Cancer-associated fibroblasts might sustain the stemness of scirrhous gastric cancer cells via transforming growth factor-ß signaling.

Cancer-associated fibroblasts (CAFs) have recently been implicated in tumor growth and metastasis in gastric cancer. Cancer stem cells (CSCs) have been proposed to have an important role in cancer progression. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric carcinoma. Scirrhous gastric cancer cell lines, OCUM-12 and OCUM-2MD3, and non-scirrhous gastric cancer cell lines, MKN-45 and MKN-74, were used. OCUM-12/side population (SP) cells and OCUM-2MD3/SP cells were sorted by flow cytometry as CSC-rich cells from the parent cells. CaF-37 was established from the tumoral gastric specimens as CAFs. Flow cytometric analysis of SP fraction, spheroid colony assay, and RT-PCR analysis of CSC markers were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM-12/SP cells was examined using nude mice. CAF CM significantly increased the percentages of the SP fraction of OCUM-12/SP and OCUM-2MD3/SP cells, but not that of MKN-45/SP and MKN-74/SP cells. Taken together, CM from CaF-37 significantly increased the number of spheroid colonies and the expression level of CSC markers of OCUM-12/SP and OCUM-2MD3/SP cells. These stimulating-activities by CM were significantly decreased by TGFß inhibitors, but not FGFR and cMet inhibitor. Tumorigenicity by subcutaneous coinoculation of OCUM-12/SP cells with CAFs was significantly high in comparison with that by OCUM-12/SP cells alone. Phospho-Smad2 expression level was significantly increased by co-inoculation with CAFs. These findings suggested that CAFs might regulate the stemness of CSCs in scirrhous gastric cancer by TGFß signaling.

Hypoxia stimulates the EMT of gastric cancer cells through autocrine TGFß signaling.

Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor ß1 (TGFß1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFß1 and TGFßR was evaluated by real time RT-PCR. The TGFß1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFß1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFßR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFßR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFß1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFß/TGFßR signaling.

Cancer-associated orthotopic myofibroblasts stimulates the motility of gastric carcinoma cells.

Tumor progression has been recognized as the product of evolving crosstalk between cancer cells and the surrounding stromal cells. Cancer-associated orthotopic myofibroblasts may be linked to the progression of gastric carcinomas. To understand the significance of orthotopic myofibroblasts, we examined the effects of cancer-associated orthotopic myofibroblasts on the malignant phenotype of gastric cancer cells. Three human gastric cancer cell lines (OCUM-2MD3, OCUM-12, MKN-45) and four human gastric fibroblast cell lines (cancer-associated orthotopic fibroblast [CaF]-29, CaF-33, normal orthotopic fibroblast [NF]-29, NF-33) were used. The cancer-associated orthotopic fibroblast cell lines CaF-29 and CaF-33 were established from a tumoral gastric wall, and normal orthotopic fibroblast NF-29 and NF-33 were established from a non-tumoral gastric wall. Fibroblasts that were α-smooth muscle actin-positive were defined as myofibroblasts. We examined the effects of cancer-associated orthotopic myofibroblasts on the aggressiveness of gastric cancer cells by wound-healing assay, invasion assay, and RT-PCR. The ratios of myofibroblasts in CaF-29 (33%) and CaF-33 (46%) were significantly (P < 0.001) greater than those in NF-29 (11%) or NF-33 (13%). Although all four orthotopic fibroblast lines increased the motility of gastric cancer cells, including migration and invasion ability, the motility-stimulating activity of cancer-associated fibroblasts (CaF-29 and CaF-33) was significantly higher than that of normal fibroblasts (NF-29 and NF-33). These motility-stimulating activities of cancer-associated orthotopic fibroblasts were downregulated by Smad2 siRNA treatment and anti-transforming growth factor-ß neutralizing antibody. These findings suggest that cancer-associated orthotopic myofibroblasts may play an important role in the progression of gastric cancers and that transforming growth factor-ß produced by myofibroblasts may be one of the factors associated with the aggressiveness of gastric carcinoma cells.

[Our experience of the treatment with XELOX±Bevacizumab for unresectable advanced colorectal cancer].

