Colorectal cancer (CRC) responses to KRAS-targeted inhibition have been limited due to low response rates, the mechanisms of which remain unknown. Herein, we explored the cancer-associated fibroblasts (CAFs) secretome as a mediator of resistance to KRAS silencing. CRC cell lines HCT15, HCT116, and SW480 were cultured either in recommended media or in conditioned media from a normal colon fibroblast cell line (CCD-18Co) activated with rhTGF-β1 to induce a CAF-like phenotype. The expression of membrane stem cell markers was analyzed by flow cytometry. Stem cell potential was evaluated by a sphere formation assay. RNAseq was performed in KRAS-silenced HCT116 colonospheres treated with either control media or conditioned media from CAFs. Our results demonstrated that KRAS-silencing up-regulated CD24 and down-regulated CD49f and CD104 in the three cell lines, leading to a reduction in sphere-forming efficiency. However, CAF-secreted factors restored stem cell marker expression and increased stemness. RNA sequencing showed that CAF-secreted factors up-regulated genes associated with pro-tumorigenic pathways in KRAS-silenced cells, including KRAS, TGFβ, NOTCH, WNT, MYC, cell cycle progression and exit from quiescence, epithelial-mesenchymal transition, and immune regulation. Overall, our results suggest that resistance to KRAS-targeted inhibition might derive not only from cell-intrinsic causes but also from external elements, such as fibroblast-secreted factors.
结直肠癌对KRAS靶向抑制的反应有限，这主要归因于低应答率，其机制尚不明确。本研究探讨了癌症相关成纤维细胞分泌组作为KRAS沉默耐药性介导因子的作用。将结直肠癌细胞系HCT15、HCT116和SW480分别培养于标准培养基或经rhTGF-β1激活的正常结肠成纤维细胞系（CCD-18Co）的条件培养基中，以诱导CAF样表型。通过流式细胞术分析膜干细胞标志物表达，采用球体形成实验评估干细胞潜能，并对经对照培养基或CAF条件培养基处理的KRAS沉默HCT116结肠球体进行RNA测序。结果显示，KRAS沉默上调了三种细胞系中CD24表达，下调了CD49f和CD104表达，导致球体形成效率降低。然而，CAF分泌因子可恢复干细胞标志物表达并增强干性特征。RNA测序表明，CAF分泌因子在KRAS沉默细胞中上调了促肿瘤通路相关基因，包括KRAS、TGFβ、NOTCH、WNT、MYC、细胞周期进程与静息态退出、上皮-间质转化及免疫调节相关基因。总体而言，本研究提示针对KRAS靶向抑制的耐药性可能不仅源于细胞内在因素，也可能来自外部因素，如成纤维细胞分泌因子。
Fibroblasts Promote Resistance to KRAS Silencing in Colorectal Cancer Cells
肿瘤(癌症)患者之家