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Immunohistochemistry is primarily employed to visualize the localization of specific molecules in tissue samples. However, there is an increasing need for software‐assisted quantitative assessment. In the present study, we performed inverted blue channel‐based pseudoimmunofluorescence image analysis using original immunohistochemistry images. In human esophageal squamous cell carcinoma tissues, various humoral factors promote the phosphorylation of signaling proteins, including protein kinase B (Akt) and/or extracellular signal‐regulated kinase 1/2 (ERK1/2), leading to tumor progression. Our method demonstrated applicability in the analysis of localized signaling proteins in histological sections. Relatively high phosphorylated Akt (p‐Akt) intensity was observed in the cancer‐stroma adjacent (Adj) and noncancerous regions of the superficial layer (SL). Furthermore, localized phosphorylated ERK1/2 (Thr202/Tyr204) was observed in the Adj of the SL and invasive front, distinct from the pattern of p‐Akt (Ser473) and p‐Akt (Thr308). In conclusion, pseudoimmunofluorescent immunohistochemistry image analysis is useful for the quantitative assessment and objective interpretation of localized signaling proteins in esophageal squamous cell carcinoma. The method can also be applied to analyze various immunohistochemistry images from diverse tissues. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

M2 macrophages contribute to the progression of oesophageal squamous cell carcinoma (ESCC); however, the roles of M2 macrophages in early ESCC remain unclear. To clarify the biological mechanisms underlying the interaction between M2 macrophages and oesophageal epithelial cells in early‐stage ESCC, in vitro co‐culture assays between the immortalised oesophageal epithelial cell line Het‐1A and cytokine‐defined M2 macrophages were established. Co‐culture with M2 macrophages promoted the proliferation and migration of Het‐1A cells via the mTOR–p70S6K signalling pathway activated by YKL‐40, also known as chitinase 3‐like 1, and osteopontin (OPN) that were hypersecreted in the co‐culture supernatants. YKL‐40 and OPN promoted the above phenotypes of Het‐1A by making a complex with integrin β4 (β4). Furthermore, YKL‐40 and OPN promoted M2 polarisation, proliferation, and migration of macrophages. To validate the pathological and clinical significances of in vitro experimental results, immunohistochemistry of human early ESCC tissues obtained by endoscopic submucosal dissection (ESD) was performed, confirming the activation of the YKL‐40/OPN–β4–p70S6K axis in the tumour area. Moreover, epithelial expression of β4 and the number of epithelial and stromal infiltrating YKL‐40‐ and OPN‐positive cells correlated with the Lugol‐voiding lesions (LVLs), a well‐known predictor of the incidence of metachronous ESCC. Furthermore, the combination of high expression of β4 and LVLs or high numbers of epithelial and stromal infiltrating YKL‐40‐ and OPN‐positive immune cells could more clearly detect the incidence of metachronous ESCC than each of the parameters alone. Our results demonstrated that the YKL‐40/OPN–β4–p70S6K axis played important roles in early‐stage ESCC, and the high expression levels of β4 and high numbers of infiltrating YKL‐40‐ and OPN‐positive immune cells could be useful predictive parameters for the incidence of metachronous ESCC after ESD. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Simple Summary: The crucial role of tumor-associated macrophages (TAMs) in the disease progression of various types of tumors, including esophageal squamous cell carcinoma (ESCC), has been well established. To investigate the close interplay between cancer cells and TAMs, we previously established a direct co-culture system. Our aim was to identify useful molecular markers whose expression is regulated by interactions with cancer cells and TAMs, to predict patient prognoses, or to gain a deeper understanding of the ESCC mechanism. Our method revealed that direct contact with macrophages stimulated matrix metalloproteinase 9 (MMP9) production in ESCC cells, and that MMP9 played an integral role in cancer cell migration and invasion in vitro. Moreover, MMP9 expression in ESCC cells was significantly correlated with high infiltration of TAMs and poor clinical outcomes in immunohistochemical analysis. Our study suggests the potential for TAMs or MMP9 as important prognostic markers or therapeutic ESCC targets. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Tumor-associated macrophages (TAMs) play significant roles in the progression of numerous types of cancers. We previously reported that the density of TAM infiltration in esophageal squamous cell carcinoma (ESCC) tissues is correlated with poor prognosis in patients with ESCC. To elucidate the significance of the direct interaction in the ESCC microenvironment, we previously established a direct co-culture assay of ESCC cells and macrophages. In the present study, direct co-culture of ESCC cells with macrophages induced interleukin 7 receptor (IL-7R) expression in ESCC. IL-7R overexpression promoted ESCC cell survival and growth via the activation of the Akt and Erk1/2 signaling pathways. Additionally, the IL-7/IL-7R axis promoted ESCC cell migration via the Akt and Erk1/2 signaling pathways. ESCC patients with high IL-7R expression in cancer nests exhibited poor disease-free survival. IL-7R could be a novel therapeutic target for ESCC. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human macrophages play important roles in tumor promotion and are called tumor-associated macrophages ( TAMs). We previously demonstrated that human esophageal squamous cell carcinomas ( ESCCs) contain TAMs and that these TAMs tend to have tumor-supporting features. Here we exposed human macrophages to conditioned media of TE-series human ESCC cell lines ( TECMs) to generate an ESCC extracellular stimuli-influenced TAM model. CD14+ peripheral blood monocytes ( PBMos) from healthy donors were treated with M-CSF and with additional IL-4 or TECM exposure. Morphological changes of the cells and the induction of CD163/ CD204 proteins were detected in the TECM-exposed model TAMs by immunofluorescence. A software-assisted immunofluorescent cell image analysis showed increased CD163/ CD204 positivity in the model TAMs and a weak to moderate positive correlation between the cytoplasmic area and the sum fluorescent intensity of CD204. Morphological changes of the cells were significantly reflected by several cytomorphometric parameters. PBMos were elongated with M-CSF treatment, then enlarged with TECM exposure. The cytoplasmic aspect ratio was decreased by M-CSF treatment and slightly increased by TECM exposure. The nuclear-cytoplasmic ratio decreased during the whole process of cell differentiation. This system is useful for quantitative assessments of TAM-like morphological changes of macrophages and the induction of CD163/ CD204 in a model ESCC microenvironment. [ABSTRACT FROM AUTHOR]

