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Abstract??Phosphatase of regenerating liver (PRL)-3, encoding a 22-kD low molecular weight tyrosine phosphatase, has been reported to be associated with metastasis of colorectal carcinoma. We assessed the levels ofPRL-3mRNA expression to know whether its up-regulation was involved in progression and metastasis of gastric carcinoma. Levels ofPRL-3expression in 94 human gastric adenocarcinomas and 54 matched lymph node metastases were detected by in situ hybridization and compared with clinicopathological characteristics including prognosis. HighPRL-3expression was detected in 36.2% of primary gastric carcinoma (with nodal metastasis, 55.6%; without nodal metastasis, 10%;P<0.001) and in 74.1% of lymph node metastases. The incidence of highPRL-3expression in lymph node metastasis was significantly higher than in primary tumors (P<0.044). Moreover, high expression ofPRL-3was closely associated with tumor size, lymphatic invasion, venous invasion, extent of lymph node metastasis, and tumor stage. These results suggest that highPRL-3expression may participate in the progression and metastasis of gastric carcinoma.PRL-3might be a novel molecular marker for aggressive gastric cancer. [ABSTRACT FROM AUTHOR]

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.

(Background) Cancer-associated fibroblasts (CAFs) are major cancer stromal components. CAFs have diverse functions and cell origins. Podoplanin (PDPN), a lymphatic vessel marker, is also a CAF marker in certain cancers. On daily diagnosis of early colorectal carcinoma (CRC), PDPN upregulation in the stroma is often encountered, suggesting PDPN-positive CAFs have emerged. However, PDPN-positive CAFs in early CRC have not been studied well. (Methods) On immunohistochemistry, PDPN expression in the lamina propria or stroma of adenomas, early CRCs, and neuroendocrine tumors, their normal neighbors, and non-neoplastic colorectal lesions were compared. Single-cell RNA sequencing (scRNA-seq) of CRC was used to explore PDPN high CAFs' cell origins. (Results) Reticular cells or pericryptal fibroblasts in the lamina propria of adenomas and early CRCs showed higher PDPN expression than did normal mucosae and non-neoplastic lesions ( p < 0.01). Pericryptal PDPN expression was a diagnostic feature of adenomas and early CRCs. scRNA-seq of CRCs highlighted that PDPN high CAFs had distinctly higher COL4A1 , COL4A2 , and WNT5A expression, unlike well-known CAFs characterized by high FAP, POSTN , or ACTA2 expression. (Conclusions) We demonstrated that pericryptal fibroblasts and reticular cells in the lamina propria are origins of early-stage CRC CAFs and thus have potential as a diagnostic marker for distinguishing colorectal non-neoplastic from neoplastic lesions.

Macrophages in the tumor microenvironment, termed tumor-associated macrophages (TAMs), promote the progression of various cancer types. However, many mechanisms related to tumor-stromal interactions in epithelial ovarian cancer (EOC) progression remain unclear. High-grade serous ovarian carcinoma (HGSOC) is the most malignant EOC subtype. Herein, immunohistochemistry was performed on 65 HGSOC tissue samples, revealing that patients with a higher infiltration of CD68 + , CD163 + , and CD204 + macrophages had a poorer prognosis. We subsequently established an indirect co-culture system between macrophages and EOC cells, including HGSOC cells. The co-cultured macrophages showed increased expression of the TAM markers CD163 and CD204, and the co-cultured EOC cells exhibited enhanced proliferation, migration, and invasion. Cytokine array analysis revealed higher YKL40 secretion in the indirect co-culture system. The addition of YKL40 increased proliferation, migration, and invasion via extracellular signal-regulated kinase (Erk) signaling in EOC cells. The knockdown of integrin β4, one of the YKL40 receptors, suppressed YKL40-induced proliferation, migration, and invasion, as well as Erk phosphorylation in some EOC cells. Database analysis showed that high-level expression of YKL40 and integrin β4 correlated with a poor prognosis in patients with serous ovarian carcinoma. Therefore, the YKL40/integrin β4 axis may play a role in ovarian cancer progression.

Causal networks are important for understanding disease signaling alterations. To reveal the network pathways affected in the epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs), which are related to the poor prognosis of cancer, the molecular networks and gene expression in diffuse- and intestinal-type gastric cancer (GC) were analyzed. The network pathways in GC were analyzed using Ingenuity Pathway Analysis (IPA). The analysis of the probe sets in which the gene expression had significant differences between diffuse- and intestinal-type GC in RNA sequencing of the publicly available data identified 1099 causal networks in diffuse- and intestinal-type GC. Master regulators of the causal networks included lenvatinib, pyrotinib, histone deacetylase 1 (HDAC1), mir-196, and erb-b2 receptor tyrosine kinase 2 (ERBB2). The analysis of the HDAC1-interacting network identified the involvement of EMT regulation via the growth factors pathway, the coronavirus pathogenesis pathway, and vorinostat. The network had RNA-RNA interactions with microRNAs such as mir-10, mir-15, mir-17, mir-19, mir-21, mir-223, mir-25, mir-27, mir-29, and mir-34. The molecular networks revealed in the study may lead to identifying drug targets for GC.

