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Abstract：
Background: Chemoresistance is a key reason for treatment failure in colorectal cancer (CRC) patients. The tumor microenvironment of chemoresistant CRC is distinctly immunosuppressive, although the underlying mechanisms are unclear. Methods: The CRC data sets GSE69657 and GSE62080 were downloaded from the GEO database, and the correlation between TRPC5 and FAP expression was analyzed by Pearson method. The in-situ expression of transient receptor potential channel 5 (TRPC5) and fibroblast activation protein (FAP) in the CRC tissues was examined by immunohistochemistry. TRPC5 expression levels in the HCT8 and HCT116 cell lines and the corresponding 5-fluorouracil (5-FU)-resistant cell lines (HCT8R and HCT116R) were analyzed by western blotting and RT-PCR. Exosomes were isolated from the HCT8R and HCT116R cells and incubated with colorectal normal fibroblasts (NFs), and cancer-associated fibroblasts (CAFs)markers were detected. NFs were also incubated with exosomes isolated from TRPC5-knockdown HCT8R cells, and the changes in intracellular Ca2+ levels and C-X-C motif chemokine ligand 12 (CXCL12) secretion were analyzed. Results: TRPC5 and FAP expression showed positive correlation in the datasets. Immunostaining of CRC tissue specimens further revealed that high TRPC5 and FAP expressions were significantly associated with worse tumor regression. Furthermore, chemoresistant CRC cells expressed higher levels of TRPC5 compared to the chemosensitive cells, and knocking down TRPC5 reversed chemoresistance. Exosomes derived from CRC cells induced the transformation of NFs to CAFs. However, TRPC5-exosomes derived from chemoresistant CRC cells can promote CAFs to secrete more CXCL12. Conclusion: Chemoresistant CRC cells can induce CAFs activation and promote CXCL12 secretion through exosomal TRPC5.
摘要：
背景：化疗耐药是结直肠癌（CRC）患者治疗失败的关键原因。化疗耐药CRC的肿瘤微环境具有明显的免疫抑制作用,尽管潜在的机制尚不清楚.方法:从GEO数据库下载CRC数据集GSE69657和GSE62080,用Pearson方法分析TRPC5与FAP表达的相关性。通过免疫组织化学检查了CRC组织中瞬时受体电位通道5（TRPC5）和成纤维细胞激活蛋白（FAP）的原位表达。通过蛋白质印迹和RT-PCR分析了HCT8和HCT116细胞系以及相应的5-氟尿嘧啶（5-FU）抗性细胞系（HCT8R和HCT116R）中的TRPC5表达水平。从HCT8R和HCT116R细胞中分离外泌体，并与结直肠正常成纤维细胞（NFs）孵育，并检测癌相关成纤维细胞（CAFs）标志物。NFs还与从TRPC5敲低HCT8R细胞分离的外泌体一起孵育，并分析了细胞内Ca2水平和C-X-C基序趋化因子配体12（CXCL12）分泌的变化。结果：TRPC5和FAP在数据集中的表达呈正相关。CRC组织标本的免疫染色进一步显示，高TRPC5和FAP表达与较差的肿瘤消退显着相关。此外，与化学敏感细胞相比，化学抗性CRC细胞表达更高水平的TRPC5，和敲低TRPC5逆转化学抗性。源自CRC细胞的外来体诱导NF向CAF的转化。然而,来自化学抗性CRC细胞的TRPC5-外泌体可以促进CAF分泌更多的CXCL12。结论:化疗耐药的CRC细胞可通过外泌体TRPC5诱导CAFs活化并促进CXCL12分泌。
