Diffuse-type gastric carcinoma is a cancer with poor prognosis that has high levels of transforming growth factor beta (TGF-beta) expression and thick stromal fibrosis. However, the association of TGF-beta signaling with diffuse-type gastric carcinoma has not been investigated in detail.

Cancer tissues have biological characteristics similar to those observed in embryos during development. Many types of cancer cells acquire pro-invasive ability through epithelial-mesenchymal transition (EMT). Similar processes (gastrulation and migration of cranial neural crest cells [CNCC]) are observed in the early stages of embryonic development in Xenopus during which cells that originate from epithelial sheets through EMT migrate to their final destinations. The present study examined Xenopus embryonic tissues to identify anti-cancer compounds that prevent cancer invasion. From the initial test of known anti-cancer drugs, AMD3100 (an inhibitor of CXCR4) and paclitaxel (a cytoskeletal drug targeting microtubules) effectively prevented migration during gastrulation or CNCC development. Blind-screening of 100 synthesized chemical compounds was performed, and nine candidates that inhibited migration of these embryonic tissues without embryonic lethality were selected. Of these, C-157 (an analog of podophyllotoxin) and D-572 (which is an indole alkaroid) prevented cancer cell invasion through disruption of interphase microtubules. In addition, these compounds affected progression of mitotic phase and induced apoptosis of SAS oral cancer cells. SAS tumors were reduced in size after intratumoral injection of C-157, and peritoneal dissemination of melanoma cells and intracranial invasion of glioma cells were inhibited by C-157 and D-572. When the other analogues of these chemicals were compared, those with subtle effect on embryos were not tumor suppressive. These results suggest that a novel chemical-screening approach based on Xenopus embryos is an effective method for isolating anti-cancer drugs and, in particular, targeting cancer cell invasion and proliferation.

A relationship exists between defects in bone morphogenetic protein (BMP) signaling and formation of hamartoma and adenoma in the gastric epithelium; however, the role of BMP signaling in the progression of diffuse-type gastric carcinoma remains unknown. We investigated whether BMP functions as a tumor suppressor in human diffuse-type gastric carcinoma using three different human diffuse-type gastric carcinoma cell lines (OCUM-12, HSC-39, and OCUM-2MLN). Overexpression of the dominant-negative form of BMP-2/4-specific type I receptor (ALK-3) in OCUM-12 and HSC-39 cells accelerated their growth in vivo. BMP-4 induced cell cycle arrest in these cells via p21 induction through the SMAD pathway. Moreover, overexpression of the constitutively active form of ALK-3 in HSC-39 and OCUM-2MLN cells suppressed the proliferation of these cells in vitro and in vivo. Our findings suggest that BMP-2 and BMP-4 function as potent tumor suppressors in diffuse-type gastric carcinoma.

Obesity is a leading risk factor for cancer. However, understanding the crosstalk between adipocytes and tumor cells in vivo, independently of dietary contributions, is a major gap in the field. Here we used a prostate cancer (PCa) mouse model in which the signaling adaptor p62/Sqstm1 is selectively inactivated in adipocytes. p62 loss in adipocytes results in increased osteopontin secretion, which mediates tumor fatty acid oxidation and invasion, leading to aggressive metastatic PCa in vivo. Furthermore, p62 deficiency triggers in adipocytes a general shutdown of energy-utilizing pathways through mTORC1 inhibition, which supports nutrient availability for cancer cells. This reveals a central role of adipocyte's p62 in the symbiotic adipose tissue-tumor collaboration that enables cancer metabolic fitness.

Thrombospondin-4 [1] is an extracellular glycoprotein involved in wound healing and tissue remodeling. Although THBS4 is reportedly frequently expressed in solid tumors, there are few reports of the clinicopathological features of carcinomas with THBS4 expression. We evaluated the clinicopathologic significance of THBS4 expression in gastric carcinoma (GC).