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, one of CAF origins, with ESCC cells. Periostin (POSTN) 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 extracellular signal-regulated kinase (Erk). Recombinant human POSTN activated Akt and Erk signaling pathways in ESCC cells, enhancing survival and migration. The suppression of POSTN in CAF-like cells by siRNA during direct co-culture also suppressed enhanced survival and migration in ESCC cells. In ESCC cells, knockdown of POSTN receptor integrin β4 inhibited Akt and Erk phosphorylation, and survival and migration increased by POSTN. POSTN also enhanced mesenchymal stem cell and macrophage migration and endowed macrophages with tumor-associated macrophage-like properties. Immunohistochemistry showed that high POSTN expression in the cancer stroma was significantly associated with tumor invasion depth, lymphatic and blood vessel invasion, higher pathologic stage, CAF marker expression, and infiltrating tumor-associated macrophage numbers. Moreover, patients with ESCC with high POSTN expression exhibited poor postoperative outcomes. Thus, CAF-secreted POSTN contributed to tumor microenvironment development. These results indicate that POSTN may be a novel therapeutic target for ESCC., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (© 2024 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment and are considered potential targets for cancer immunotherapy. To examine the antitumor effects of agents targeting human TAMs in vivo , we here established preclinical tumor xenograft models based on immunodeficient mice that express multiple human cytokines and have been reconstituted with a human immune system by transplantation of human CD34 + hematopoietic stem and progenitor cells (HIS-MITRG mice). HIS-MITRG mice supported the growth of both human cell line (Raji)- and patient-derived B cell lymphoma as well as the infiltration of human macrophages into their tumors. We examined the potential antitumor action of an antibody to human SIRPα (SE12C3) that inhibits the interaction of CD47 on tumor cells with SIRPα on human macrophages and thereby promotes Fcγ receptor-mediated phagocytosis of the former cells by the latter. Treatment with the combination of rituximab (antibody to human CD20) and SE12C3 inhibited Raji tumor growth in HIS-MITRG mice to a markedly greater extent than did rituximab monotherapy. This enhanced antitumor effect was dependent on human macrophages and attributable to enhanced rituximab-dependent phagocytosis of lymphoma cells by human macrophages. Treatment with rituximab and SE12C3 also induced reprogramming of human TAMs toward a proinflammatory phenotype. Furthermore, the combination treatment essentially prevented the growth of patient-derived diffuse large B cell lymphoma in HIS-MITRG mice. Our findings thus support the study of HIS-MITRG mice as a model for the preclinical evaluation in vivo of potential therapeutics, such as antibodies to human SIRPα, that target human TAMs., Competing Interests: TM and YM hold a patent on the antitumor drug “anti-SIRPα Ab” Japan patent number P6923942. TM has received royalties from Daiichi Sankyo. He also has received a research grant from JCR Pharmaceuticals Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Saito, Iida-Norita, Afroj, Refaat, Hazama, Komori, Ohata, Takai, Oduori, Kotani, Funakoshi, Koma, Murata, Yakushijin, Matsuoka, Minami, Yokozaki, Manz and Matozaki.)

The precise mechanism of resistance to anti-cancer drugs such as platinum drugs is not fully revealed. To reveal the mechanism of drug resistance, the molecular networks of anti-cancer drugs such as cisplatin, carboplatin, oxaliplatin, and arsenic trioxide were analyzed in several types of cancers. Since diffuse-type stomach adenocarcinoma, which has epithelial-mesenchymal transition (EMT)-like characteristics, is more malignant than intestinal-type stomach adenocarcinoma, the gene expression and molecular networks in diffuse- and intestinal-type stomach adenocarcinomas were analyzed. Analysis of carboplatin revealed the causal network in diffuse large B-cell lymphoma. The upstream regulators of the molecular networks of cisplatin-treated lung adenocarcinoma included the anti-cancer drug trichostatin A (TSA), a histone deacetylase inhibitor. The upstream regulator analysis of cisplatin revealed an increase in FAS, BTG2, SESN1, and CDKN1A, and the involvement of the tumor microenvironment pathway. The molecular networks were predicted to interact with several microRNAs, which may contribute to the identification of new drug targets for drug-resistant cancer. Analysis of oxaliplatin, a platinum drug, revealed that the SPINK1 pancreatic cancer pathway is inactivated in ischemic cardiomyopathy. The study showed the importance of the molecular networks of anti-cancer drugs and tumor microenvironment in the treatment of cancer resistant to anti-cancer drugs.

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.

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., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

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.

Tumor-associated macrophages are associated with more malignant phenotypes of esophageal squamous cell carcinoma (ESCC) cells. Previously, an indirect co-culture assay of ESCC cells and macrophages was used to identify several factors associated with ESCC progression. Herein, a direct co-culture assay of ESCC cells and macrophages was established, which more closely simulated the actual cancer microenvironment. Direct co-cultured ESCC cells had significantly increased migration and invasion abilities, and phosphorylation levels of Akt and p38 mitogen-activated protein kinase (MAPK) compared with monocultured ESCC cells. According to a cDNA microarray analysis between monocultured and co-cultured ESCC cells, both the expression and release of S100 calcium binding protein A8 and A9 (S100A8 and S100A9), which commonly exist and function as a heterodimer (herein, S100A8/A9), were significantly enhanced in co-cultured ESCC cells. The addition of recombinant human S100A8/A9 protein induced migration and invasion of ESCC cells via Akt and p38 MAPK signaling. Both S100A8 and S100A9 silencing suppressed migration, invasion, and phosphorylation of Akt and p38 MAPK in co-cultured ESCC cells. Moreover, ESCC patients with high S100A8/A9 expression exhibited significantly shorter disease-free survival (P = 0.005) and cause-specific survival (P = 0.038). These results suggest that S100A8/A9 expression and release in ESCC cells are enhanced by direct co-culture with macrophages and that S100A8/A9 promotes ESCC progression via Akt and p38 MAPK signaling pathways., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background/aims: The anastomotic site after distal gastrectomy is the area most affected by duodenogastric reflux. Different reconstruction methods may affect the lesion characteristics and treatment outcomes of remnant gastric cancers at the anastomotic site. We retrospectively investigated the clinicopathologic and endoscopic submucosal dissection outcomes of remnant gastric cancers at the anastomotic site., Methods: We recruited 34 consecutive patients who underwent endoscopic submucosal dissection for remnant gastric cancer at the anastomotic site after distal gastrectomy. Clinicopathology and treatment outcomes were compared between the Billroth II and non-Billroth II groups., Results: The tumor size in the Billroth II group was significantly larger than that in the non-Billroth II group (22 vs. 19 mm; p=0.048). More severe gastritis was detected endoscopically in the Billroth II group (2 vs. 1.33; p=0.0075). Moreover, operation time was longer (238 vs. 121 min; p=0.004) and the frequency of bleeding episodes was higher (7.5 vs. 3.1; p=0.014) in the Billroth II group., Conclusion: Compared to remnant gastric cancers in non-Billroth II patients, those in the Billroth II group had larger lesions with a background of severe remnant gastritis. Endoscopic submucosal dissection for remnant gastric cancers in Billroth II patients involved longer operative times and more frequent bleeding episodes than that in patients without Billroth II.

