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.

Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and significantly contribute to the progression of various cancers, including esophageal squamous cell carcinoma (ESCC). Our previous study established a direct co-culture system of human bone marrow-derived mesenchymal stem cells (progenitors of CAFs) and ESCC cell lines, which facilitates the generation of CAF-like cells and enhances malignancy in ESCC cells. In this study, we further elucidated the mechanism by which CAFs promote ESCC progression using cDNA microarray analysis of monocultured ESCC cells and those co-cultured with CAFs. We observed an increase in the expression and secretion of amphiregulin (AREG) and the expression and phosphorylation of its receptor EGFR in co-cultured ESCC cells. Moreover, AREG treatment of ESCC cells enhanced their survival and migration via the EGFR-Erk/p38 MAPK signaling pathway. Immunohistochemical analysis of human ESCC tissues showed a positive correlation between the intensity of AREG expression at the tumor-invasive front and the expression level of the CAF marker FAP. Bioinformatics analysis confirmed significant upregulation of AREG in ESCC compared with normal tissues. These findings suggest that AREG plays a crucial role in CAF-mediated ESCC progression and could be a novel therapeutic target for ESCC.

Tumor-associated macrophages (TAMs) contribute to disease progression in various cancers, including esophageal squamous cell carcinoma (ESCC). We have previously used an indirect co-culture system between ESCC cell lines and macrophages to analyze their interactions. Recently, we established a direct co-culture system to closely simulate actual ESCC cell-TAM contact. We found that matrix metalloproteinase 9 (MMP9) was induced in ESCC cells by direct co-culture with TAMs, not by indirect co-culture. MMP9 was associated with ESCC cell migration and invasion, and its expression was controlled by the Stat3 signaling pathway in vitro. Immunohistochemical analyses revealed that MMP9 expression in cancer cells at the invasive front (“cancer cell MMP9”) was related to high infiltration of CD204 positive M2-like TAMs (p < 0.001) and was associated with worse overall and disease-free survival of patients (p = 0.036 and p = 0.038, respectively). Furthermore, cancer cell MMP9 was an independent prognostic factor for disease-free survival. Notably, MMP9 expression in cancer stroma was not associated with any clinicopathological factors or patient prognoses. Our results suggest that close interaction with TAMs infiltrating in cancer stroma or cancer nests induces MMP9 expression in ESCC cells, equipping them with more malignant features.

High infiltration of tumor-associated macrophages (TAMs), which contribute to the progression of several cancer types, is correlated with poor prognosis of esophageal squamous cell carcinoma (ESCC). In addition to the previously reported increase in migration and invasion, ESCC cells co-cultured directly with macrophages exhibited enhanced survival and growth. Furthermore, interleukin-related molecules are associated with ESCC; however, the precise mechanism underlying this association is unclear. Therefore, we explored the role of interleukin-related molecules in ESCC progression. A cDNA microarray analysis of monocultured and co-cultured ESCC cells revealed that the interleukin 7 receptor (IL-7R) was upregulated in ESCC cells co-cultured with macrophages. Overexpression of IL-7R promoted the survival and growth of ESCC cells by activating the Akt and Erk1/2 signaling pathways. The IL-7/IL-7R axis also contributed to the promotion of ESCC cell migration via the Akt and Erk1/2 signaling pathways. Furthermore, immunohistochemistry showed that ESCC patients with high IL-7R expression in cancer nests exhibited a trend toward poor prognosis in terms of disease-free survival, and showed significant correlation with increased numbers of infiltrating macrophages and cancer-associated fibroblasts. Therefore, IL-7R, which is upregulated when directly co-cultured with macrophages, may contribute to ESCC progression by promoting the development of various malignant phenotypes in cancer cells.

Although a therapeutic response to neoadjuvant chemoradiotherapy (NACRT) is important to improve oncological outcomes after surgery in patients with locally advanced rectal cancer, there is no reliable predictor for this. The Wnt/β-catenin signal is known to be crucial for the tumorigenesis of colorectal cancer. This study aimed to investigate the association of Wnt/β-catenin signal activation with a pathological response to NACRT. The immunohistochemical expression of nuclear and membranous β-catenin was analyzed in biopsy samples obtained from 60 patients with locally advanced rectal cancer who received curative surgery following NACRT. The association of Wnt/β-catenin signal activation with their clinical outcomes was investigated. Notably, the body mass index of these patients was significantly higher in the low nuclear β-catenin expression group. Moreover, patients in the high nuclear β-catenin expression group tended to have more advanced disease and a higher rate of positive vascular invasion than those in the low expression group. Furthermore, the rate of good histological responses was significantly higher in the low nuclear β-catenin expression group (72% vs. 37.1%, p < 0.01). Overall, relapse-free survival tended to be better in patients with low nuclear/high membranous β-catenin expression (n = 9) than in other individuals (n = 51) (p = 0.093 and p = 0.214, respectively). Activation of the Wnt/β-catenin signal pathway represented by nuclear β-catenin accumulation was significantly associated with a poor response to NACRT in patients with rectal cancer. Analysis of nuclear β-catenin accumulation before starting treatment might help predict the therapeutic response to NACRT.

Tumor-associated macrophages (TAMs), one of the major components of the tumor microenvironment, contribute to the progression of esophageal squamous cell carcinoma (ESCC). We previously established a direct co-culture system of human ESCC cells and macrophages and reported the promotion of malignant phenotypes, such as survival, growth, and migration, in ESCC cells. These findings suggested that direct interactions between cancer cells and macrophages contribute to the malignancy of ESCC, but its underlying mechanisms remain unclear. In this study, we compared the expression levels of the interferon-induced genes between mono- and co-cultured ESCC cells using a cDNA microarray and found that interferon-inducible protein 16 (IFI16) was most significantly upregulated in co-cultured ESCC cells. IFI16 knockdown suppressed malignant phenotypes and also decreased the secretion of interleukin-1α (IL-1α) from ESCC cells. Additionally, recombinant IL-1α enhanced malignant phenotypes of ESCC cells through the Erk and NF-κB signaling. Immunohistochemistry revealed that high IFI16 expression in human ESCC tissues tended to be associated with disease-free survival and was significantly associated with tumor depth, lymph node metastasis, and macrophage infiltration. The results of this study reveal that IFI16 is involved in ESCC progression via IL-1α and imply the potential of IFI16 as a novel prognostic factor for ESCC.