Amphiregulin Upregulated in Esophageal Squamous Cell Carcinoma Cells upon Direct Contact with Cancer-Associated Fibroblasts

Cancer-associated fibroblasts (CAFs) are a pivotal component of the tumor microenvironment, significantly contributing to the progression of esophageal squamous cell carcinoma (ESCC). Direct co-culture of human bone marrow-derived mesenchymal stem cells (MSCs), one of the origins of CAFs, with ESCC cell line, led to increased amphiregulin (AREG) expression and secretion in the ESCC cells. Consequently, the expression and phosphorylation of the AREG receptor EGFR were upregulated in co-cultured ESCC cells. Moreover, AREG treatment enhanced ESCC cell survival and migration through the EGFR-Erk/p38 signaling pathway. Immunohistochemical analysis of AREG using human ESCC tissues showed a positive correlation between the intensity of AREG expression in tumor invasive front and the expression level of the CAF marker FAP. Furthermore, bioinformatics database analysis confirmed a significant upregulation of AREG expression in ESCC tissues compared to normal tissues. These findings suggest that AREG is involved in CAF-mediated ESCC progression and could be a novel therapeutic target for ESCC. MSCs and ESCC cells were seeded in the same dish and co-cultured for 4 days. Monocultures of both cell types served as controls. Following co-culture or monoculture, cells were washed with phosphate-buffered saline (FUJIFILM Wako Pure Chemical Corporation) and detached themselves from the dish using trypsin (FUJIFILM Wako Pure Chemical Corporation) treatment. The harvested cells were then mixed with anti-CD326 (epithelial cell adhesion molecule; EpCAM) microbeads (130-061-101; Miltenyi Biotec) and subjected to magnetic separation using the autoMACS Pro Separator (Miltenyi Biotec) to isolate high-purity tumor cells. cDNA microarray analysis was performed on monocultured and co-cultured TE-9 cells.

癌症相关成纤维细胞（Cancer-associated fibroblasts, CAFs）是肿瘤微环境的关键组成部分，对食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）的进展具有显著促进作用。将作为CAFs来源之一的人骨髓来源间充质干细胞（mesenchymal stem cells, MSCs）与ESCC细胞系直接共培养后，ESCC细胞内双调蛋白（amphiregulin, AREG）的表达与分泌水平均显著升高。相应地，共培养后的ESCC细胞中，AREG受体表皮生长因子受体（epidermal growth factor receptor, EGFR）的表达与磷酸化水平均被上调。此外，AREG处理可通过EGFR-Erk/p38信号通路增强ESCC细胞的存活能力与迁移能力。针对人类ESCC组织的AREG免疫组化分析显示，肿瘤侵袭前沿的AREG表达强度与CAF标志物成纤维细胞活化蛋白（fibroblast activation protein, FAP）的表达水平呈正相关。进一步的生物信息学数据库分析证实，相较于正常组织，ESCC组织中AREG的表达水平显著上调。上述研究结果表明，AREG参与了CAF介导的ESCC进展过程，有望成为ESCC的新型治疗靶点。本研究中，将MSCs与ESCC细胞共同接种于同一培养皿，共培养4天；以两种细胞的单独培养作为对照。共培养或单独培养结束后，使用磷酸盐缓冲液（phosphate-buffered saline, PBS，购自富士胶片和光纯药株式会社，FUJIFILM Wako Pure Chemical Corporation）洗涤细胞，随后使用胰蛋白酶（购自富士胶片和光纯药株式会社，FUJIFILM Wako Pure Chemical Corporation）消化以从培养皿中分离细胞。收集得到的细胞与抗CD326（上皮细胞黏附分子，epithelial cell adhesion molecule, EpCAM）磁珠（货号130-061-101，Miltenyi Biotec）混合后，采用autoMACS Pro全自动磁珠分选仪（Miltenyi Biotec）进行磁分离以获取高纯度肿瘤细胞。最后对单独培养与共培养的TE-9细胞开展cDNA微阵列分析。