Background: Pancreatic ductal adenocarcinoma (PDA) is associated with very poor prognoses. Therefore, new therapies and preclinical models are urgently needed. In the present study, we sought to develop more realistic experimental models for use in PDA research., Methods: We developed patient-derived xenografts (PDXs), established PDX-derived cell lines (PDCLs), and generated cell line-derived xenografts (CDXs), which we integrated to create 13 matched "trios" - i.e., patient-derived tumor models of PDA. We then compared and contrasted histological and molecular alterations between these three model systems., Results: Orthotopic implantation (OI) of the PDCLs resulted in tumorigenesis and metastases to the liver and peritoneum. Morphological comparisons of OI-CDXs and OI-PDXs with passaged tumors revealed that the histopathological features of the original tumor were maintained in both models. Molecular alterations in PDX tumors (including those to KRAS, TP53, SMAD4, and CDKN2A) were similar to those in the respective PDCLs and CDX tumors. When gene expression levels in the PDCLs, ectopic tumors, and OI tumors were compared, the distant metastasis-promoting gene CXCR4 was specifically upregulated in OI tumors, whose immunohistochemical profiles suggested epithelial-mesenchymal transition and adeno-squamous trans-differentiation., Conclusion: These patient-derived tumor models provide useful tools for monitoring responses to antineoplastic agents and for studying PDA biology., (© 2022 S. Karger AG, Basel.)

Dynamic regulation in molecular networks including cell cycle regulation and DNA damage response play an important role in cancer. To reveal the feature of cancer malignancy, gene expression and network regulation were profiled in diffuse- and intestinal-type gastric cancer (GC). The results of the network analysis with Ingenuity Pathway Analysis (IPA) showed that the activation states of several canonical pathways related to cell cycle regulation were altered. The G 1 /S checkpoint regulation pathway was activated in diffuse-type GC compared to intestinal-type GC, while canonical pathways of the cell cycle control of chromosomal replication, and the cyclin and cell cycle regulation, were activated in intestinal-type GC compared to diffuse-type GC. A canonical pathway on the role of BRCA1 in the DNA damage response was activated in intestinal-type GC compared to diffuse-type GC, where gene expression of BRCA1, which is related to G 1 /S phase transition, was upregulated in intestinal-type GC compared to diffuse-type GC. Several microRNAs (miRNAs), such as mir-10, mir-17, mir-19, mir-194, mir-224, mir-25, mir-34, mir-451 and mir-605, were identified to have direct relationships in the G 1 /S cell cycle checkpoint regulation pathway. Additionally, cell cycle regulation may be altered in epithelial-mesenchymal transition (EMT) conditions. The alterations in the activation states of the pathways related to cell cycle regulation in diffuse- and intestinal-type GC highlighted the significance of cell cycle regulation in EMT.

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.

Tumor-associated macrophages (TAMs) are the most abundant cancer stromal cells and are directed by the tumor microenvironment to acquire trophic functions facilitating angiogenesis, matrix breakdown and cancer cell motility. TAMs have anti-inflammatory or alternatively activated (M2) phenotypes expressing CD204 and/or CD163. We previously reported that infiltration of a large number of CD204-positive TAMs are associated with angiogenesis, progression and poor disease-free survival of human esophageal squamous cell carcinomas (ESCCs). In this study, we investigated the initraepithelial distribution of TAMs in the early human esophageal carcinogenesis. We found that the numbers of CD68-, CD163- or CD204-positive macrophages within the unit length of 38 lesions of carcinoma in situ (CIS) excised by endoscopic mucosal dissection were significantly higher than those in the corresponding non-neoplastic squamous epithelia. Mapping of the infiltrating number of CD204-positive macrophages per 5 mm unit length within the whole epithelial area of 5 resected cancer laden esophagi demonstrated that the areas with high CD204-positive macrophage infiltration were significantly associated with CIS or squamous intraepithelial neoplasia. These results may suggest that macrophages with the M2-skewed phenotype have some biological roles in the early squamous carcinogenesis of the esophagus.