Tumor-associated macrophages ( TAMs) are known to be involved in the progression of various human malignancies. We previously demonstrated that CD204 was a useful marker for TAMs contributing to the angiogenesis, progression, and prognosis of human esophageal squamous cell carcinoma ( ESCC). We also showed that conditioned media of ESCC cell lines induced CD204 expression in THP-1 human monocytic leukemia cells. Here, we performed a cDNA microarray analysis between THP-1 cells stimulated with TPA (macrophage [MΦ]-like THP-1 cells) treated with and without conditioned medium of ESCC cell line to clarify the molecular characteristics of TAMs in ESCC. From the microarray data, we discovered that Cyr61 was induced in CD204-positive-differentiated THP-1 cells ( TAM-like THP-1 cells). In the ESCC microenvironment, not only cancer cells but also TAMs expressed Cyr61. Interestingly, the expression levels of Cyr61 showed a significant positive correlation with the number of CD204-positive macrophages in ESCCs by immunohistochemistry. Recombinant human Cyr61 (rhCyr61) promoted cell migration and induced the expression of CD204 along with the activation of the MEK/ ERK pathway in MΦ-like THP-1 cells. Pretreatment with a MEK1/2 inhibitor significantly inhibited not only the Cyr61-mediated migration but also the CD204 expression in the MΦ-like THP-1 cells. These results suggest that Cyr61 may contribute to the expression of CD204 and the promotion of cell migration via the MEK/ ERK pathway in TAMs in the ESCC microenvironment. [ABSTRACT FROM AUTHOR]

Summary: Claudins are transmembrane proteins that seal tight junctions, bind with peripheral protein zonula occludens (ZO)-1, and are known to play an important role in several normal tissues and cancers. However, the role of claudin-1 and claudin-7 expressions in esophageal squamous cell carcinoma remains to be clarified. In the present study, we confirmed the expressions of claudin-1, claudin-7, and ZO-1 in the prickle cell layer of the normal human esophageal squamous epithelium. The expressions of claudin-1 and claudin-7 at the invasive front of the esophageal squamous cell carcinoma were analyzed immunohistochemically to clarify their role in tumor progression. Reduced expression of claudin-7 at the invasive front of the esophageal cancer was significantly associated with the depth of invasion (P = .004), stage (P = .038), lymphatic vessel invasion (P = .001), and lymph node metastasis (P = .014). In contrast, significant association was not detected between claudin-1 expression and clinicopathologic factors except for histologic differentiation of the tumor (P = .0029). Comparison of claudin-7 expression at the invasive front of the primary tumor and its corresponding metastatic lymph nodes revealed significant reduction in claudin-7 expression in the metastatic lymph nodes (P = .007). These results suggest that the reduced expression of claudin-7 at the invasive front of esophageal squamous cell carcinoma may lead to tumor progression and subsequent metastatic events. Thus, claudin-7 can be a novel marker for the prediction of lymph node metastasis. [Copyright &y& Elsevier]

In the present study, we examined the expression of CD44 variant exons in human oral squamous cell carcinoma (OSCC). Of ten cell lines from OSCCs, two (KB and H357), as well as the HeLa cell line, failed to express CD44 variant exons. In surgical specimens, all normal mucosa expressed CD44v9 (both mRNA and protein). Of 40 primary OSCCs, 19 (47.5%) showed downregulation of CD44v9, which correlated with tumor cell differentiation, primary metastasis to lymph nodes and secondary metastasis to lymph nodes. The results suggest that the downregulation of CD44v9 may play a role in lymphatic metastasis of OSCC and changes in its expression may be a useful diagnostic tool to determine the metastatic potential of OSCC to lymph nodes. Moreover, three cell lines that failed to express CD44 variant exons might become a useful experimental model to study the role of variant exons in the progression of OSCC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]