Bladder cancer cells re-educate TAMs through lactate shuttling in the microfluidic cancer microenvironment
Bladder cancer cells re-educate TAMs through lactate shuttling in the microfluidic cancer microenvironment
复制标题
膀胱癌细胞通过微流体癌症微环境中的乳酸穿梭来重新教育 TAM
DOI：
10.18632/oncotarget.5538
复制
发表时间：
2015-11-17
期刊：
影响因子：
--
通讯作者：
Niu, Haitao
中科院分区：
文献类型：
--
作者：
Zhao, Yang;Wang, Degui;Niu, Haitao
在本研究中，我们旨在研究乳酸穿梭对膀胱移行细胞癌（TCCB）细胞和巨噬细胞功能极化和空间分布的影响。方法设计微流控共培养芯片进行实时综合检测。通过总NO测定试剂盒检测NO水平，通过细胞免疫荧光和western blotting检测iNOS、p-NFkB-p65、Arg-1和HIF-1α蛋白表达，探讨乳酸穿梭对TCCB细胞巨噬细胞再教育的影响。此外，我们使用吖啶橙/溴化乙啶（AO/EB）和MitoTracker染色检测TCCB细胞活力。此外，使用4 '，6-二氨基-2-苯基吲哚（DAPI）和异硫氰酸酯-葡聚糖荧光素（fitc -葡聚糖）测量培养室中乳酸和大信号蛋白的浓度分布。光镜下观察巨噬细胞募集情况及巨噬细胞对BC转移的影响。结果我们证实TCCB细胞将巨噬细胞重编程为M2表型。乳酸抑制了巨噬细胞的M1极化，诱导了M2极化，但阻断癌细胞后，巨噬细胞乳酸通量明显抑制了TCCB细胞对巨噬细胞的再教育。此外，在微流控肿瘤微环境中，乳酸比大信号蛋白扩散得更快、更深。此外，乳酸单独诱导巨噬细胞的迁移，巨噬细胞M1而非M2降低了TCCB细胞的运动性。结论TCCB细胞以依赖于癌细胞- tam乳酸通量的方式将巨噬细胞重编程为M2表型。此外，乳酸穿梭可能是肿瘤组织中tam密度的决定因素。
Background In the present study, we aimed to investigate the influence of lactate shuttling on the functional polarization and spatial distribution of transitional cell carcinoma of the bladder (TCCB) cells and macrophages. Methods We designed a microfluidic coculture chip for real-time integrative assays. The effect of lactate shuttling on the re-education of macrophages by TCCB cells was explored by measuring the levels of NO using a total NO assay kit and by evaluating the protein expression of iNOS, p-NFkB-p65, Arg-1 and HIF-1α via cell immunofluorescence and western blotting. Additionally, we examined TCCB cell viability using acridine orange/ethidium bromide (AO/EB) and MitoTracker staining. Moreover, the concentration distributions of lactate and large signaling proteins in the culture chambers were measured using 4′,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate-dextran (FITC-dextran). Furthermore, the recruitment of macrophages and the influence of macrophages on BC metastasis were observed via light microscopy. Results We confirmed that TCCB cells reprogrammed macrophages into an M2 phenotype. Moreover, lactate inhibited M1 polarization and induced M2 polarization of macrophages, but blockade of cancer cell-macrophage lactate flux significantly inhibited the re-education of macrophages by TCCB cells. In addition, lactate diffused faster and deeper than large signaling proteins in the microfluidic tumor microenvironment. Furthermore, lactate alone induced the migration of macrophages, and M1, but not M2, macrophages reduced the motility of TCCB cells. Conclusions TCCB cells reprogrammed macrophages into an M2 phenotype in a manner that depended on cancer cell-TAM lactate flux. Furthermore, the lactate shuttle may be a determinant of the density of TAMs in tumor tissue.