Background: CD163-positive macrophages contribute to the aggressiveness of oral squamous cell carcinoma. We showed in a previous report that CD163-positive macrophages infiltrated not only to the cancer nest but also to its surrounding epithelium, depending on the presence of stromal invasion in tongue carcinogenesis. However, the role of intraepithelial macrophages in tongue carcinogenesis remains unclear. In this study, we assessed the biological behavior of intraepithelial macrophages on their interaction with cancer cells., Materials and Methods: We established the indirect coculture system (intraepithelial neoplasia model) and direct coculture system (invasive cancer model) of human monocytic leukemia cell line THP-1-derived CD163-positive macrophages with SCC25, a tongue squamous cell carcinoma (TSCC) cell line. Conditioned media (CM) harvested from these systems were analyzed using cytokine array and enzyme-linked immunosorbent assay and extracted a specific upregulated cytokine in CM from the direct coculture system (direct CM). The correlation of both this cytokine and its receptor with various clinicopathological factors were evaluated based on immunohistochemistry using clinical samples from 59 patients with TSCC. Moreover, the effect of this cytokine in direct CM on the phenotypic alterations of THP-1 was confirmed by real-time polymerase chain reaction, western blotting, immunofluorescence, and transwell migration assay., Results: It was shown that CCL20 was induced in the direct CM specifically. Interestingly, CCL20 was produced primarily in SCC25. The expression level of CCR6, which is a sole receptor of CCL20, was higher than the expression level of SCC25. Our immunohistochemical investigation showed that CCL20 and CCR6 expression was associated with lymphatic vessel invasion and the number of CD163-positive macrophages. Recombinant human CCL20 induced the CD163 expression and promoted migration of THP-1. We also confirmed that a neutralizing anti-CCL20 antibody blocked the induction of CD163 expression by direct CM in THP-1. Moreover, ERK1/2 phosphorylation was associated with the CCL20-driven induction of CD163 expression in THP-1., Conclusions: Tongue cancer cell-derived CCL20 that was induced by interaction with macrophages promotes CD163 expression on macrophages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shigeoka, Koma, Kodama, Nishio, Akashi and Yokozaki.)

Tumor-associated macrophages (TAMs) promote tumor progression. The number of infiltrating TAMs is associated with poor prognosis in esophageal squamous cell carcinoma (ESCC) patients; however, the mechanism underlying this phenomenon is unclear. cDNA microarray analysis indicates that the expression of chemokine (C-C motif) ligand 1 (CCL1) is up-regulated in peripheral blood monocyte-derived macrophages stimulated using conditioned media from ESCC cells (TAM-like macrophages). Here, we evaluated the role of CCL1 in ESCC progression. CCL1 was overexpressed in TAM-like macrophages, and CCR8, a CCL1 receptor, was expressed on ESCC cell surface. TAM-like macrophages significantly enhanced the motility of ESCC cells, and neutralizing antibodies against CCL1 or CCR8 suppressed this increased motility. Recombinant human CCL1 promoted ESCC cell motility via the Akt/proline-rich Akt substrate of 40 kDa/mammalian target of rapamycin pathway. Phosphatidylinositol 3-kinase or Akt inhibitors, CCR8 silencing, and neutralizing antibody against CCR8 could significantly suppress these effects. The overexpression of CCL1 in stromal cells or CCR8 in ESCC cells was significantly associated with poor overall survival (P = 0.002 or P = 0.009, respectively) and disease-free survival (P = 0.009 or P = 0.047, respectively) in patients with ESCC. These results indicate that the interaction between stromal CCL1 and CCR8 on cancer cells promotes ESCC progression via the Akt/proline-rich Akt substrate of 40 kDa/mammalian target of rapamycin pathway, thereby providing novel therapeutic targets., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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.

Background: The mortality of oral squamous cell carcinoma (OSCC) has remained high for decades; therefore, methods for early detection of OSCC are warranted. However, in the oral cavity, various mucosal diseases may be encountered, including reactive lesions and oral potentially malignant disorders, and it is difficult to differentiate OSCC from these lesions based on both clinical and histopathological findings. It is well known that chronic inflammation contributes to oral cancer development. Macrophages are among the most common inflammatory cells in cancer stromal tissue and have various roles in cancer aggressiveness. Although the roles of macrophages in cancer development have attracted attention, only a few studies have linked macrophages to carcinogenesis, particularly, oral precancerous lesions., Summary: This review article consists of 3 parts: first, we summarize current knowledge on macrophages in human various epithelial precancerous lesions, excluding the oral cavity, to show the importance and gaps in knowledge regarding macrophages in carcinogenesis; second, we review published data related to the role of macrophages in oral carcinogenesis; finally, we present a novel view on oral carcinogenesis, focusing on crosstalk between epithelial cells and macrophages. Key Messages: The biological features of macrophages in oral carcinogenesis differ drastically depending on the anatomical compartment that they infiltrate. Focusing on the alteration of macrophage infiltrating compartment may serve as a useful novel approach for studying the role of the macrophages in oral carcinogenesis and for gaining further insight into cancer prevention and early detection., (© 2021 S. Karger AG, Basel.)

