食管癌相关纤维细胞对食管癌放疗敏感性的影响及其作用机制研究

Radiotherapy has been often used as the primary curative modality for esophageal squamous cell carcinoma (ESCC), one of the most common malignancies with high morbidity and mortality in China. However, due to acquired radioresistance, many patients with ESSC suffered from local recurrence after radiotherapy. There is thus an urgent need to clarify the molecular mechanism leading to the development of radioresistance in ESCC. We and other researchers have previously focused on what influences radiation treatment induced on esophageal cancer cells, but the effect tumour microenvironment exerted on radiation response in ESCC has never been studied. In fact, the tumour microenvironment has been demonstrated to play significant roles in modulating cancer cell response to ionizing radiation. CAFs (cancer-associated fibroblasts), the major actor of the impact of tumour microenvironment on tumour radiation response, were involved in the development of radioresistance via multiple mechanisms. In our lab, CAFs were successfully isolated from surgical specimens from ESCC patients and cultured for further studies. We found esophageal cancer cell KYSE-150 displayed much less double-srand breads (DSBs) and cell apoptosis, but much higher level of cell survival after radiation exposure when cultured in conditioned medium from CAFs compared with that cultured in normal medium. There results suggested that CAFs may confer KYSE-150 cells radioresistance in a direct manner and deserved in-depth study. In this study, multiple esophageal cancer cells were employed to further investigate the effect of CAFs on radiation response in ESCC and meanwhile, the precise molecular mechanism by which CAFs mediated the development of radioresistance would be uncovered. This work holds promise in discovering innovative molecular target for improving the radiotherapy effect for ESCC.

食管癌是我国常见的高发病率、高死亡率的恶性肿瘤，且以食管鳞癌为主。放疗为食管癌的主要治疗手段，然而由于获得性放疗抵抗性的存在，多数病例治疗后易出现局部复发。以往关于食管癌放疗抵抗性的研究均着眼于癌细胞本身，而未关注肿瘤微环境. 研究证实肿瘤微环境对肿瘤的治疗敏感性亦起着至关重要的作用。尤其是肿瘤相关纤维细胞为肿瘤微环境发挥影响作用的主要执行者，逐渐受到重视。我们前期通过采用原代培养的方法成功地从食管鳞癌患者的手术标本中分离、培养了食管癌相关纤维细胞。研究发现食管癌相关纤维细胞可增加食管癌细胞KYSE-150放疗后的细胞存活率，降低其DNA损伤程度及细胞凋亡率，提示食管癌相关纤维细胞可能直接介导了KYSE-150细胞放疗抵抗性的形成。本研究中，我们拟通过采用多个食管癌细胞株系统地研究食管癌相关纤维细胞对食管癌放疗抵抗性的影响以及具体的作用机制，从而筛选出能够逆转食管癌放疗抵抗性的分子靶点。

肿瘤微环境对肿瘤的发生发展具有至关重要的影响。肿瘤相关纤维细胞作为肿瘤微环境发挥其对癌细胞影响作用的主要执行者，可通过激活自分泌或旁分泌信号通路调控肿瘤细胞的DNA损伤修复响应、促结缔组织增生反应等，从而诱导肿瘤组织产生放疗抵抗性。放疗作为食管鳞癌患者的主要治疗手段，患者的五年生存率则不足20%。究其主要原因为肿瘤组织产生了放疗抵抗性。本项目率先在国内外开展了食管癌相关纤维细胞对食管癌放疗抵抗性的影响及其作用机制研究。通过采用原代培养的方法从食管癌患者手术切除的癌组织标本中分离培养了食管癌相关纤维细胞（cancer-associated fibroblasts，CAFs）及配对的正常纤维细胞（normal fibroblasts, NFs）。细胞因子/趋化因子检测显示，较配对的NFs相比，CAFs可显著地高表达趋化因子1（The chemokine (C-X-C motif) ligand 1, CXCL1）。采用克隆形成实验，DNA损伤修复检测及在裸鼠体内建立移植瘤模型系统地在体内外水平研究了CAFs分泌的CXCL1对食管癌细胞放疗抵抗性的影响。结果显示CAFs分泌的CXCL1可在体内外水平介导食管癌细胞产生显著的放疗抵抗性。抑制CAFs中CXCL1的表达可显著地逆转食管癌细胞的放疗抵抗性。进一步的机制研究显示CAFs分泌的CXCL1通过以SOD1/ROS依赖的方式调控食管癌细胞的DNA损伤修复响应及激活MEK/Erk信号通路，从而介导食管癌细胞产生显著的放疗抵抗性。C此外，研究显示食管癌相关纤维细胞中CXCL1的表达量与食管癌患者放疗后总生存显著相关。上述研究成果率先从肿瘤微环境的角度阐明了食管癌产生放疗抵抗性的分子机制，对于临床上如何设计有效的逆转食管癌放疗抵抗性的策略提供了重要的基础。