The aim of this study was to analyze the significance of glucose metabolism-related enzymes in the proliferation of gastric cancer under hypoxia. Four hypoxia-resistant gastric cancer cell lines and four parent cell lines were used. Reverse transcription-PCR was used to evaluate the mRNA expression levels of the following metabolism-related enzymes: pyruvate kinase isozyme M2 (PKM2), glutaminase (GLS), enolase 1 (ENO1), glucose-6-phosphate dehydrogenase (G6PDH), and PKM1. The effects of these enzymes on the proliferation of gastric cancer cells were examined using siRNAs, shikonin as a PKM2 inhibitor, or BPTES as a GLS inhibitor, in vitro and in vivo. Levels of both PKM2 and GLS mRNA were significantly high in all hypoxia-resistant cell lines, compared with those of their parent cells. Knockdown of PKM2 and GLS significantly decreased the proliferation of all hypoxia-resistant cells. The combination of siPKM2 and siGLS significantly decreased proliferation compared with treatment by siPKM2 or siGLS alone. The knockdown of ENO1, G6PDH, or PKM1 did not decrease the proliferation of all hypoxia-resistant cells. Combination treatment using shikonin and BPTES inhibited the proliferation of all hypoxia-resistant cancer cells more than that by either agent alone. The in vivo study indicated that the tumor size treated by the combination of shikonin and BPTES was significantly smaller than that of vehicle-treated group. These findings suggested that PKM2 and GLS might play important roles in the proliferation of hypoxic gastric cancer cells. A combination of PKM2 and GLS inhibitors could be therapeutically promising for the treatment of gastric cancer.

Although many studies have identified several inflammation-based and/or nutritional markers with prognostic value for patients with various types of cancer, the optimal markers and cut-off values for these markers remain obscure. Therefore, this retrospective study aimed to identify optimal markers and their cutoffs.

Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-β receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.

Fibroblast growth factor receptor 2 (FGFR2) has two isoforms: IIIb type and IIIc type. Clinicopathologic significance of these two FGFR2 subtypes in gastric cancer remains to be known. This study aimed to clarify the clinicopathologic difference of FGFR2IIIb and/or FGFR2IIIc overexpression. A total of 562 patients who underwent gastrectomy was enrolled. The expressions of FGFR2IIIb and FGFR2IIIc were retrospectively examined by immunohistochemistry or fluorescence in situ hybridization (FISH) using the 562 gastric tumors. We evaluated the correlation between clinicopathologic features and FGFR2IIIb overexpression and/or FGFR2IIIc overexpression in gastric cancer. FGFR2IIIb overexpression was observed in 28 cases (4.9%), and FGFR2IIIc overexpression was observed in four cases (0.7%). All four FGFR2IIIc cases were also positive for FGFR2IIIb, but not in the same cancer cells. FGFR2IIIb and/or FGFR2IIIc overexpression was significantly correlated with lymph node metastasis and clinical stage. Both FGFR2IIIb and FGFR2IIIc were significantly associated with poor overall survival. A multivariate analysis showed that FGFR2IIIc expression was significantly correlated with overall survival. FISH analysis indicated that FGFR2 amplification was correlated with FGFR2IIIb and/or FGFR2IIIc overexpression. These findings suggested that gastric tumor overexpressed FGFR2IIIc and/or FGFR2IIIb at the frequency of 4.9%. FGFR2IIIc overexpression might be independent prognostic factor for patients with gastric cancer.

Peritoneal metastasis of gastric cancer (GC) results in extremely poor prognoses. The peritoneal cavity is covered by a monolayer of peritoneal mesothelial cells (PMCs). Interactions between GC cells and PMCs might play a pivotal role in peritoneal metastasis. Extracellular vesicles (EVs) correlate with intercellular communication. Although intercellular communication between cancer cells and PMCs might be associated with the peritoneal metastatic process, the role of EVs from PMCs remains unclear. We investigated the effects of EVs from PMCs on GC cells. Three GC cell lines (OCUM-12, NUGC-3, and MKN74) and four mesothelial cell lines were used. The effects of EVs derived from the PMCs on the invasion and migration of GC cells were evaluated by Matrigel invasion assay. Factors contained in the PMC EVs were analyzed; extra-cellular matrix metalloproteinase inducer (EMMPRIN) was detected in the EVs. The effects of an EMMPRIN inhibitor on the invasion-stimulating activity of EVs were examined. The EMMPRIN expressions of 110 GCs were evaluated by immunohistochemistry. PMC EVs significantly promoted the invasion of diffuse-type GC cells, i.e., OCUM-12 and NUGC-3 cells. EMMPRIN in the EVs stimulated the invasion of OCUM-12 and NUGC-3 cells. The invasion-stimulating activity of PMC EVs was inhibited by the EMMPRIN inhibitor. A high EMMPRIN expression in PMCs was significantly associated with worse cancer-specific survival and peritoneal-recurrence-free survival. EMMPRIN in EVs from PMCs might stimulate the malignant progression of diffuse-type GC. EMMPRIN might be a useful prognostic marker of recurrence in GC patients.

