阻断肿瘤微环境中TGF-β增强三阴乳腺癌脑转移瘤放疗敏感性及其机制研究

Brain metastatic triple negative breast cancer frequently progressed due to radiation resistance. Our previous study identified that blocking stromal TGF-β in orthotopic breast carcinoma could inhibit angiogenesis, and improve drug delivery via tumor vascular normalization. Preliminary results showed that blocking tumor microenvironmental TGF-β in the brain metastatic tumor of triple-negative breast cancer inhibited angiogenesis, decreased the infiltration of tumor associated stromal cells, and suppressed tumor growth; and TGF- β blockade could significantly down-regulate the miR-10a expression of vascular endothelial cells in vitro. Therefore, we hypothesize that blocking tumor microenvironmental TGF-β in brain metastatic tumor of triple-negative breast cancer could suppress tumor growth via inhibition of angiogenesis and regulation of stromal cells infiltration, and increase the radiosensitivity of brain metastatic tumor by inducing tumor vascular normalization and consequently improving tumor hypoxia. The mechanism of TGF-β blockade-induced vascular normalization might be due to inducement of pericyte recruitment to vascular endothelium by regulation of the expressions and functions of specific microRNAs and their target genes in endothelial cells. We plan to use brain metastatic triple-negative breast cancer nude mice xenografts, vascular imaging, pimonidazole experiment, and in vitro. molecular biology methods to prove the hypothesis. Our study will help to find novel therapy target for triple negative breast cancer with brain metastasis, and may provide strong preclinical evidence for conducting clinical trials on combining anti-TGF-β antibodies and radiation therapy in treating triple-negative breast cancer patients with brain metastasis.

三阴乳腺癌脑转移常因放疗抵抗而进展。我们前期发现阻断原位乳腺癌微环境中TGF-β抑制血管生成，诱导肿瘤血管正常化而增强化疗药物的运输。预实验显示阻断三阴乳腺癌脑转移瘤微环境中TGF-β抑制肿瘤生长、血管生成和减少肿瘤间质细胞浸润；并且阻断细胞培养液中TGF-β能下调血管内皮细胞的miR-10a。据此提出假设：阻断三阴乳腺癌脑转移瘤微环境中TGF-β能通过抑制血管生成和调控间质细胞浸润而抑制肿瘤生长，且能诱导肿瘤血管正常化以改善组织缺氧增强肿瘤对放疗的敏感性，其诱导血管正常化的机制可能是调控内皮细胞特异microRNAs和下游靶基因的表达及其功能以诱导血管旁细胞向内皮聚集。我们拟应用裸鼠三阴乳腺癌脑转移模型及血管三维成像、缺氧标记物实验和一系列分子生物学方法，从体内、体外两方面证明上述假设，从而为治疗三阴乳腺癌脑转移提供新的靶点，为TGF-β抑制剂与放疗联用治疗三阴乳腺癌脑转移提供理论依据。

三阴乳腺癌的预后在乳腺癌所有分子亚型中最差，发生脑转移后患者的中位生存期小于6个月，并且脑转移常因放疗抵抗而进展。我们的预实验显示阻断MDA-MB-231-BR-stb（简称231-BR-stb）三阴乳腺癌脑转移瘤微环境中TGF-β抑制肿瘤生长、血管生成和减少肿瘤间质细胞浸润。本课题在预实验231-BR-stb三阴乳腺癌模型的基础上，成功建立了MDA- MB-468-stb-Fluc脑转移模型，发现阻断肿瘤微环境中TGF-β能显著抑制肿瘤生长；并且在三阴乳腺癌患者临床标本中验证了高TGF-β表达与不良预后正相关。进一步用231-BR-stb裸鼠脑转移放疗模型证明阻断微环境中TGF-β能逆转肿瘤放疗抵抗；肿瘤血管免疫荧光染色示231-BR-stb血管旁细胞覆盖率比对照组增加，即发生了肿瘤血管正常化；并且分离两组肿瘤的血管内皮细胞提取 RNA，筛选出表达显著不同的miRNA为miR-10a。在小鼠血管内皮细胞SVEC4-10中下调miR-10a, 发现下调后的SVEC4-10细胞分泌的血管旁细胞趋化相关因子IL-6及IL-8显著增多。该项目首次发现了阻断三阴乳腺癌脑转移瘤微环境中TGF-β 能通过下调血管内皮细胞的miR-10a而诱导肿瘤血管正常化以增强脑转移瘤对放疗的敏感性，为今后进一步开展放疗与抗TGF-β抗体联用治疗晚期脑转移性三阴乳腺癌的临床试验提供临床前证据。