Periostin in Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression by Enhancing Cancer and Stromal Cell Migration.

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 ß4 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.

Pseudoimmunofluorescent immunohistochemistry image analysis of phosphorylated signaling proteins in human esophageal squamous cell carcinoma tissue.

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.

Colostomy-site carcinoma with primitive phenotype in a rectal cancer patient after achieving pathological complete response with neoadjuvant chemoradiotherapy.

Herein, we report a rare case of a carcinoma with primitive phenotype (enteroblastic and/or hepatoid differentiation) occurring at a colostomy site. The patient was an elderly male who underwent neoadjuvant chemoradiotherapy for rectal cancer, followed by abdominoperineal resection. A biopsy specimen for the rectal carcinoma before neoadjuvant chemoradiotherapy was conventional tubular adenocarcinoma. Moreover, a pathological complete response was confirmed in the proctectomy specimen. However, a colostomy-site tumor appeared 6 months after the proctectomy, and it was resected 1 year after the initial proctectomy. The colostomy-site tumor comprised solid to focal glandular growth of atypical polygonal cells with clear to pale eosinophilic cytoplasm and was immunohistochemically positive for cytokeratin, spalt-like transcription factor 4, glypican-3, caudal type homeobox 2, and special AT-rich sequence-binding protein 2. Thus, the tumor was diagnosed as poorly differentiated adenocarcinoma with primitive phenotype, with suggested origin from the colorectal epithelium. Additionally, a multilocular cystic lesion comprising various types of epithelia was found adjacent to the tumor, suggestive of metaplasia or heterotopia. Changes in the histology and immunophenotype, and the findings of an adjacent cystic lesion suggest a metachronous tumor rather than a recurrence of the primary tumor.

IFI16 Induced by Direct Interaction between Esophageal Squamous Cell Carcinomas and Macrophages Promotes Tumor Progression via Secretion of IL-1α.

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.

Molecular Networks of Platinum Drugs and Their Interaction with microRNAs in Cancer.

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.

Biological and clinical significance of the YKL-40/osteopontin-integrin ß4-p70S6K axis induced by macrophages in early oesophageal squamous cell carcinoma.

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 ß4 (ß4). 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-ß4-p70S6K axis in the tumour area. Moreover, epithelial expression of ß4 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 ß4 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-ß4-p70S6K axis played important roles in early-stage ESCC, and the high expression levels of ß4 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.

Matrix Metalloproteinase 9 Induced in Esophageal Squamous Cell Carcinoma Cells via Close Contact with Tumor-Associated Macrophages Contributes to Cancer Progression and Poor Prognosis.

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.

Roles of IL-7R Induced by Interactions between Cancer Cells and Macrophages in the Progression of Esophageal Squamous Cell Carcinoma.

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.

Preclinical evaluation of the efficacy of an antibody to human SIRPα for cancer immunotherapy in humanized mouse models.

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.

A Comparative Study of Patient-Derived Tumor Models of Pancreatic Ductal Adenocarcinoma Involving Orthotopic Implantation.

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.

S100A8/A9 Induced by Interaction with Macrophages in Esophageal Squamous Cell Carcinoma Promotes the Migration and Invasion of Cancer Cells via Akt and p38 MAPK Pathways.

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.

Clinical Impact of Different Reconstruction Methods on Remnant Gastric Cancer at the Anastomotic Site after Distal Gastrectomy.

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.

Cell Cycle Regulation and DNA Damage Response Networks in Diffuse- and Intestinal-Type Gastric Cancer.

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 G1/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 G1/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 G1/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.

Metallothionein 2A Expression in Cancer-Associated Fibroblasts and Cancer Cells Promotes Esophageal Squamous Cell Carcinoma Progression.

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.

Close Association of Intraepithelial Accumulation of M2-Skewed Macrophages with Neoplastic Epithelia of the Esophagus.

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.

Tongue Cancer Cell-Derived CCL20 Induced by Interaction With Macrophages Promotes CD163 Expression on Macrophages.

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.

Alteration of Macrophage Infiltrating Compartment: A Novel View on Oral Carcinogenesis.

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.

Chemokine (C-C Motif) Ligand 1 Derived from Tumor-Associated Macrophages Contributes to Esophageal Squamous Cell Carcinoma Progression via CCR8-Mediated Akt/Proline-Rich Akt Substrate of 40 kDa/Mammalian Target of Rapamycin Pathway.

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.

PAI-1 derived from cancer-associated fibroblasts in esophageal squamous cell carcinoma promotes the invasion of cancer cells and the migration of macrophages.

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.