Tumor-associated macrophages (TAMs) contribute to the progression and mortality of various malignancies. We reported that high numbers of infiltrating TAMs were significantly associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma (ESCC). In our previous investigation of TAMs’ actions in ESCC, we compared gene expression profiles between peripheral blood monocyte (PBMo)-derived macrophages and TAM-like macrophages stimulated with conditioned media of ESCC cell lines. Among the upregulated genes in the TAM-like macrophages, we focused on CC chemokine ligand 3 (CCL3), which was reported to contribute to tumor progression in several malignancies. Herein, we observed that not only TAMs but also ESCC cell lines expressed CCL3. A CCL3 receptor, CC chemokine receptor 5 (CCR5) was expressed in the ESCC cell lines. Treating the ESCC cell lines with recombinant human (rh)CCL3 induced the phosphorylations of Akt and ERK, which were suppressed by CCR5 knockdown. Migration and invasion of ESCC cells were promoted by treatment with rhCCL3 and co-culture with TAMs. TAMs/rhCCL3-promoted cell migration and invasion were suppressed by inhibition of the CCL3–CCR5 axis, PI3K/Akt, and MEK/ERK pathways. Treatment with rhCCL3 upregulated MMP2 and VEGFA expressions in ESCC cell lines. Our immunohistochemical analysis of 68 resected ESCC cases showed that high expression of CCL3 and/or CCR5 in ESCC tissues was associated with poor prognosis. High CCR5 expression was associated with deeper invasion, presence of vascular invasion, higher pathological stage, higher numbers of infiltrating CD204⁺ TAMs, and higher microvascular density. High expression of both CCL3 and CCR5 was an independent prognostic factor for disease-free survival. These results suggest that CCL3 derived from both TAMs and cancer cells contributes to the progression and poor prognosis of ESCC by promoting cell migration and invasion via the binding of CCR5 and the phosphorylations of Akt and ERK. The CCL3–CCR5 axis could become the target of new therapies against ESCC.

Cancer-associated fibroblasts (CAFs) contribute to the progression of various cancers. Previously, we reported the significance of CAFs in esophageal squamous cell carcinoma (ESCC); however, the functions of CAFs in the ESCC microenvironment remain unknown. To investigate CAFs’ function, we established an indirect coculture assay between human bone marrow-derived mesenchymal stem cells (MSCs) and ESCC cells. Cocultured MSCs expressed more fibroblast activation protein, one of the markers of CAFs, compared with monocultured MSCs. Therefore, we defined cocultured MSCs as CAF-like cells. To identify molecules associated with the ESCC progression in CAFs, we conducted a cDNA microarray analysis on monocultured MSCs and CAF-like cells to compare their gene expression profiles. We found that SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), was more abundant in CAF-like cells than in monocultured MSCs, and the PAI-1 derived from CAF-like cells induced the abilities of migration and invasion in both ESCC cells and macrophages by the Akt and Erk1/2 signaling pathways via the low-density lipoprotein receptor-related protein 1 (LRP1), which is a PAI-1 receptor. Based on immunohistochemistry assays of ESCC tissues, higher expression levels of PAI-1 and LRP1 were correlated with poor prognosis in ESCC patients. These results suggest that the PAI-1/LRP1 axis contributes to the progression of ESCC, making it a potential target for ESCC therapy.

Tumor-associated macrophages (TAMs) have important roles in the angiogenesis and tumor immunosuppression of various cancers, including esophageal squamous cell carcinomas (ESCCs). To elucidate the roles of TAMs in ESCCs, we compared the gene expression profiles between human peripheral blood monocyte-derived macrophage-like cells (Macrophage_Ls) and Macrophage_Ls stimulated with conditioned medium of the TE series human ESCC cell line (TECM) (TAM_Ls) using cDNA microarray analysis. Among the highly expressed genes in TAM_Ls, we focused on neural cell adhesion molecule (NCAM). NCAM knockdown in TAM_Ls revealed a significant decrease of migration and survival via a suppression of PI3K-Akt and fibroblast growth factor receptor 1 (FGFR1) signaling. Stimulation by TECM up-regulated the level of FGFR1 in Macrophage_Ls. Recombinant human fibroblast growth factor-2 (rhFGF-2) promoted the migration and survival of TAM_Ls and TE-cells through FGFR1 signaling. Our immunohistochemical analysis of 70 surgically resected ESCC samples revealed that the up-regulated FGF-2 in stromal cells, including macrophages, was associated with more aggressive phenotypes and a high number of infiltrating M2 macrophages. These findings may indicate a novel role of NCAM- and FGF-2-mediated FGFR1 signaling in the tumor microenvironment of ESCCs.

Esophageal cancer has the sixth highest mortality rate worldwide. Cancer-associated fibroblasts (CAFs) are involved in the progression of various cancers. Previously, we demonstrated an association between high expression of the CAF marker, fibroblast activation protein, and poor prognosis of esophageal squamous cell carcinoma (ESCC). We also established CAF-like cells by indirect co-culture of bone marrow-derived mesenchymal stem cells with ESCC cell lines and found metallothionein 2A (MT2A) to be highly expressed in them. Here, to explore the function of MT2A in CAFs, we silenced MT2A in the CAF-like cells and ESCC cell lines using small interfering RNA. MT2A knockdown in the CAF-like cells suppressed expression and secretion of insulin-like growth factor binding protein 2 (IGFBP2); recombinant IGFBP2 promoted migration and invasiveness of ESCC cells via NFκB, Akt, and Erk signaling pathways. Furthermore, MT2A knockdown in the ESCC cell lines inhibited their growth, migration, and invasiveness. Immunohistochemistry demonstrated that high MT2A expression in the cancer stroma and cancer nest of ESCC tissues correlated with poor prognosis of ESCC patients. Hence, we report that MT2A in CAFs and cancer cells contributes to ESCC progression. MT2A and IGFBP2 are potential novel therapeutic targets in ESCC.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment are involved in the progression of various cancers, including esophageal squamous cell carcinoma (ESCC). CAF-like cells were generated through direct co-culture of human bone marrow-derived mesenchymal stem cells (MSCs), one of CAF origins, with ESCC cells, and periostin was found to be highly expressed in CAF-like cells. After direct co-culture, ESCC cells showed increased malignant phenotypes such as survival, growth, and migration, as well as increased phosphorylation of Akt and Erk. Recombinant human periostin activated Akt and Erk signaling pathways in ESCC cells, enhancing survival and migration. The suppression of periostin in CAF-like cells by small interfering RNA during direct co-culture also suppressed enhanced survival and migration in ESCC cells. In ESCC cells, knockdown of periostin receptor integrin β4 inhibited Akt and Erk phosphorylation, as well as survival and migration increased by periostin. Periostin also enhanced MSC and macrophage migration and endowed macrophages with tumor-associated macrophage (TAM)-like properties. Immunohistochemistry showed that high periostin expression in the cancer stroma was significantly associated with tumor invasion depth, lymphatic and blood vessel invasion, higher pathological stage, CAF marker expression, and infiltrating TAM numbers. Moreover, patients with ESCC with high periostin expression exhibited poor postoperative outcomes. Thus, CAF-secreted periostin contributed to tumor microenvironment development. These results indicate that periostin may be a novel therapeutic target for ESCC.