Tumor Associated Macrophages And Microglia Drive Tumor Progression And Are Transcriptionally Shaped By Histone Mutations In Pediatric Diffuse Midline Glioma

Pediatric high-grade gliomas, including diffuse midline gliomas (DMG), harbor mutually exclusive tumor location specific histone mutations. Using immunocompetent genetically engineered mouse models, we demonstrate the predominant non-neoplastic cell population are infiltrating myeloid cells, including peripheral monocytes and brain resident microglia. Single cell RNA sequencing, flow cytometry, and immunohistochemistry demonstrate the presence of unique myeloid cell populations distinctly shaped by the specific histone mutation. Disease associated myeloid cell phenotypes are identified, resembling those found in other neurodegenerative diseases, and demonstrate immune permissive characteristics. H3.3K27M DMGs, the most aggressive DMG subtype, demonstrate enrichment of disease associated myeloid cells as well as proliferating myeloid and tumor cells. Overall design: Using immunocompetent de novo mouse models of pediatric high grade glioma (pHGG), we generated tumors in both the cortical hemisphere and midline to represent each major subtype of pHGG including H3WT, H3.1K27M, or H3.3K27M pHGGs. Each tumor subtype contains the relevant driver mutations found in their human counterparts. Further experiments utilizing H3.1K27M DMGs in Ccl8/12 total knockout mice were performed, using H3.1K27M samples as wild type controls.Brain tumor cells were isolated at the humane endpoint of these mice, and sc-RNA seq was conducted to compare their functional differences.

儿童高级别胶质瘤（pediatric high-grade glioma, pHGG）涵盖弥漫性中线胶质瘤（diffuse midline glioma, DMG），此类肿瘤携带有与肿瘤位置特异性相关的互斥组蛋白突变。
本研究利用免疫健全型基因工程小鼠模型（immunocompetent genetically engineered mouse models），证实肿瘤中占比最高的非肿瘤细胞群体为浸润性髓系细胞，包括外周单核细胞与脑内常驻小胶质细胞。
通过单细胞RNA测序（single cell RNA sequencing, scRNA-seq）、流式细胞术（flow cytometry）与免疫组化（immunohistochemistry）实验，我们证实存在由特定组蛋白突变塑造的独特髓系细胞亚群。
本研究鉴定出疾病相关性髓系细胞表型，此类表型与其他神经退行性疾病中发现的髓系表型相似，且呈现免疫许可性特征。
H3.3K27M型弥漫性中线胶质瘤作为侵袭性最强的DMG亚型，其肿瘤内疾病相关性髓系细胞、增殖性髓系细胞与肿瘤细胞均呈现富集现象。

实验整体设计：
本研究利用免疫健全型基因工程小鼠模型构建原发儿童高级别胶质瘤，在大脑皮层半球与中线位置分别植入肿瘤，以覆盖儿童高级别胶质瘤的各主要亚型，包括H3WT、H3.1K27M与H3.3K27M型儿童高级别胶质瘤。
各肿瘤亚型均携带对应人类肿瘤中发现的功能性驱动突变。
后续实验以H3.1K27M型DMG样本作为野生型对照，在Ccl8/12全基因敲除小鼠（Ccl8/12 total knockout mice）中开展相关研究。
在实验小鼠达到人道终点时分离脑肿瘤细胞，并通过单细胞RNA测序比较各组间的功能差异。