Epithelial-mesenchymal transition (EMT) plays an important role in the acquisition of cancer stem cell (CSC) feature and drug resistance, which are the main hallmarks of cancer malignancy. Although previous findings have shown that several signaling pathways are activated in cancer progression, the precise mechanism of signaling pathways in EMT and CSCs are not fully understood. In this study, we focused on the intestinal and diffuse-type gastric cancer (GC) and analyzed the gene expression of public RNAseq data to understand the molecular pathway regulation in different subtypes of gastric cancer. Network pathway analysis was performed by Ingenuity Pathway Analysis (IPA). A total of 2815 probe set IDs were significantly different between intestinal- and diffuse-type GC data in cBioPortal Cancer Genomics. Our analysis uncovered 10 genes including male-specific lethal 3 homolog (Drosophila) pseudogene 1 ( MSL3P1 ), CDC28 protein kinase regulatory subunit 1B ( CKS1B ), DEAD-box helicase 27 ( DDX27 ), golgi to ER traffic protein 4 ( GET4 ), chromosome segregation 1 like ( CSE1L ), translocase of outer mitochondrial membrane 34 ( TOMM34 ), YTH N6-methyladenosine RNA binding protein 1 ( YTHDF1 ), ribonucleic acid export 1 ( RAE1 ), par-6 family cell polarity regulator beta ( PARD6B ), and MRG domain binding protein ( MRGBP ), which have differences in gene expression between intestinal- and diffuse-type GC. A total of 463 direct relationships with three molecules (MYC, NTRK1, UBE2M) were found in the biomarker-filtered network generated by network pathway analysis. The networks and features in intestinal- and diffuse-type GC have been investigated and profiled in bioinformatics. Our results revealed the signaling pathway networks in intestinal- and diffuse-type GC, bringing new light for the elucidation of drug resistance mechanisms in CSCs.

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.

Gastric adenocarcinoma with enteroblastic differentiation (GAED) is a very rare variant of alpha-fetoprotein-producing gastric cancer (AFPGC). GAED is histologically characterized by cuboidal or columnar cells, which resemble those found in the primitive gut and have clear cytoplasm. In previously reported cases, GAED exhibit more aggressive behavior, as well as AFPGC, than conventional gastric cancer, such as marked lymphovascular invasion, lymph node metastasis, and liver metastasis. And also GAED was usually located in a deep mucosal layer and was covered by a conventional adenocarcinoma (CA) component. Based on these findings, GAED is considered to develop from CA during the process of tumor invasion and proliferation. We present a very rare case of early-stage GAED achieved curatively resected via endoscopic submucosal dissection, in which the lesion was composed of a pure enteroblastic differentiation component without a CA component.

Objective: Oral leukoplakia has mixed and differing histopathological features, and it is thus difficult to reach an accurate histological diagnosis of oral leukoplakia based on a local biopsy alone. We recently demonstrated the significance of CD163 + macrophages in oral carcinogenesis. Herein we sought to determine whether CD163 + macrophages in biopsy specimens of oral leukoplakia help identify the overall histological nature of the lesion., Patients and Methods: Twenty-six patients with tongue leukoplakia who underwent a histological examination by both a preoperative local biopsy and consecutive total excision were enrolled. We evaluated clinicopathological factors and the expression of CD163 + macrophages based on a retrospective comparison of the histological diagnostic concordance between the biopsies and excisions., Results: Seventeen patients (diagnostic-agreement group) were diagnosed with squamous intraepithelial lesion based on both the biopsy and the excision. Nine patients (diagnostic-discrepancy group) were diagnosed with invasive cancer by excision, although invasive cancer was not observed in their biopsy specimens. Compared to the diagnostic-agreement group, the diagnostic-discrepancy group had more tongue leukoplakia with non-homogenous or high numbers of intraepithelial CD163 + macrophages., Conclusion: The evaluation of intraepithelial CD163 + macrophages in local biopsy specimens from tongue leukoplakia patients is a promising tool for cancer screening., (© 2019 The Authors. Oral Diseases published by John Wiley & Sons Ltd.)

Oral verruciform xanthoma (OVX) is an uncommon benign lesion that is characterized histologically by the accumulation of several foamy macrophages in the lamina propria papillae. The pathogenesis of OVX has not been completely elucidated, although the significance of macrophage polarization (M1, tumor suppression; and M2, tumor promotion) and the contribution of M2 macrophages to angiogenesis are well established. This study investigated the role of foamy macrophages in OVX, with a focus on angiogenesis. Four patients who underwent surgical excision or total excisional biopsy for OVXs were enrolled in this study. We evaluated the expression of the macrophage markers CD68 (broad) and CD163 (M2) and the CD34-positive microvessel density (MVD) of OVXs. The foamy macrophages of all patients exhibited positivity to CD68 and CD163. We evaluated the MVD and the expression of the vascular endothelial growth factor (VEGF) based on histological architecture. The MVD of all OVX cases was significantly higher than that of the corresponding normal epithelia. Interestingly, the MVD of verrucous-type OVX cases was higher than that of the other type. VEGF was expressed on foamy macrophages in all cases. Overall, the foamy macrophages expressing CD163 were associated with the morphogenesis of OVX through the process of angiogenesis by VEGF expression., Competing Interests: The authors declare that they have no competing interest.