XELOX for unresectable advanced colorectal cancer has been approved in Japan. We report here an experience of the treatment with XELOX±bevacizumab in our department. We thought that the regimen has some merits in the patient's quality of life. Forty-five patients were treated with XELOX±bevacizumab. In evaluable 23 cases, partial response (PR) was obtained in 13 cases and stable disease (SD) and progressive disease (PD) were in 5 and 5 cases, so that the response rate and disease control rate were 56.5% and 78.2%, respectively. In the first-line administration of 19 cases, the response rate and disease control rate were respectively 80.0% and 90.0%. Hand-foot syndrome was observed in 13.2% (grade 2) and 6 .6% (grade 3). We think that it is important to control hand-foot syndrome for a continuation of XELOX±bevacizumab.

Effects of acute and chronic hypoxia on the radiosensitivity of gastric and esophageal cancer cells.

AIM: The aim of this study was to examine the effects of hypoxia on radiosensitivity and to analyze the mechanisms responsible for radiation resistance in gastric and esophageal cancer. MATERIALS AND METHODS: A gastric cancer cell line, OCUM-12, and an esophageal cancer cell line, TE-6, were used. The effects of hypoxia with irradiation on the growth-activity, cell cycle distribution, and gene expression were examined. RESULTS: Both acute and chronic hypoxia decreased radiosensitivity of cancer cells. The radiosensitivity of chronic hypoxic cells was significantly enhanced by reoxygenation. Acute and chronic all hypoxia reduced the percentage of cells in the G(2)/M and S phases, respectively. In acute hypoxia, the mRNA expression of BRCA1 and BRCA2 was reduced in cancer cells. Reoxygenation increased the expression of BRCA1 and BRCA2. CONCLUSION: Hypoxia is associated with radiation resistance. Therefore, reoxygenation may enhance the radiosensitivity of hypoxic cells. BRCA1 and BRCA2 may be associated with factors for radiation resistance by regulation of cell cycle progression.

[Pilot study of preoperative mFOLFOX6 chemotherapy for advanced rectal cancers which were difficult to ensure surgical margins].

Seven patients with rectal cancers which were difficult to ensure surgical margins for because of huge tumors(over 60mm in diameter), invasion to other organs, or severe nodal metastases, were treated with preoperative chemotherapy consisting of 2-10 courses of mFOLFOX6.The response rate was 85. 7%.Complete response was observed in one patient, and partial response was observed in 5 patients.Four to 5 weeks after chemotherapy, surgery was performed for all patients.Following surgical procedures, abdominoperineal resections were performed in 4 cases, low anterior resections in 3 cases, and removal of the liver metastases(not diagnosed preoperatively)in 2 cases.R0 resections were also performed in all patients. According to the histological regression grading of the resected specimens, one patient had a complete disappearance of tumor, 5 had grade 1a regression, and one had grade 1b regression.One of the 7 patients had recurrence at the lung. However, another patient survived without recurrence. In this study, preoperative mFOLFOX6 chemotherapy was expected to be an effective treatment for improving the curative resection rate of patients with tumors which were difficult to ensure surgical margins because of huge tumors, invasion to other organs, or severe nodal metastases.

Clinical significance of vimentin-positive gastric cancer cells.

BACKGROUND/AIM: Vimentin expression in epithelial cells is reported to be associated with the malignant phenotype of cancer cells in vitro. However, the clinical significance of vimentin expression in carcinomas is not well understood. The aim of this study was to clarify the significance of vimentin-positive gastric cancer (GC). PATIENTS AND METHODS: A total of 265 GCs were examined by immunofluorescent staining with antibodies against vimentin and carcinoembryonic antigen. GCs were determined to be vimentin-positive when cells were positive for both vimentin and carcinoembryonic antigen staining. RESULTS: A total of 86 (32%) of 265 GCs were vimentin positive. There was a statistically significant correlation between vimentin-positive GCs and advanced clinical stage (p<0.001), macroscopic scirrhous-type (p < 0.001), histological diffuse-type (p < 0.001), lymph node metastasis (p = 0.008) and lymphatic invasion (p = 0.013). The prognosis of patients with vimentin-positive GCs was significantly (p < 0.001) worse than that with vimentin-negative GCs. CONCLUSION: Vimentin expression might contribute to the high invasive phenotype of GC, and may be a useful biomarker to determine the biological aggressiveness of GC.

Expression of a hypoxia-associated protein, carbonic anhydrase-9, correlates with malignant phenotypes of gastric carcinoma.

