Background The most threatening metastases in breast cancer are brain metastases, which correlate with a very poor overall survival, but also a limited quality of life. A key event for the metastatic progression of breast cancer into the brain is the migration of cancer cells across the blood–brain barrier (BBB). Methods We adapted and validated the CD34 + cells-derived human in vitro BBB model (brain-like endothelial cells, BLECs) to analyse the effects of patient serum on BBB properties. We collected serum samples from healthy donors, breast cancer patients with primary cancer, and breast cancer patients with, bone, visceral or cerebral metastases. We analysed cytokine levels in these sera utilizing immunoassays and correlated them with clinical data. We used paracellular permeability measurements, immunofluorescence staining, Western blot and mRNA analysis to examine the effects of patient sera on the properties of BBB in vitro. Results The BLECs cultured together with brain pericytes in transwells developed a tight monolayer with a correct localization of claudin-5 at the tight junctions (TJ). Several BBB marker proteins such as the TJ proteins claudin-5 and occludin, the glucose transporter GLUT-1 or the efflux pumps PG-P and BCRP were upregulated in these cultures. This was accompanied by a reduced paracellular permeability for fluorescein (400 Da). We then used this model for the treatment with the patient sera. Only the sera of breast cancer patients with cerebral metastases had significantly increased levels of the cytokines fractalkine (CX3CL1) and BCA-1 (CXCL13). The increased levels of fractalkine were associated with the estrogen/progesterone receptor status of the tumour. The treatment of BLECs with these sera selectively increased the expression of CXCL13 and TJ protein occludin. In addition, the permeability of fluorescein was increased after serum treatment. Conclusion We demonstrate that the CD34 + cell-derived human in vitro BBB model can be used as a tool to study the molecular mechanisms underlying cerebrovascular pathologies. We showed that serum from patients with cerebral metastases may affect the integrity of the BBB in vitro, associated with elevated concentrations of specific cytokines such as CX3CL1 and CXCL13.

中文翻译：

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乳腺癌脑转移患者的血清衍生因子改变人血脑屏障模型的通透性


背景乳腺癌中最具威胁性的转移是脑转移，它与非常差的总体生存率相关，但也与有限的生活质量相关。乳腺癌转移至大脑的一个关键事件是癌细胞穿过血脑屏障（BBB）的迁移。方法 我们采用并验证了 CD34 + 细胞衍生的人体外 BBB 模型（脑样内皮细胞，BLEC），以分析患者血清对 BBB 特性的影响。我们收集了健康捐献者、患有原发性癌症的乳腺癌患者以及患有骨、内脏或脑转移的乳腺癌患者的血清样本。我们利用免疫测定分析了这些血清中的细胞因子水平，并将其与临床数据相关联。我们使用细胞旁通透性测量、免疫荧光染色、蛋白质印迹和 mRNA 分析来检查患者血清对体外 BBB 特性的影响。结果 BLEC 与脑周细胞一起在 Transwell 中培养，形成紧密的单层，claudin-5 在紧密连接 (TJ) 处正确定位。一些 BBB 标记蛋白，如 TJ 蛋白claudin-5 和 occludin、葡萄糖转运蛋白 GLUT-1 或外排泵 PG-P 和 BCRP 在这些培养物中上调。这伴随着荧光素（400 Da）的细胞旁通透性降低。然后我们使用该模型对患者血清进行治疗。只有乳腺癌脑转移患者的血清中细胞因子 fractalkine (CX3CL1) 和 BCA-1 (CXCL13) 水平显着升高。 fratalkine 水平的增加与肿瘤的雌激素/孕激素受体状态相关。 用这些血清处理 BLEC 可选择性增加 CXCL13 和 TJ 蛋白 occludin 的表达。此外，血清处理后荧光素的通透性增加。结论 我们证明 CD34 + 细胞衍生的人体外 BBB 模型可用作研究脑血管病理的分子机制的工具。我们发现，脑转移患者的血清可能会影响体外 BBB 的完整性，与特定细胞因子（如 CX3CL1 和 CXCL13）浓度升高相关。