Objectives: Growth differentiation factor 15 (GDF15), which is derived from tumor-associated macrophages (TAM) and cancer cells, promotes progression of esophageal squamous cell carcinomas (ESCC). However, its role in the ESCC microenvironment remains unclear. Here, we examined the effects of GDF15 on ESCC cell lines and tissues., Methods: Western blotting, MTS, and Transwell migration/invasion assays were used to evaluate cell signaling, proliferation, and migration/invasion, respectively, in ESCC cell lines treated with recombinant human GDF15 (rhGDF15). ESCC cell lines were administered a TGF-βRI/II inhibitor (LY2109761), small interfering RNA against TGF-β type II receptor (TGF-βRII), or neutralizing antibody against TGF-βRII to study the role of TGF-βRII in mediating the effects of rhGDF15. The localization of GDF15 and TGF-βRII in ESCC cell lines was observed by immunofluorescence. TGF-βRII expression in ESCC tissues was analyzed by immunohistochemistry, and the relationship between clinicopathological factors and prognosis in ESCC patients was evaluated., Results: rhGDF15 increased levels of phosphorylated Akt, Erk1/2, and TGF-βRII in ESCC cell lines. Inhibition/knockdown of TGF-βRII suppressed rhGDF15-induced activation of Akt and Erk1/2 and enhancement of cellular proliferation, migration, and invasion. Immunofluorescence revealed that TGF-βRII and GDF15 were colocalized in ESCC cell lines. High TGF-βRII expression in ESCC tissues, as determined by immunohistochemistry, correlated with depth of invasion and increased number of infiltrating TAMs. ESCC patients with high TGF-βRII expression showed a tendency toward poor prognosis., Conclusions: GDF15 promotes ESCC progression by increasing cellular proliferation, migration, and invasion via TGF-βRII signaling., (© 2020 S. Karger AG, Basel.)

Interleukin-6 (IL-6) is one of the pleiotropic cytokines and has received attention as a critical factor implicated in the invasion and the angiogenesis of various cancers. In glioma, IL-6 is known to be associated with the prognosis; however, the roles of IL-6 in cerebrospinal fluid (CSF) has not been studied sufficiently. We examined the concentration of CSF IL-6 using 75 CSF samples of glioma (54 glioblastomas (GBMs) and 21 other grades of gliomas) and analyzed the association CSF IL-6 with infiltration levels of tumor-associated macrophages (TAMs) and prognosis. The concentration of CSF IL-6 in GBM patients was significantly higher than that in other grades of gliomas. CSF IL-6 levels were associated with the infiltration rate of TAMs in GBMs, and IL-6 levels were increased in the GBM cells co-cultured with TAM-like macrophages. The CSF of GBM patients, which contained high concentration of IL-6, promoted the migration ability of GBM cells, and neutralization antibodies of IL-6 inhibited its migration ability. Finally, in both univariate and multivariate analysis, higher CSF IL-6 levels were associated with poorer prognosis in GBM patients. These results indicated that the concentration of CSF IL-6 is associated with TAMs' infiltration level and may be a useful prognostic biomarker for the GBM patients., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: Accumulating evidence suggests that macrophages are involved in the immunoediting of oral squamous cell carcinoma but the role of macrophages in oral carcinogenesis is unclear. We aimed to clarify the role of macrophages in oral leukoplakia, which is the most common oral potentially malignant disorder from immunotolerance viewpoint., Materials and Methods: The study included 24 patients who underwent surgical resection for tongue leukoplakia. The relationships between macrophage markers and clinicopathological factors were assessed. Conditioned medium was harvested from the CD163 + human monocytic leukaemia cell line, THP-1. The phenotypic alteration of human oral keratinocytes by the conditioned medium treatment was assessed using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Moreover, the clinical samples were evaluated using immunohistochemistry., Results: Tongue leukoplakia tissues with high CD163 + macrophage infiltration were associated with significantly higher degrees of epithelial dysplasia, abnormal Ki-67 expression and cytokeratin13 loss when compared with the tissues with low CD163 + macrophage infiltration. In vitro, CD163 + THP-1 conditioned medium induced immunosuppressive molecules, especially interleukin-10 (IL-10) in human oral keratinocytes. The IL-10 expression levels showed significant positive correlations with not only the numbers of FOXP3 + regulatory T cells but also that of CD163 + macrophages., Conclusions: In tongue leukoplakia, CD163 + macrophages infiltration correlates with immunosuppressive cytokine IL-10 expression., Competing Interests: The authors have no conflict of interest., (©2019 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

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 marrow-derived 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.

Cancer cachexia interferes with therapy and worsens patients' quality of life. Therefore, for a better understanding of cachexia, we aimed to establish a reliable cell line to develop a cachexia model. We recently established and characterized the TCC-NECT-2 cell line, derived from a Japanese patient with poorly differentiated neuroendocrine carcinoma of the duodenum (D-NEC). Subcutaneous xenograft of TCC-NECT-2 cells in mice resulted in tumor formation, angiogenesis, and 20% incidence of body weight (BW)-loss. Subsequently, we isolated a potent cachexia-inducing subline using stepwise selection and designated as AkuNEC. Orthotopic and s.c. implantation of AkuNEC cells into mice led to diminished BW, anorexia, skeletal muscle atrophy, adipose tissue loss, and decreased locomotor activity at 100% incidence. Additionally, orthotopic implantation of AkuNEC cells resulted in metastasis and angiogenesis. Serum IL-8 overproduction was observed, and levels were positively correlated with BW-loss and reduced adipose tissue and muscle volumes in tumor-bearing mice. However, shRNA knockdown of the IL-8 gene did not suppress tumor growth and cachexia in the AkuNEC model, indicating that IL-8 is not directly involved in cachexia induction. In conclusion, AkuNEC cells may serve as a useful model to study cachexia and D-NEC., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

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., (© 2019 The Authors Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Objectives: Peritoneal dissemination (PD) is an important cause of morbidity and mortality among patients with pancreatic ductal adenocarcinoma (PDAC). We sought to develop and characterized a novel PD mouse model by using a previously established PDAC cell line TCC-Pan2., Methods: TCC-Pan2 cell line was characterized for growth rate, tumor markers, histology, and somatic mutations. TCC-Pan2 cells were implanted orthotopically to produce PD. TCC-Pan2 cells from these metastatic foci were expanded in vitro and then implanted orthotopically in mice. This PD model was used for comparing the antitumor effect of paclitaxel and NK105., Results: Orthotopically implanted TCC-Pan2 cells caused tumor formation and PD with high frequency in mice. A potent metastatic subline-Pan2M-was obtained. NK105 exerted a stronger antitumor effect than paclitaxel against Pan2M cells harboring a luciferase gene (Pan2MmLuc). Notably, the survival rate on day 80 in the Pan2MmLuc mouse model was 100% for the NK105 group and 0% for the paclitaxel group., Conclusion: TCC-Pan2 cell line and Pan2MmLuc PD model can serve as useful tools for monitoring the responses to antineoplastic agents and for studying PDAC biology.

