Cellular Neurosciences, Max Delbrueck Center for Molecular Medicine in the Helmholtz Association and Berlin Institute of Health, Berlin, Germany.
Department of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
Neuro Oncol. 2018 Feb 19;20(3):355-366. doi: 10.1093/neuonc/nox165.
Microglia and periphery-derived monocytes infiltrate human and mouse glioblastoma and their density is positively correlated with malignancy. Using microarray and RNA sequencing, we have previously shown that glioblastoma-associated microglia/monocytes (GAMs) express osteopontin/SPP1.
We used quantitative reverse transcriptase PCR, immunofluorescence stainings, western blot, and flow cytometry to identify the various sources of osteopontin (OPN) expression in human and mouse glioblastoma. We implanted wild type GL261 glioblastoma cells, which do not express significant levels of OPN, into wild type and OPN-/- mice to investigate the role of microenvironment-derived OPN on glioblastoma progression.
Our data indicate that GAMs are the predominant source of OPN in both human and mouse glioblastoma and express only the secreted form of OPN. Loss of microenvironment-derived OPN enhanced tumor progression. Staining by Ki67 and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling showed no difference in overall cell proliferation but a decreased apoptosis rate in tumors in OPN-/- mice. CD31 staining showed a significantly decreased number of microvessels in tumors in OPN-/- mice, accompanied by reduced coverage of vessels with platelet derived growth factor receptor β+ pericytes. Flow cytometry analysis revealed a significant increase of CD11b+/CD45low microglia but not of CD11b+/CD45high macrophages/monocytes in tumors in OPN-/- mice. Sorted CD11b+ cells from wild type and OPN-/- naïve brains and tumors did not show a significant difference in the expression pattern of activation marker genes.
Our results show that in tested human and mouse glioblastoma samples, OPN is predominantly expressed and secreted by GAMs and that, in contrast to OPN expression in the tumor cells per se, loss of stroma-derived OPN creates a glioblastoma-promoting microenvironment.
小胶质细胞和外周来源的单核细胞浸润人类和小鼠的胶质母细胞瘤，其密度与恶性程度呈正相关。我们之前使用微阵列和 RNA 测序技术表明，胶质母细胞瘤相关的小胶质细胞/单核细胞（GAMs）表达骨桥蛋白/ SPP1。
我们使用定量逆转录 PCR、免疫荧光染色、Western blot 和流式细胞术来鉴定人类和小鼠胶质母细胞瘤中各种骨桥蛋白（OPN）表达的来源。我们将不表达显著水平 OPN 的野生型 GL261 胶质母细胞瘤细胞植入野生型和 OPN-/- 小鼠中，以研究微环境衍生的 OPN 对胶质母细胞瘤进展的作用。
我们的数据表明，GAMs 是人类和小鼠胶质母细胞瘤中 OPN 的主要来源，仅表达分泌型 OPN。微环境衍生的 OPN 缺失增强了肿瘤的进展。Ki67 和末端脱氧核苷酸转移酶脱氧尿苷三磷酸末端标记染色显示总体细胞增殖无差异，但 OPN-/- 小鼠肿瘤中的细胞凋亡率降低。CD31 染色显示 OPN-/- 小鼠肿瘤中的微血管数量显著减少，同时血小板衍生生长因子受体β+周细胞覆盖的血管减少。流式细胞术分析显示 OPN-/- 小鼠肿瘤中 CD11b+/CD45low 小胶质细胞显著增加，但 CD11b+/CD45high 巨噬细胞/单核细胞没有增加。从野生型和 OPN-/- 未致敏大脑和肿瘤中分选的 CD11b+细胞在激活标志物基因的表达模式上没有显著差异。
我们的结果表明，在测试的人类和小鼠胶质母细胞瘤样本中，OPN 主要由 GAMs 表达和分泌，与肿瘤细胞本身的 OPN 表达相反，基质衍生的 OPN 缺失会产生促进胶质母细胞瘤的微环境。
