNA测序，阐明肿瘤相关血管与肿瘤源性内皮细胞之间的关联，并在患者来源原位异种移植模型中验证了上述研究发现。