Periostin Upregulated in Cancer-Associated Fibroblasts upon Direct Contact with Cancer Cells Promotes Esophageal Squamous Cell Carcinoma Progression by Enhancing the Migration of Cancer Cells and Stromal Cells

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment are involved in the progression of esophageal squamous cell carcinoma (ESCC). We generated CAF-like cells by direct co-culture of human bone marrow-derived mesenchymal stem cells (MSCs), one of the origins of CAFs, with ESCC cell lines and found that periostin is highly expressed in CAF-like cells. Periostin activated Akt and Erk signaling pathways in ESCC cells, enhancing survival and migration via integrin β4, one of the receptors for periostin. Periostin also enhanced migration of MSCs and macrophages and caused macrophages to acquire tumor-associated macrophage (TAM)-like properties. High periostin expression in cancer stroma was associated with several clinicopathological factors and expressions of CAF markers and numbers of infiltrating TAMs. Moreover, ESCC patients with high periostin expression exhibited significantly poor postoperative outcomes. Thus, periostin secreted from CAFs enhanced the migration of ESCC cells, MSCs, and macrophages and contributed to developing the tumor microenvironment. These results indicate that periostin may be a novel therapeutic target for ESCC. To induce the CAF-like cells, MSCs and TE cells were seeded in the same dish. Direct co-culture was performed for 4 days in DMEM high-glucose (Wako) with 10% FBS and 1% antibiotic-antimycotic. MSCs and TE cells were seeded and cultured similarly without co-culture as controls. Cell suspensions were mixed with CD326 (EpCAM) microbeads (#130-061-101; Miltenyi Biotec) and incubated at 4°C for 30 minutes. Prepared cell suspensions were separated from EpCAM-positive and -negative cells using the autoMACS Pro Separator. Mono-cultured TE cells and mono-cultured MSCs were collected using autoMACS Pro Separator as well as cells after direct co-culture. We performed cDNA microarray analysis between mono-cultured MSCs and CAF9 (MSC co-cultured with TE-9 cells) cells.