Clinical sequencing using a panel of genes has recently been applied worldwide for patients with refractory solid tumors, but the significance of clinical sequencing using gene panel testing remains uncertain. Here we sought to clarify the feasibility and utility of clinical sequencing in the treatment of refractory tumors at our hospital.

Trophoblast cell-surface antigen 2 (TROP2) is a transmembrane glycoprotein expressed in epithelial cells. Increased TROP2 expression has been reported to be associated with malignant progression in most carcinomas; however, TROP2 has a tumor-suppressive function in certain types of cancer. Since the function of TROP2 is controversial, the present study subsequently aimed to clarify the clinicopathologic significance of TROP2 and pTROP2 expression in human gastric cancer (GC). The cases of 704 patients with GC who underwent gastrectomy were retrospectively analyzed. The expression levels of TROP2 and pTROP2 in each tumor were evaluated by immunohistochemistry. The association between the clinicopathologic features of patients with GC and the levels of TROP2 and pTROP2 in their tumors was analyzed. Increased TROP2 and pTROP2 expression was identified in 330 (46.9%) and 306 (43.5%) of the 704 patients with GC, respectively. Increased TROP2 expression was associated with the histological intestinal type, high tumor invasion depth (T3/T4), lymph node metastasis, lymphatic invasion and venous invasion. By contrast, increased pTROP2 expression was associated with intestinal type, low tumor invasion depth (T1/2), no lymph node metastasis and no lymphatic invasion. Increased TROP2 expression was associated with poorer overall survival (OS) (P<0.01; log rank test), whereas increased pTROP2 expression was significantly associated with improved OS (P<0.01; log rank test). In conclusion, increased expression levels of TROP2, but not pTROP2, may be associated with the metastatic ability of GC, resulting in poor prognosis of patients with GC.

In vivo interrogation of the functional role of genes implicated in colorectal cancer (CRC) is limited by the need for physiological models that mimic the disease. Here, we describe a protocol that provides the steps required for the orthotopic co-implantation of tumoral and stromal cells into the cecum and rectum to investigate the crosstalk between the tumor and its microenvironment. This protocol recapitulates metastases to the lymph nodes, liver, and lungs observed in human CRC. For complete details on the use and execution of this protocol, please refer to Kasashima et al. (2020).

Lung cancer is associated with a high morbidity and mortality rate worldwide; however, no reliable and independent prognostic predictor for non-small cell lung cancer (NSCLC) after curative surgery is available. Glucose metabolism is correlated with cancer cell proliferation. Pyruvate dehydrogenase E1α (PDH-E1α) catalyzes the conversion of pyruvate to acetyl-CoA and promotes aerobic glucose metabolism. In this study, we examined the relationship between PDH-E1α expression and clinicopathological factors associated with NSCLC to identify a reliable prognostic predictor of NSCLC after curative surgery.

Lung cancer has a poor prognosis despite recent progresses being made regarding its treatment. In addition, there is a paucity of reliable and independent prognostic predictors for non-small cell lung cancer (NSCLC) following curative resection. Glycolysis is associated with the malignancy and proliferation of cancer cells. Glucose transporter 1 (GLUT1) promotes glucose uptake, whereas pyruvate kinase M2 (PKM2) promotes anaerobic glycolysis. The present study aimed to evaluate the relationship between the expression of GLUT1 and PKM2 and the clinicopathological features of patients with NSCLC, and to identify a reliable prognostic factor for NSCLC following curative resection. Patients with NSCLC who underwent curative surgery were retrospectively enrolled to the present study. GLUT1 and PKM2 expression was assessed using immunohistochemistry. Subsequently, the association between the clinicopathological features of patients with NSCLC and the expression of GLUT1 and PKM2 was assessed. Of the 445 patients with NSCLC included in the present study, 65 (15%) were positive for both GLUT1 and PKM2 expression (G+/P+ group). GLUT1 and PKM2 positivity was significantly associated with sex, absence of adenocarcinoma, lymphatic invasion and pleural invasion. Furthermore, patients with NSCLC in the G+/P+ group presented significantly poorer survival rates than those expressing other markers. G+/P+ expression was significantly associated with poor disease-free survival. In conclusion, the findings of the present study indicated that the combination of GLUT1 and PKM2 may be considered a reliable prognostic factor for patients with NSCLC following curative resection, especially in patients with stage I NSCLC.

