Esophageal squamous cell carcinoma (ESCC) is a highly aggressive tumor with frequent recurrence even after curative resection. The tumor microenvironment, which consists of non-cancer cells, such as cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs), was recently reported to promote several cancers, including ESCC. However, the role of CAF as a coordinator for tumor progression in ESCC remains to be elucidated. In our immunohistochemical investigation of ESCC tissues, we observed that the intensity of expression of two CAF markers-alpha smooth muscle actin (αSMA) and fibroblast activation protein (FAP)-in the tumor stroma was significantly correlated with the depth of tumor invasion, lymph node metastasis, advanced pathological stage, and poor prognosis. We co-cultured human bone marrowderived mesenchymal stem cells (MSCs) with ESCC cells and confirmed the induction of FAP expression in the co-cultured MSCs. These FAP-positive MSCs (which we defined as CAF-like cells) promoted the cell growth and migration of ESCC cells and peripheral blood mononuclear cell-derived macrophage-like cells. CAF-like cells induced the M2 polarization of macrophage-like cells. A cytokine array and ELISA revealed that CAF-like cells secreted significantly more CCL2, Interleukin-6, and CXCL8 than MSCs. These cytokines promoted the migration of tumor cells and macrophage-like cells. The silencing of FAP in CAF-like cells attenuated cytokine secretion. We compared cell signaling of MSCs, CAF-like cells, and FAP-silenced CAF-like cells; PTEN/Akt and MEK/Erk signaling were upregulated and their downstream targets, NF-κB and β-catenin, were also activated with FAP expression. Silencing of FAP attenuated these effects. Cytokine secretion from CAF-like cells were attenuated by inhibitors against these signaling pathways. These findings indicate that the collaboration of CAFs with tumor cells and macrophages plays a pivotal role in tumor progression, and that FAP expression is responsible for the tumor promotive and immunosuppressive phenotypes of CAFs.

Tumor-associated macrophages (TAMs) are involved in tumor progression and poor prognosis in several malignancies. We previously demonstrated the interaction between high numbers of infiltrating TAMs and poor prognosis in esophageal squamous cell carcinomas (ESCCs). To investigate the significance of TAMs in ESCC, we conducted a cDNA microarray analysis of peripheral blood monocytes (PBMo)-derived macrophages and PBMo-derived macrophages stimulated with conditioned media of TE-series ESCC cell lines (TAM-like PBMo-derived macrophages). C-X-C motif chemokine ligand 8 (CXCL8) was up-regulated in the TAM-like PBMo-derived macrophages. Here we confirmed a high expression level of CXCL8 in TAM-like PBMo-derived macrophages and the expression of CXCR1/2, known as CXCL8 receptors, in TE-series ESCC cell lines. Recombinant human CXCL8 induced the ESCC cell lines’ migration and invasion by the phosphorylation of Akt and Erk1/2. In indirect co-cultures, not only signal pathway inhibitors but also neutralizing antibodies against CXCL8, CXCR1 and CXCR2 suppressed these phenotypes induced by TAM-like PBMo-derived macrophages. Immunohistochemical analysis of 70 resected ESCC samples showed that high expression levels of CXCL8 in ESCC tissues were significantly associated with lymph node metastasis and poor prognosis. These results suggest that CXCL8 up-regulated in the microenvironment may contribute to ESCC progression by promoting cancer cells’ migration and invasion.

Tumor‐associated macrophages (TAMs) have important roles in the growth, angiogenesis and progression of various tumors. Although we have demonstrated the association of an increased number of infiltrating CD204+ TAMs with poor prognosis in esophageal squamous cell carcinomas (ESCCs), the roles of TAMs in ESCC remain unclear. Here, to study the effects of TAMs on the tumor microenvironment of ESCCs, we established a co‐culture assay using a human ESCC cell line and TAM‐like peripheral blood monocyte‐derived macrophages and performed a cDNA microarray analysis between monocultured and co‐cultured ESCC cell lines. Our qRT‐PCR confirmed that in the co‐cultured ESCC cell lines, CYP1A1, DHRS3, ANXA10, KLK6 and CYP1B1 mRNA were highly up‐regulated; AMTN and IGFL1 mRNA were down‐regulated. We observed that the high expression of a calcium‐dependent phospholipid‐binding protein ANXA10 was closely associated with the depth of invasion and high numbers of infiltrating CD68+ and CD204+ TAMs and poor disease‐free survival (P = 0.0216). We also found ANXA10 promoted the cell growth of ESCC cell lines via the phosphorylation of Akt and Erk1/2 pathways in vitro. These results suggest that ANXA10 induced by the interaction with TAMs in the tumor microenvironment is associated with cell growth and poor prognosis in human ESCC tissues.

Tumor-associated macrophages (TAMs) have important roles in the angiogenesis, tumor immunosuppression and tumor infiltration of various cancers. Previously, we reported that a large number of infiltrated tumor promoting CD204+ TAMs within esophageal squamous cell carcinomas (ESCCs) contributed to the significant association of clinicopathological malignancy and poor disease free survival. However, the relevant malignant progression mechanisms have not been studied explicitly yet. To elucidate roles of TAMs in ESCCs, we established an in vitro macrophage model using human peripheral blood acquired by healthy volunteer donors. Peripheral blood monocytes were treated with 25 ng/mL recombinant human M-CSF for 6 days to induce macrophage-like differentiation (macrophage-like cells), then exposed to the conditioned media of TE series ESCC cell lines (TECM) for 2 days to induce TAM-like cells. From the cDNA microarray data between macrophages-like cells treated with and without TECM, we interested in upregulated genes. Among highly expressed genes in TAM-like cells, we focused on neural cell adhesion molecule (NCAM) which was a possibility to act on the regulation between tumor and its surrounding stroma. Gene knockdown of NCAM by siRNA in TAM-like cells revealed significant suppression of cell survival and migration via decrease of PI3K/Akt signaling and downregulation of fibroblast growth factor receptor 1 (FGFR1) expression. Interestingly, fibroblast growth factor-2 (FGF-2) derived from ESCCs promotes FGF2/FGFR1 autocrine loop, and activates FGFR1 signaling in TAM-like cells. Furthermore, we analyzed 70 ESCC tissue samples by immunohistochemistry whether the expression levels of FGF-2 were associated with clinicopathological background factors of ESCC patients. Relationships of the expression levels of FGF-2 in human ESCC stromal cells including macrophages had a correlation with the depth of invasion, blood vessel invasion, TNM classification UICC cancer staging and high number of infiltrating CD163 positive and CD204 positive macrophages as a marker for the M2 macrophage phenotype. These findings may indicate novel roles of FGF-2-NCAM/FGFR1 interplay in ESCCs and its microenvironment including TAMs. Citation Format: NOBUHISA TAKASE, Yuichiro Koma, Noriaki Arai, Himiko Kodaira, Masayoshi Hosono, Yumi Ichihara, Mari Nishio, Manabu Shigeoka, Hiroshi Yokozaki. The roles of FGF-2-NCAM/FGFR1 interplay in esophageal squamous cell carcinomas and its microenvironment including tumor-associated macrophages. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 730.