BACKGROUND Glioblastoma multiforme is an aggressive primary brain tumor that is characterized by local invasive growth and resistance to therapy. The role of the microenvironment in glioblastoma invasiveness remains unclear. While carcinomas release CD147, a protein that signals for increased matrix metalloproteinase (MMP) release by fibroblasts, glioblastoma does not have a significant fibroblast component. We hypothesized that astrocytes release MMPs in response to CD147 contained in glioblastoma-derived extracellular vesicles (EVs) and that ionizing radiation, part of the standard treatment for glioblastoma, enhances this release. METHODS Astrocytes were incubated with EVs released by irradiated or non-irradiated human glioblastoma cells wild-type, knockdown, or knockout for CD147. Levels of CD147 in glioblastoma EVs and MMPs secreted by astrocytes were quantified. Levels of proteins in the mitogen activated protein kinase (MAPK) pathway, which can be regulated by CD147, were measured in astrocytes incubated with EVs from glioblastoma cells wild-type or knockdown for CD147. Immunofluorescence was performed on the glioblastoma cells to identify changes in CD147 localization in response to irradiation, and to confirm uptake of the EVs by astrocytes. RESULTS Immunoblotting and mass spectrometry analyses showed that CD147 levels in EVs were transiently increased when the EVs were from glioblastoma cells that were irradiated with γ rays. Specifically, the highly-glycosylated 45 kDa form of CD147 was preferentially present in the EVs relative to the cells themselves. Immunofluorescence demonstrated that astrocytes incorporate glioblastoma EVs and subsequently increase their secretion of active MMP9. The increase was greater if the EVs were from irradiated glioblastoma cells. Testing MAPK pathway activation, which also regulates MMP expression, showed that JNK signaling, but not ERK1/2 or p38, was increased in astrocytes incubated with EVs from irradiated compared to non-irradiated glioblastoma cells. Knockout of CD147 in glioblastoma cells blocked the increased JNK signaling and the rise in secreted active MMP9 levels. CONCLUSIONS The results support a tumor microenvironment-mediated role of CD147 in glioblastoma invasiveness, and reveal a prominent role for ionizing radiation in enhancing the effect. They provide an improved understanding of glioblastoma intercellular signaling in the context of radiotherapy, and identify pathways that can be targeted to reduce tumor invasiveness. Video abstract.

中文翻译：

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源自胶质母细胞瘤细胞的细胞外囊泡增加星形胶质细胞释放的金属蛋白酶：CD147（EMMPRIN）和电离辐射的作用。

背景技术多形胶质母细胞瘤是侵袭性的原发性脑肿瘤，其特征在于局部侵袭性生长和对治疗的抵抗力。微环境在胶质母细胞瘤侵袭中的作用仍不清楚。虽然癌症会释放CD147，CD147是一种信号，指示成纤维细胞释放的基质金属蛋白酶（MMP）增加，但成胶质细胞瘤却没有明显的成纤维细胞成分。我们假设星形胶质细胞响应于胶质母细胞瘤衍生的细胞外囊泡（EVs）中所含的CD147释放MMP，并且电离辐射是胶质母细胞瘤标准治疗的一部分，可增强这种释放。方法将星形胶质细胞与野生型，敲除或敲除CD147的经照射或未照射的人胶质母细胞瘤细胞释放的EV一起孵育。胶质母细胞瘤EV和星形胶质细胞分泌的MMP中的CD147水平被定量。在星形胶质细胞中，用来自成胶质母细胞瘤细胞野生型或敲除CD147的EV孵育，测量可通过CD147调节的促分裂原活化蛋白激酶（MAPK）途径中的蛋白水平。在胶质母细胞瘤细胞上进行了免疫荧光，以鉴定响应于辐射的CD147定位变化，并确认星形胶质细胞对EV的摄取。结果免疫印迹和质谱分析表明，当电动汽车来自胶质母细胞瘤细胞并受到γ射线照射时，电动汽车中的CD147水平会瞬时升高。具体而言，相对于细胞本身，EV中优先存在高度糖基化的45 kDa形式的CD147。免疫荧光显示星形胶质细胞掺入胶质母细胞瘤电动汽车，并随后增加其活性MMP9的分泌。如果电动车来自照射的胶质母细胞瘤细胞，则增加更大。测试MAPK途径活化（也调节MMP表达）表明，与未照射的胶质母细胞瘤细胞相比，用照射的EV孵育的星形胶质细胞中JNK信号传导增加，但ERK1 / 2或p38却没有增加。胶质母细胞瘤细胞中CD147的敲除阻止了JNK信号的增加和分泌的活性MMP9水平的增加。结论该结果支持CD147在胶质母细胞瘤侵袭中的肿瘤微环境介导的作用，并揭示了电离辐射在增强作用中的重要作用。他们在放射疗法的背景下提供了对胶质母细胞瘤细胞间信号转导的更好理解，并确定了可靶向减少肿瘤侵袭性的途径。录像摘要。