应用胶质母细胞瘤类器官探究放化疗及缺氧诱导因子2α抑制剂对肿瘤细胞的抑制作用

The inhibitory effect of radiotherapy,chemotherapy,and hypoxia inducible factor-2α inhibitor on tumor cells studied on glioblastoma organoids

目的构建胶质母细胞瘤(GBM)类器官模型,使用该模型探讨放化疗、缺氧诱导因子2α(HIF-2α)抑制剂对GBM的抑制作用。方法脑胶质瘤及非脑胶质瘤患者的脑组织标本均来源于中南大学湘雅医院神经外科行手术治疗的患者,肿瘤细胞来源于GBM患者的脑组织标本和U-251细胞株。构建GBM类器官、GBM神经球体模型。细胞培养30 d,观察GBM类器官及GBM神经球体细胞的生长情况;采用免疫荧光染色检测肿瘤干细胞标志物的表达;采用免疫组织化学染色法检测脑胶质瘤标本及非胶质瘤脑组织标本、贴壁培养的GBM细胞、GBM类器官中HIF-2α的表达。细胞培养第30天,将GBM类器官分为未放疗组和放疗组,每组又分为二甲基亚砜(DMSO)组、替莫唑胺(TMZ)组及HIF-2α抑制剂组;放疗组给予6 Gy的X射线照射,继续培养30 d,观察各组细胞的传代率和HIF-2α的表达。结果细胞培养30 d,GBM类器官的直径约为2 mm,外缘细胞密集,中央排列松散;而GBM神经球体直径约为200μm,中心与边缘细胞分布均匀。免疫荧光染色结果显示,GBM类器官外缘的SOX2、CD133及表皮生长因子受体(EGFR)表达水平均高于其中心部位。GBM神经球体SOX2表达阳性,整个球体表达无分布差异;CD133和EGFR的表达均为阴性。非肿瘤脑组织不表达HIF-2α;高级别胶质瘤HIF-2α的表达强于低级别胶质瘤。GBM类器官中HIF-2α的表达主要分布在坏死细胞附近;贴壁培养的GBM细胞中,HIF-2α的表达分布较均匀。放疗组及未放疗组中,HIF-2α抑制剂组、TMZ组的细胞传代效率均比DMSO组降低(均P<0.05);与未放疗组中的DMSO组、TMZ组比较,放疗组中的DMSO组、TMZ组的细胞传代效率均降低,差异均有统计学意义(均P<0.05),而HIF-2α抑制剂组的差异无统计学意义(P=0.301)。免疫组织化学染色显示,未放疗组和放疗组中,除HIF-2α抑制剂组HIF-2α的表达降低外,DMSO组、TMZ组HIF-2α的表达均未降低。结论与GBM神经球体模型比较,GBM类器官肿瘤异质性更接近于肿瘤组织;HIF-2α与脑胶质瘤的恶性程度有关;放化疗对肿瘤的抑制作用不是通过抑制HIF-2α起作用的。

Objective To generate glioblastoma(GBM)organoid models and to investigate the inhibitory effect of radiotherapy,chemotherapy and hypoxia inducible factor-2α(HIF-2α)inhibitor on GBM using the models.Methods The brain tissue samples of glioma and non-glioma patients were obtained from patients undergoing surgical treatment at the Department of Neurosurgery,Xiangya Hospital,Central South University,and the primary tumor cells were derived from the brain tissue samples of GBM patients and U-251 cell line.The GBM organoids and GBM neurosphere models were generated.The cells were cultured for 30 days,and the growth of GBM organoids and GBM neurosphere cells was observed;immunofluorescence staining was used to detect the expression of tumor stem cell markers,and immunohistochemical staining was used to detect glioma specimens and non-glioma brain tissue specimens,HIF-2αexpression in GBM adherent cells and GBM organoids.On the 30th day of cell culture,GBM organoids were divided into non-radiotherapy group and radiotherapy group.Each group was further divided into dimethyl sulfoxide(DMSO)group,temozolomide(TMZ)group and HIF-2αinhibitor group.In the radiotherapy group,the cells were irradiated with 6 Gy X-rays and cultured for 30 d.The passage efficiency and HIF-2αexpression of cells in each group were observed.Results After 30 days of cell culture,GBM organoids were about 2 mm in diameter,with dense cells at the outer edge and loosely arranged in the center;while GBM neurospheres were about 200μum in diameter,with evenly ditributed cells in the center and edge.Immunofluorescence staining showed that the expression levels of SOX2,CD133 and epidernal growth factor(EGFR)in the outer edge of GBM organoids were higher than those in the central part.The expression of S0X2 was positive in CBM neurospheres,and there was no diference in the expression of the whole spheroids;the expressions of CD133 and EGFR were both negative.Non-tumor brain tissue does not express HIF-2α;high-grade gliomas have stronger HIF-2αexpression than low grade gliomas.The expression of HIF-2αin GBM organoids was mainly ditributed near necrotic cells;the expression of HIF-2αin GBM adherent cultured cells was more evenly distributed.In the radiotherapy group and the non-radiotherapy group,the cell passage efficiency of the HIF-2αinhibitor group and the TMZ group was lower than that of the DMSO group(both P<0.05).The cell passage efficiency in the DMSO group and TMZ group was decreased,and the diference was staistically significant(both P<0.05),while the difference in the HIF-2αinhibitor group was not staitically significant(P=0.301).Immunohistochemical staining showed that in the non-radiotherapy group and the radiotherapy group,the expression of HIF-2αin the DMSO group and TMZ group did not decrease except the HIF-2αinhibitor group.Conclusions The tumor heterogeneity of GBM organoids is closer to the tumor tissue.HIF-2αis related to the malignancy of glioma.The tumor suppressive effects of radiotherapy and chemotherapy are not through the inhibition of HIF-2α.