Rheumatoid vasculitis is a rare etiology for pulmonary hypertension (PH) in patients with connective tissue disease. We encountered a case of acute PH crisis in a case with rheumatoid vasculitis eight months after undergoing adalimumab reduction. Since no repetition of arthralgia occurred after the adalimumab reduction, we decided to not increase the dose of adalimumab. However, hemodynamic collapse thereafter developed and even though steroid pulse therapy was administered, the patient nevertheless died. The autopsy showed clusters of acute and chronic inflammation around the remodeled pulmonary arteries along with micro-thrombi in the vessel lumen. We should consider the possibility of critical worsening of PH as a phenotype of vasculitis related to immunosuppressive therapy reduction.

Poorly differentiated neuroendocrine carcinoma of the duodenum (D-NEC) is a rare cancer with poor prognosis. However, a D-NEC cell line has not yet been established to study the disease. We established a cell line, TCC-NECT-2, from the ascites tumor of a 59-year-old male Japanese patient with D-NEC. TCC-NECT-2 was positive for neuroendocrine markers, chromogranin A (CGA), cluster of differentiation 56 (CD56/NCAM), synaptophysin (SYN/p38), and neuron specific enolase (NSE). Cells exhibited retinoblastoma (RB) protein loss. Orthotopic implantation of TCC-NECT-2 cells into nu/nu mice resulted in tumor formation (incidence = 83.3%) with neuroendocrine characteristics, metastasis, and weight loss. BRAF V600E and TP53 mutations and C-MYC gene amplification were also observed in TCC-NECT-2. BRAF V600E -expressing TCC-NECT-2 cells were sensitive to BRAF inhibitor vemurafenib, and especially dabrafenib, in vitro , and were strongly inhibited in a dose-dependent manner. Dabrafenib treatment (30 mg/kg) in a xenograft model for 14 days significantly suppressed tumor growth (percent tumor growth inhibition, TGI% = 48.04). An enhanced therapeutic effect (TGI% = 95.81) was observed on combined treatment of dabrafenib and irinotecan (40 mg/kg). Therefore, TCC-NECT-2, the first reported cell line derived from D-NEC, might serve as a useful model to study the basic biology of D-NEC and translational applications for treatment., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

We review the significance of cancer-stromal interactions (CSIs) in the development, morphogenesis and progression of human gastric and esophageal cancer based on the data obtained from co-culture experiments. Orthotopic fibroblasts in the gastric cancer stroma not only promoted their growth by cancer cells but were also responsible for the mobility, morphogenesis and epithelial-to-mesenchymal transition (EMT) of the cancer cells through CSI. Bone marrow-derived mesenchymal stem cells could be part of the origin of cancer-associated fibroblasts (CAFs) of the gastric cancer providing an advantageous microenvironment for the restoration of cancer stem cells with the induction of the EMT. Tumor-associated macrophages (TAMs) may differentiate from bone marrow-derived monocytes/macrophages within the tumor microenvironment of esophageal cancer and participate in the growth and the progression of esophageal squamous cell carcinomas (ESCCs). Macrophages infiltrated into the intraepithelial neoplastic lesions of the esophagus may function as a biological promoter by promoting the growth and motility of squamous epithelia. Tumor cells build up "cancer as a tissue" by taking advantage of the existing network of growth factors, cytokines and chemokines through the interactions of TAMs, CAFs and cancer cells themselves., (© 2018 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Background and Study Aims: Endoscopic submucosal dissection (ESD) is a reliable method that can replace surgery under certain conditions. However, limited information is available on the clinical course of T1b colorectal cancer (CRC) after ESD. The aim of the study was to clarify the feasibility of ESD for T1b CRC., Patients and Methods: Three hundred and two patients with 312 T1 CRC were identified in this retrospective cohort study. All patients were treated with ESD, other endoscopic treatments, or surgery. In this study, we (I) investigated the en bloc resection rate of ESD and (II) compared the overall survival (OS) rate for patients who underwent ESD with additional surgery (Group A) and surgery without upfront endoscopic resection (Group B) for T1b CRC., Results: No significant differences were observed in the en bloc resection rates between T1b and T1a CRC (100 vs. 98.7%), but the en bloc R0 resection rate was significantly lower in T1b CRC than in T1a CRC (64.7 vs. 97.4%). Regarding complications, perforations occurred in 2.9% of patients with T1b CRC, which was not significantly different from the rate of 5.3% in patients with T1a CRC. No significant differences were observed in the OS or recurrence-free survival (RFS) curves between Groups A and B (OS rates at 5 years: 92.3 vs. 88.9%, RFS rates at 5 years: 81.4 vs. 85.3%). Similarly, the 5-year disease-specific survival (DSS) rate of Group A was identical to that of Group B (both 100%)., Conclusions: ESD for T1b CRC before surgery is a possible strategy because of the low rate of complications and favorable long-term outcomes.

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., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Background/aim: Amrubicin (AMR) has shown promising activity for lung cancer. However, little is known about the mechanism underlying resistance to this agent. The aim of this study was to elucidate the mechanism underlying resistance to AMR., Materials and Methods: We first developed amrubicinol (AMR-OH)-resistant cell lines (H520/R and DMS53/R) by exposing lung cancer cell lines (H520 and DMS53) to increasing concentrations of AMR-OH and performed functional analysis by using these cell lines., Results: Transcriptome analyses showed that amphiregulin (AREG) was the most highly up-regulated gene in both AMR-OH-resistant cell lines compared to parent cells. Conditioned medium from DMS53/R cells reduced the sensitivity to AMR-OH in DMS53 cells. In contrast, DMS53/R cells transfected with siRNA directed against AREG recovered their sensitivity to AMR-OH. An additional administration of cetuximab with amrubicinol also restored the sensitivity to AMR-OH., Conclusion: Amphiregulin plays an important role in resistance to AMR-OH., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Aim: To investigate β-catenin (CTNNB1) signaling in cancer and stem cells, the gene expression and pathway were analyzed using bioinformatics., Methods: The expression of the catenin β 1 ( CTNNB1 ) gene, which codes for β-catenin, was analyzed in mesenchymal stem cells (MSCs) and gastric cancer (GC) cells. Beta-catenin signaling and the mutation of related proteins were also analyzed using the cBioPortal for Cancer Genomics and HOMology modeling of Complex Structure (HOMCOS) databases., Results: The expression of the CTNNB1 gene was up-regulated in GC cells compared to MSCs. The expression of EPH receptor A8 (EPHA8), synovial sarcoma translocation chromosome 18 (SS18), interactor of little elongation complex ELL subunit 1 (ICE1), patched 1 (PTCH1), mutS homolog 3 (MSH3) and caspase recruitment domain family member 11 (CARD11) were also shown to be altered in GC cells in the cBioPortal for Cancer Genomics analysis. 3D complex structures were reported for E-cadherin 1 (CDH1), lymphoid enhancer binding factor 1 (LEF1), transcription factor 7 like 2 (TCF7L2) and adenomatous polyposis coli protein (APC) with β-catenin., Conclusion: The results indicate that the epithelial-mesenchymal transition (EMT)-related gene CTNNB1 plays an important role in the regulation of stem cell pluripotency and cancer signaling., Competing Interests: Conflict-of-interest statement: To the best of the authors’ knowledge, no conflict of interest exists.

Neuroblastoma is the most common extracranial solid tumour in children and is histologically classified by its Schwannian stromal cells. Although having fewer Schwannian stromal cells is generally associated with more aggressive phenotypes, the exact roles of other stromal cells (mainly macrophages and fibroblasts) are unclear. Here, we examined 41 cases of neuroblastoma using immunohistochemistry for the tumour-associated macrophage (TAM) markers CD68, CD163, and CD204, and a cancer-associated fibroblast (CAF) marker, alpha smooth muscle actin (αSMA). Each case was assigned to low/high groups on the basis of the number of TAMs or three groups on the basis of the αSMA-staining area for CAFs. Both the number of TAMs and the area of CAFs were significantly correlated with clinical stage, MYCN amplification, bone marrow metastasis, histological classification, histological type, and risk classification. Furthermore, TAM settled in the vicinity of the CAF area, suggesting their close interaction within the tumour microenvironment. We next determined the effects of conditioned medium of a neuroblastoma cell line (NBCM) on bone marrow-derived mesenchymal stem cells (BM-MSCs) and peripheral blood mononuclear cell (PBMC)-derived macrophages in vitro. The TAM markers CD163 and CD204 were significantly up-regulated in PBMC-derived macrophages treated with NBCM. The expression of αSMA by BM-MSCs was increased in NBCM-treated cells. Co-culturing with CAF-like BM-MSCs did not enhance the invasive ability but supported the proliferation of tumour cells, whereas tumour cells co-cultured with TAM-like macrophages had the opposite effect. Intriguingly, TAM-like macrophages enhanced not only the invasive abilities of tumour cells and BM-MSCs but also the proliferation of BM-MSCs. CXCL2 secreted from TAM-like macrophages plays an important role in tumour invasiveness. Taken together, these results indicate that PBMC-derived macrophages and BM-MSCs are recruited to a tumour site and activated into TAMs and CAFs, respectively, followed by the formation of favourable environments for neuroblastoma progression. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

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&#95;Ls) and Macrophage&#95;Ls stimulated with conditioned medium of the TE series human ESCC cell line (TECM) (TAM&#95;Ls) using cDNA microarray analysis. Among the highly expressed genes in TAM&#95;Ls, we focused on neural cell adhesion molecule (NCAM). NCAM knockdown in TAM&#95;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&#95;Ls. Recombinant human fibroblast growth factor-2 (rhFGF-2) promoted the migration and survival of TAM&#95;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., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Recent research has shown that the alteration of combinations in gene expression contributes to cellular phenotypic changes. Previously, it has been demonstrated that the combination of cadherin 1 and cadherin 2 expression can identify the diffuse-type and intestinal-type gastric cancers. Although the diffuse-type gastric cancer has been resistant to treatment, the precise mechanism and phenotypic involvement has not been revealed. It may be possible that stem cells transform into gastric cancer cells, possibly through the involvement of a molecule alteration and signaling mechanism. In this review article, we focus on the role of catenin beta 1 (CTNNB1 or β-catenin) and describe the regulation of CTNNB1 signaling in gastric cancer and stem cells.