BACKGROUND/AIMS: Gastric cancers are characterized by a heterogeneously hypoxic environment. Hypoxia might stimulate the malignant potential of cancer cells. The purpose of our study was to clarify the significance of hypoxia in gastric carcinoma by evaluating the expression of a hypoxic marker, namely carbonic anhydrase-9 (CA-9). METHODS: A total of 265 patients who had undergone a resection of the primary tumor and were confirmed histologically to have sporadic gastric cancer were enrolled in this study. We evaluated the immunohistochemical expression of CA-9 in the paraffin-embedded specimens of 265 gastric adenocarcinomas. RESULTS: The CA-9 expression was positive in 88 (33%) of 265 gastric carcinomas. The CA-9 expression level was significantly high in cases of type 4 carcinoma (60%, p < 0.001) and diffuse type carcinoma (41%, p < 0.001), and significantly correlated with the invasion depth (p < 0.001), lymph node metastasis (p < 0.001) and lymphatic invasion p = 0.002). The prognosis for CA-9-positive patients was significantly poorer than that of CA-9-negative patients (p = 0.003, log-rank). CONCLUSION: Hypoxia might be associated with aggressive tumor phenotypes of gastric carcinomas. The hypoxic marker CA-9 may be a useful prognostic indicator.

Significance of phospho-vascular endothelial growth factor receptor-2 expression in pancreatic cancer.

Vascular endothelial growth factor receptors (VEGFRs) are mainly expressed by endothelial cells, but they are also expressed by some cancer cells, including pancreatic cancer. The objective of this study was to evaluate the significance of VEGFRs expression in pancreatic cancer cells. A total of 107 primary pancreatic tumors were stained with antibodies against VEGFR-1, VEGFR-2, phospho-VEGFR-2 (pVEGFR-2), VEGFR-3, VEGF-A, VEGF-C, and VEGF-D. VEGFR-2 and pVEGFR-2 expression were positive in 74 (69%) and 54 (50%) of 107 pancreatic cancers. There was a significant correlation (P < 0.001) between VEGFR-2 expression and pVEGFR-2 expression. pVEGFR-2 was significantly associated with invasion to the anterior capsule of pancreas (P = 0.032) and arterial invasion (P = 0.012). In contrast, VEGFR-1 and VEGFR-3 expression was only observed in 13 (12%) and 15 (14%) of 107 pancreatic cancers, and was not associated with any clinicopathological features. The prognosis of pVEGFR-2 positive patients with stage IIA tumors was significantly (P = 0.0441) poorer than that of pVEGFR-2-negative patients. VEGF-A, VEGF-C, and VEGF-D expression was positive in 42 (39%), 82 (77%), and 39 (36%) of 107 pancreatic cancers, respectively. The prognosis for VEGF-A-positive patients was significantly (P = 0.0425) poor, but not for VEGF-C-positive and VEGF-D-positive patients. A multivariate analysis indicated pVEGFR-2 expression to be an independent prognostic factor, but not VEGF-A. These findings suggested that VEGFR-2 signaling might therefore be associated with the prognosis of patients with pancreatic cancer. The expression of pVEGFR-2 might be a novel predictive prognostic marker for patients with pancreatic cancers, especially at clinical stage IIA.

Myofibroblasts are associated with the progression of scirrhous gastric carcinoma.

Fibroblasts, particularly myofibroblasts, affect the malignant progression of cancer cells in vitro. However, to date few reports have addressed the clinical significance of myofibroblasts in the gastric cancer microenvironment. This study examined the correlation between myofibroblast expression and clinicopathological features in gastric carcinoma. A total of 265 primary gastric tumors resected by gastrectomy were stained with antibodies against α-smooth muscle actin and vimentin. Stromal cells positive for vimentin were considered to be fibroblasts. Myofibroblasts were defined as fibroblasts positive for α-smooth muscle staining. Myofibroblast-positive gastric carcinoma was established when myofibroblasts accounted for more than 25% of fibroblasts in the cancer stroma. Myofibroblast expression was positive in 92 (35%) of the 265 gastric carcinomas. Myofibroblast expression showed a significantly (p<0.001) high frequency in advanced gastric cancers (76 of 146), in comparison to the early stage cancers (16 of 119). Taken together, there was a statistically significant correlation between myofibroblast expression and scirrhous type gastric cancer (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p<0.001) and peritoneal dissemination (p=0.005). The prognosis of patients with tumors positive for myofibroblast expression was significantly (p<0.001) worse, while a multivariate analysis revealed that myofibroblast expression was not an independent prognostic factor. These findings suggest that myofibroblasts are associated with scirrhous gastric cancer. Overexpression of myofibroblasts may therefore be a useful prognostic indicator of gastric carcinoma.