Diffuse-type gastric carcinoma (DGC) has a poor prognosis due to its rapid diffusive infiltration and frequent peritoneal dissemination. DGC is associated with massive fibrosis caused by aberrant proliferation of cancer-associated fibroblasts (CAFs). Previously, we reported that direct heterocellular interaction between cancer cells and CAFs is important for the peritoneal dissemination of DGC. In this study, we aimed to identify and target the molecules that mediate such heterocellular interactions. Monoclonal antibodies (mAbs) against intact DGC cells were generated and subjected to high-throughput screening to obtain several mAbs that inhibit the adhesion of DGC cells to CAFs. Immunoprecipitation and mass spectrometry revealed that all mAbs recognized integrin α5 complexed with integrin β1. Blocking integrin α5 in DGC cells or fibronectin, a ligand of integrin α5β1, deposited on CAFs abrogated the heterocellular interaction. Administration of mAbs or knockout of integrin α5 in DGC cells suppressed their invasion led by CAFs in vitro and peritoneal dissemination in a mouse xenograft model. Altogether, these findings demonstrate that integrin α5 mediates the heterotypic cancer cell-fibroblast interaction during peritoneal dissemination of DGC and may thus be a therapeutic target.

Air-liquid organotypic culture models enable the study of the cellular crosstalk in the tumor microenvironment. This 3D assay recapitulates the tumor niche more faithfully than 2D culture systems and represents a versatile platform that can be easily adapted to different types of cancer cells, stromal components, or ECM composition. Here, we detail the steps to build an organotypic culture including the preparation of the organotypic structure, organotypic gels, cell seeding, gel casting, membrane processing, and image and data analysis. For complete details on the use and execution of this protocol, please refer to Linares et al. (2022).

Gastric cancer (GC) with microsatellite instability (MSI) has been reported to be sensitive to immunotherapy, however some of GC cases with MSI remain resistant to immunotherapy. Cancer cell lines showing MSI might be useful for the analysis of mechanisms of immunotherapy, while only a few GC cell lines with MSI are available so far. In this study, we established a unique GC cell line with MSI, OCUM-13, from a primary GC with abundant tumor-infiltrating lymphocytes. MSI assay indicated that OCUM-13 cells as well as the primary tumor showed a band shift in more than 3 of 5 microsatellite loci, suggesting that OCUM-13 did have high MSI. The subcutaneous inoculation of OCUM-13 cells into mice performed tumor formation. Insulin-like growth factor 1 receptor inhibitor decreased the growth of OCUM-13 cells. The newly established cell line with MSI, OCUM-13, might be useful for the analysis of cancer therapy for GC with MSI.

MicroRNAs (miRNAs) play pivotal roles in the tumor microenvironment. Here, we analyzed miRNAs in tumor stromal fibroblasts. Expression of miR-224-3p in cancer-associated fibroblasts (CAF) from scirrhous gastric cancer patients was lower than in normal fibroblasts (NF). Introduction of a miR-224-3p mimic attenuated migration and invasion of CAF. Coiled-coil domain containing 85A (CCDC85A), whose function in tumors is not understood, was the target gene of miR-224-3p. Immunohistological analysis revealed that CCDC85A is expressed to varying degrees by cancer cells and CAFs in gastric and pancreatic carcinomas. Downregulation of CCDC85A in cancer cells revealed that these cells are vulnerable to endoplasmic reticulum (ER) stress induced by thapsigargin or tunicamycin, which were ameliorated after addback of CCDC85A. Injection of NF-derived exosomes containing miR-224-3p into the xenograft tumor increased tumor shrinkage by cisplatin treatment. Mechanistically, CCDC85A associated with the molecular chaperone GRP78 and GRP94, thereby inhibiting association of these negative regulators of the unfolded protein response (UPR), leading to sustained activation of PERK and downstream eIF2〈 and ATF4 upon ER stress. These data suggest a novel miR-224-3p-mediated function for CCDC85A: protection from ER stress and cisplatin resistance.

Cell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood.