Selective cyclooxygenase-2 inhibitor downregulates the paracrine epithelial-mesenchymal interactions of growth in scirrhous gastric carcinoma

The importance of cancer-mesenchymal interactions in the aggressive behavior of scirrhous gastric cancer is supported by experimental and clinical evidences. We have previously reported that gastric fibroblasts secretion of keratinocyte growth factor (KGF) underline the remarkable proliferation of scirrhous gastric cancer cells. Cyclooxygenase-2 (COX-2) is not only expressed in cancer cells, but also in interstitial fibroblasts in gastric carcinoma. To clarify the mechanisms responsible for the antiproliferation effect of COX-2 inhibitors, effect of COX-2 inhibitor on the paracrine epithelial-mesenchymal interactions of growth was examined. Scirrhous gastric cancer cell line, OCUM-2M, gastric fibroblasts, NF-21, and COX-2 inhibitor, JTE-522, were used. Growth-interaction was examined by calculating the number of cancer cells or by measuring [(3)H] thymidine incorporation of cancer cells. Effect of JTE-522 on KGF expression from NF-21 cells and OCUM-2M cells was analyzed by ELISA and RT-PCR. The conditioned medium from gastric fibroblasts significantly stimulated the growth of scirrhous gastric cancer cells. JTE-522 at the concentrations of 10(-5) and 10(-6) M significantly decreased the growth-stimulating activity of gastric fibroblasts. JTE-522 reduced the expression of KGF mRNA and the production of KGF from gastric fibroblasts. Oral administration of JTE-522 significantly decreased the size of xenografted tumor coinoculated with OCUM-2M cells and NF-21 cells in nude mice. JTE-522 decreased COX-2 expression and Ki67 labeling index within the coinoculated tumor. These findings suggested that a selective COX-2 inhibitor, JTE-522, downregulates KGF production from gastric fibroblasts, resulting in the inhibition of paracrine epithelial-mesenchymal interactions of proliferation between scirrhous gastric cancer cells and gastric fibroblasts.     

胃硬化型癌的特征与治疗

胃硬化型癌是进展期胃癌中的特殊类型,伴随间质高度纤维化和癌细胞广泛浸润的胃癌。大体类型主要是Bor? rmann4型癌,组织学类型以低分化腺癌、印戒细胞癌为主体。以胃壁肥厚、狭窄、易产生腹膜转移和高度的淋巴结转移为特征,恶性程度高,预后不佳。手术疗法效果有限,但腹膜转移所致的肠梗阻、尿路梗阻需行姑息性手术。可治愈性切除的病例,积极的R0切除和S-1的术后化疗是必要的。S-1/CDDP术前化疗将是可期待的方法。非治愈切除的病例化疗为第一选择。除此之外,针对胃硬化型癌的生物学恶性度,尤其是腹膜转移的控制,腹腔内化疗的有用性倍受期待。另外,bevacizumab分子靶向治疗,ad- enovirus的基因治疗等研究性的治疗期待其结果。      

Inhibitory effect of a selective cyclooxygenase inhibitor on the invasion-stimulating activity of orthotopic fibroblasts for scirrhous gastric cancer cells

The cyclooxygenase (COX)-2 inhibitor has been reported to impede the progression of gastric cancer, but underlying mechanisms remain unclear. We therefore investigated the effect of a COX-2 inhibitor, JTE-522, on the ability of orthotopic fibroblasts to stimulate invasion of scirrhous gastric carcinoma cells. The human scirrhous gastric cancer cell lines OCUM-2D or OCUM-2M, and human gastric fibroblasts (NF-21) were cultured in the absence or presence of JTE-522 at various concentrations. Cancer cells were then assayed for invasiveness in vitro by invasion assay. The effect of prostaglandins (PG) on growth factor production in NF-21 cells was examined by ELISA. Finally, the effects of orally administrated JTE-522 on orthotopically transplanted tumors were examined in nude mice. NF-21 cells stimulated invasion by OCUM-2D cells, an effect suppressed by JTE-522 at 5 x 10(-6) M. Hepatocyte growth factor (HGF) and PGE2 production by NF-21 cells were suppressed by JTE-522 (P < 0.01). PGE2 stimulated HGF production by NF-21 cells in a dose-dependent manner. JTE-522 significantly suppressed orthotopic tumor growth and lymph node metastasis, and also decreased HGF expression by fibroblasts within the gastric tumor. In conclusion, we found that gastric fibroblasts stimulated invasiveness in scirrhous gastric cancer cells, whereas a selective COX-2 inhibitor inhibited this paracrine effect by decreasing fibroblast PGE2 production, resulting in downregulation of HGF production.     

A novel transforming growth factor beta receptor kinase inhibitor, A-77, prevents the peritoneal dissemination of scirrhous gastric carcinoma

PURPOSE: Transforming growth factor beta receptor (TGFbeta-R) is reported to correlate with the malignant potential of scirrhous gastric carcinoma. The aim of the current study is to clarify the possibility of molecular target therapy with a TGFbeta-R inhibitor, A-77, for the treatment of peritoneal dissemination of scirrhous gastric cancer. EXPERIMENTAL DESIGN: Three scirrhous gastric cancer cell lines and two fibroblasts were used. For in vivo experiments, the A-77 was administered i.p. to mouse models of peritoneal dissemination. The influences of A-77 on the adhesion ability, invasion ability, and the expression of adhesion molecules were examined in vitro. RESULTS: The A-77 administration resulted in a significantly (P < 0.01) better prognosis for the mice with peritoneal dissemination (median survival time, 51 days), compared with the control (median survival time, 25 days). A-77 therefore significantly (P < 0.01) decreased the weight and number of metastatic nodes. The adhesive ability and invasion ability of cancer cells were significantly decreased by A-77. A-77 decreased the expression of alpha(2), alpha(3), and alpha(5) integrins in gastric cancer cells. The histologic findings showed the degree of fibrosis to be less in the tumors treated by A-77. A-77 decreased the growth of fibroblast and invasion-stimulating activity of fibroblasts on cancer cells. CONCLUSION: The TGFbeta-R inhibitor, A-77, decreased the expression of integrins in cancer cells and the proliferation of fibroblasts, which resulted in the decreased adhesive and invasive abilities of scirrhous gastric cancer cells to peritoneum. A-77 is thus considered to be useful for the inhibition of peritoneal dissemination of scirrhous gastric carcinoma.     

Dihydropyrimidine dehydrogenase inhibitory fluoropyrimidine S-1 may be effective against peritoneal dissemination in gastric cancer

The effects of a novel oral fluoropyrimidine derivative S-1 on peritoneal metastasis from gastric cancer were investigated. OCUM-2MD3 cells, a highly peritoneal-metastatic cell line, were injected intraperitoneally in nude mice. These mice were allocated to the following three groups (each group, n=10): the S-1 group, to which 10 mg/kg body weight of S-1 was administered per os daily; the FT group, to which 100 mg/kg body weight of tegafur (FT) was administered per os daily; the control group, to which no anticancer drug was administered. Drug administration was starting the day after inoculation. The median survival time of the S-1 group was found to be significantly longer than that of the FT group (30 days vs. 23 days; P<0.005) and the control group (vs. 24 days; P<0.005). The mean values of 5-fluorouracil (5-FU) concentrations in ascites of the S-1 group at 1-4 h were 414-580 ng/ml (n=5), and those of FT group were 70-87 ng/ml (n=5), with significant differences between the two groups at each observation time. The high CDHP concentrations in ascites of the S-1 group were observed at 1-6 h after drug administration. DPD was expressed strongly in fibrous tissue around peritoneal metastasis and weakly in tumor cells of peritoneal metastasis themselves. The high concentrations and long duration of 5-FU in the peritoneal cavity after S-1 administration suggest that S-1 may be effective against peritoneal dissemination. High concentrations of CDHP may prevent 5-FU degradation in peritoneal dissemination and its surrounding fibrous tissue.     

胃癌腹膜高转移潜能细胞系的建立及其生物学特性

目的利用人胃癌细胞系(GC9811)在裸小鼠体内反复接种建立一株具有腹膜高转移潜能的胃癌细胞系(GC9811P),并对其生物学特性进行观察,为实验研究提供模型。方法采用胃癌细胞系(GC9811)在裸小鼠腹腔内反复接种,行体外培养腹膜转移灶筛选高转移亚系,绘制细胞生长曲线,光镜、电镜下观察细胞形态,利用流式细胞仪、染色体分析等方法,研究该高转移亚系的细胞周期、增殖、染色体核型等生物学特性。结果母本细胞系GC9811腹膜转移形成率为33.3%(3/10),而GC9811P腹膜转移形成率为100%。两种细胞系腹膜结节组织学形态大体相似。增殖速度较母系加快。细胞周期分析G1期53.5%、G2期12.5%、S期37.1%。且遗传学特性包括染色体形态仍为人类核型,众数维持在104～126之间,占70%。结论具有腹膜高转移的胃癌细胞系GC9811P的建立及裸小鼠体内实验模型,为研究胃癌腹膜转移机制及探索新的治疗途径提供了极为有用的工具。     

ICAM-2 gene therapy for peritoneal dissemination of scirrhous gastric carcinoma.

PURPOSE: Human scirrhous gastric carcinoma develops peritoneal dissemination with high frequency, and the prognosis of patients with peritoneal metastasis is poor. There have been few reports of an immunogene therapy for peritoneal dissemination. Intercellular adhesion molecule (ICAM)-2 is a second ligand of leukocyte function-associated antigen-1, which functions as a costimulatory molecule for effector cells. In the present study, we examined whether ICAM-2 transfection using adenovirus vector is effective gene therapy for peritoneal metastasis of gastric cancer. EXPERIMENTAL DESIGN: We constructed an adenovirus vector, AdICAM-2, that encodes the full-length human ICAM-2 gene under control of the cytomegalovirus promoter. This vector expresses high levels of ICAM-2 on the human gastric cancer cell line OCUM-2MD3, which has high peritoneal metastatic ability in nude mice. We investigated the antitumor effects of gene transfer of ICAM-2 using the adenovirus vector AdICAM-2 in vitro and in vivo. RESULTS: ICAM-2 expressed on OCUM-2MD3 cells by AdICAM-2 demonstrated significantly high adhesiveness to and cytotoxicity against peripheral blood mononuclear cells in vitro compared with the control adenovirus vector AdlacZ. Intratumoral injection of AdICAM-2 significantly inhibited the growth of s.c. tumor. Mice with peritoneal metastasis survived for a significantly longer time after AdICAM-2 injection, compared with injection of AdlacZ. Histopathological findings revealed that many natural killer cells infiltrated the peritoneal metastatic lesions after AdICAM-2 injection. CONCLUSIONS: These findings suggest that transduction of ICAM-2 into cancer cells enhances the adhesion and activation of natural killer cells, resulting in a reduction of peritoneal metastasis. ICAM-2 transfection using adenovirus vector might be an effective form of gene therapy for peritoneal metastasis of gastric cancer.     

Combination effect of a TGF‐β receptor kinase inhibitor with 5‐FU analog S1 on lymph node metastasis of scirrhous gastric cancer in mice

Transforming growth factor‐β (TGF‐β) signals are closely associated with the distant metastases of gastric cancer. The aim of this study was to clarify the effect of a TGF‐β receptor I (TβR‐I) phosphorylation inhibitor, Ki26894, in combination with anticancer drugs, on the lymph node (LN) metastasis of scirrhous gastric cancer. A novel TβR‐I kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at the ATP binding site of TβR‐I. S1 is a 5‐fluorouracil analog. The human scirrhous gastric cancer cell line OCUM‐2MLN and the human gastric fibroblasts NF‐33 were used. OCUM‐2MLM cells in the upper well and NF‐33 cells in the lower well were co‐incubated with or without Ki26894. The proliferation of OCUM‐2MLN cells was significantly stimulated by co‐culture with NF‐33 cells. Ki26894 significantly suppressed the growth interactions between OCUM‐2MLN cells and NF‐33 cells. Gastric cancer models established by orthotopic inoculation of OCUM‐2MLN cells showed diffusely infiltrating gastric adenocarcinoma accompanied by LN metastases. We divided these mice into four groups, (control vehicle, Ki26894, S1, Ki26894 plus S1), and examined the effect of Ki26894 and/or S1 on phosphorylation of Smad2, tumor size, LN metastases, and lymphatic involvements. Ki26894 inhibited the Smad2 phosphorylation of cancer cells and decreased the extent of lymphatic involvement, compared with the control or S1 only group. The Ki26894 plus S1 administration group significantly suppressed tumor growth and decreased LN metastasis more effectively than either alone. These findings suggested that the TβR‐I kinase inhibitor with S1 is useful for the treatment of scirrhous gastric carcinoma with LN metastasis. (Cancer Sci 2010)     

Establishment of a new scirrhous gastric cancer cell line with loss of heterozygosity at E-cadherin locus

Scirrhous gastric carcinoma, characterized by carcinoma cell proliferation and infiltration with extensive fibrosis in the stroma, frequently causes peritoneal metastasis. We describe here a newly established cell line, OCUM-6, derived from ascites effusion of a scirrhous gastric cancer patient. The cells are floating and round shape, similar to other scirrhous gastric carcinoma cell lines previously reported. Histologic findings of xenografted tumor obtained from OCUM-6 cells showed medullary growth with a poorly differentiated adenocarcinoma containing signet ring cells. LOH at E-cadherin locus 16q22 was observed in the OCUM-6 cells. LOH at E-cadherin locus might be closely associated with histologic findings and metastatic process of scirrhous gastric cancer. The scirrhous gastric cancer cell line, OCUM-6, may be useful for investigation of the mechanisms of peritoneal dissemination and carcinogenesis.     

Suppression of gastric cancer dissemination by ephrin-B1-derived peptide

Interaction of the Eph family of receptor protein tyrosine kinases and their ligands, ephrin family members, induces bidirectional signaling through cell–cell contacts. High expression of B-type ephrin is associated with high invasion potential of tumors, and we previously observed that signaling through the C-terminus of ephrin-B1 mediates the migration and invasion of cells, and is involved in the promotion of carcinomatous peritonitis in vivo . Here we show that the intracellular introduction of a synthetic peptide derived from ephrin-B1 C-terminus blocks ephrin-B1 mediated signaling in scirrhous gastric cancer cells. Treatment of cancer cells with a fusion peptide consisting of HIV-TAT and amino acids 331–346 of ephrin-B1 (PTD-EFNB1-C) suppressed the activation of RhoA, mediated by the association of ephrin-B1 with an adaptor protein Dishevelled, and also inhibited extracellular secretion of metalloproteinase. Moreover, injection of PTD-EFNB1-C peptide into the peritoneal cavity of nude mice suppressed carcinomatous peritonitis of intraperitoneally transplanted scirrhous gastric cancer cells. These results indicate the possible application of ephrin-B1 C-terminal peptide to develop novel protein therapy for scirrhous gastric carcinoma, especially in the stage of tumor progression, including peritoneal dissemination. ( Cancer Sci 2009)     

Anti-stromal therapy with imatinib inhibits growth and metastasis of gastric carcinoma in an orthotopic nude mouse model

Recent studies have revealed that platelet-derived growth factor (PDGF) plays a role in promoting progressive tumor growth in several organs; however, whether PDGF plays such a role in gastric carcinoma is undetermined. We examined whether inhibition of PDGF receptor (PDGF-R) tyrosine kinase signaling by imatinib affects tumor growth and metastasis in an orthotopic nude mouse model of human gastric carcinoma. TMK-1 human gastric carcinoma cells were injected into the gastric wall of nude mice. Groups of mice (n = 10 each) received sterile water (control), low-dose imatinib (50 mg/kg/day), high-dose imatinib (200 mg/kg/day), cancer chemotherapeutic agent irinotecan (5 mg/kg/week), or imatinib (50 mg/kg/day or 200 mg/kg/day) and irinotecan (5 mg/kg/week) in combination for 28 days. Tumor growth and metastasis were assessed. Resected tumors were analyzed immunohistochemically. Carcinoma-associated fibroblasts, pericytes and lymphatic endothelial cells in stroma expressed high levels of PDGF-R; carcinoma cells did not. Treatment with imatinib alone did not inhibit tumor growth and metastasis; however, treatment with irinotecan alone or combined with imatinib significantly inhibited tumor growth. Only treatment with high-dose imatinib and irinotecan in combination inhibited lymph node and peritoneal metastases. Immunohistochemically, only imatinib alone or in combination with irinotecan was shown to significantly decrease the stromal reaction, microvessel area and pericyte coverage of tumor microvessels. These effects were marked with high-dose imatinib. In conclusion, administration of PDGF-R tyrosine kinase inhibitor in combination with irinotecan appears to impair the progressive growth of gastric carcinoma by blockade of PDGF-R signaling pathways in stromal cells.     

A case of non-curatively resected scirrhous gastric cancer successfully treated over 2 years with weekly administration of paclitaxel

We report a patient with far-advanced gastric cancer treated by weekly administration of paclitaxel (TXL) over 2 years. The patient was a 66-year-old female with peritoneal metastasis and remarkable lymph node metastasis of scirrhous gastric cancer. She underwent a non-curative resection with total gastrectomy and splenectomy in May 2002. Postoperative chemotherapy with TS-1 (80 mg/body) was performed. Due to grade 4 neutropenia and grade 2 anorexia, this treatment could not be continued. Three months after surgery, the tumor marker (CA19-9) had elevated to an abnormal level. Alternatively, TXL was administered at a weekly dose of 70 mg/m2 for 3 weeks followed by 6 weeks rest from September 2002. The tumor marker (CA19-9) gradually decreased to the normal level. Because of the long rest interval, 10 courses of treatment could be continued, and the patient has been alive over 2 years with the cancer controlled. There have been few effective chemotherapies for gastric cancer with peritoneal metastasis. Weekly paclitaxel therapy is considered to be effective for the treatment of advanced scirrhous gastric cancer with peritoneal metastasis.     

Matrix metalloproteinase inhibitor, marimastat, decreases peritoneal spread of gastric carcinoma in nude mice.

Marimastat, a matrix metalloproteinese inhibitor, was examined for the ability to prevent peritoneal dissemination of a human gastric cancer xenograft, TMK-1. Even with novel approaches such as molecular targeting of cancer chemotherapy, peritoneal dissemination of gastric cancer has little sensitivity to anticancer drugs, and it is impossible to inhibit its growth completely. Intraperitoneal injection of TMK-1 into nude mice at 5 x 10( 5) cells / body resulted in carcinomatous peritonitis that mimicked clinical cases. Continuous administration of marimastat (18 mg / kg / day) from 24 h after the tumor inoculation successfully inhibited the growth of peritoneal dissemination nodules. Combined administration of marimastat (18 mg / kg / day) and mitomycin C (MMC, 2 mg / kg) showed synergistic inhibition of growth of peritoneal dissemination, being superior to MMC alone (2 mg / kg). Although marimastat alone could not increase survival time with statistical significance, combined administration of marimastat and MMC had a survival benefit with statistical significance. The combination of marimastat and MMC increased the preventive effect on peritoneal dissemination. Marimastat seems to be a candidate for the prevention of peritoneal spread of gastric carcinoma.     

Role of Orthotopic Fibroblasts in the Development of Scirrhous Gastric Carcinoma

We examined the interaction between scirrhous gastric cancer cells and organ-specific fibroblasts in vivo and in vitro. Co-inoculation of scirrhous gastric cancer cells with gastric fibroblasts into nude mice specifically increased tumorigenicity, compared with that of gastric cancer cells alone. Furthermore, the histologic findings of the xenograft produced by co-inoculation with gastric fibroblasts was similar to that of human scirrhous gastric carcinoma. Conditioned medium from gastric fibroblasts significantly stimulated the growth of gastric cancer cells. These findings suggest that the growth of scirrhous gastric cancer cells was affected by orthotopic fibroblasts.     

Phase II trial of S-1 for neoadjuvant chemotherapy against scirrhous gastric cancer (JCOG 0002)

Background  The prognosis of scirrhous gastric cancer remains poor despite extended surgery or adjuvant or neoadjuvant chemotherapy. A pilot study of S-1 (TS-1; Taiho Pharmaceutical, Tokyo, Japan), an oral 5-fluorouracil derivative, for neoadjuvant chemotherapy unexpectedly showed good response and a promising effect on survival. Therefore, the Japan Clinical Oncology Group conducted a phase II trial to confirm the efficacy of S-1 for neoadjuvant chemotherapy against resectable scirrhous gastric cancer. Methods  Patients were eligible if they had typical scirrhous gastric cancer invading more than half of the stomach, and resectable disease confirmed by laparoscopic staging. The treatment schedule consisted of two courses (each, 4-week administration and 2-week withdrawal) of S-1 (100–120 mg/body per day), followed by radical surgery. Results  Fifty-five eligible patients were registered. Three completed only one course of the neoadjuvant chemotherapy, whereas 52 completed two courses. Toxicity was acceptable, with a few grade 3 (5.5%) events, but no grade 4 adverse events. The response rate was 32.6% in 43 evaluable patients. Of the 55 patients, 2 refused operation, 1 developed lung metastasis, and 52 underwent laparotomy. The curative resection rate was 80.8%, with acceptable morbidity and no mortality. The survival curve at 2 years’ follow up showed a better survival rate than that of the historical controls, but did not reach the expected survival rate. Conclusion  S-1 neoadjuvant chemotherapy appeared feasible and showed positive effects against scirrhous gastric cancer; however, the survival rate with S-1 did not reach the expected rate required when selecting an agent for a phase III trial to confirm the effectiveness of neoadjuvant chemotherapy against scirrhous gastric cancer. (on behalf of the Gastric Cancer Surgery Study Group of the Japan Clinical Oncology Group)     

Matrix Metalloproteinase Inhibitor, Marimastat, Decreases Peritoneal Spread of Gastric Carcinoma in Nude Mice

Marimastat, a matrix metalloproteinese inhibitor, was examined for the ability to prevent peritoneal dissemination of a human gastric cancer xenograft, TMK–1. Even with novel approaches such as molecular targeting of cancer chemotherapy, peritoneal dissemination of gastric cancer has little sensitivity to anticancer drugs, and it is impossible to inhibit its growth completely. Intraperitoneal injection of TMK–1 into nude mice at 5 × 10⁵ cells/body resulted in carcinomatous peritonitis that mimicked clinical cases. Continuous administration of marimastat (18 mg/kg/day) from 24 h after the tumor inoculation successfully inhibited the growth of peritoneal dissemination nodules. Combined administration of marimastat (18 mg/kg/day) and mitomycin C (MMC, 2 mg/kg) showed synergistic inhibition of growth of peritoneal dissemination, being superior to MMC alone (2 mg/kg). Although marimastat alone could not increase survival tune with statistical significance, combined administration of marimastat and MMC had a survival benefit with statistical significance. The combination of marimastat and MMC increased the preventive effect on peritoneal dissemination. Marimastat seems to be a candidate for the prevention of peritoneal spread of gastric carcinoma.     

Paracrine activation of MET promotes peritoneal carcinomatosis in scirrhous gastric cancer

Scirrhous gastric cancer is associated with abundant stroma and frequently develops into peritoneal carcinomatosis with malignant ascites. Although malignant ascites is among the most deadly diseases worldwide, its molecular pathogenesis is poorly understood. We investigated the role of hepatocyte growth factor (HGF) in the production of peritoneal carcinomatosis with malignant ascites. We examined three scirrhous and three non‐scirrhous human gastric cancer cell lines for the production of peritoneal carcinomatosis in vivo and responses to HGF in vitro. Furthermore, clinical scirrhous gastric cancer specimens were examined for HGF production. Among the six cell lines examined, only two scirrhous cell lines (NUGC4 and GCIY) produced peritoneal carcinomatosis with massive ascites after intraperitoneal injection in nude mice. Their proliferation was stimulated by exogenous HGF in vitro. On the other hand, a non‐scirrhous cell line, MKN45, with MET amplification generated peritoneal tumors but not ascites. MET tyrosine kinase inhibitors, crizotinib and TAS‐115, inhibited HGF‐stimulated proliferation of NUGC4 and GCIY as well as constitutive proliferation of MKN45. Furthermore, crizotinib and TAS‐115 prolonged the survival of mice bearing established tumors by NUGC4 or MKN45. In clinical specimens, HGF was markedly produced by stromal fibroblasts. Malignant ascitic fluids from patients with peritoneal carcinomatosis contained high levels of HGF. Our results strongly suggest that paracrine HGF‐induced activation of MET‐mediated signaling pathways plays an important role in the pathogenesis of peritoneal carcinomatosis in scirrhous gastric cancer. Thus, MET signaling pathway may be a potential therapeutic target for peritoneal carcinomatosis of gastric cancer, even without MET amplification.     

Role of alpha 2 beta 1- and alpha 3 beta 1-integrin in the peritoneal implantation of scirrhous gastric carcinoma.

We established a highly peritoneal-seeding cell line, OCUM-2MD3, from a poorly peritoneal-seeding cell line, OCUM-2M, of human scirrhous gastric carcinoma. The intraperitoneal inoculation of OCUM-2MD3 cells produced peritoneal dissemination in nude mice, whereas that of OCUM-2M cells did not. We then investigated the correlation between seeding potential and adhesion molecule beta 1-integrins or alpha 6 beta 4-integrin. alpha 2 beta 1- and alpha 3 beta 1-integrin expression on OCUM-2MD3 cells (91.6% and 93.6%) was increased compared with that of OCUM-2M cells (47.8% and 34.3%) by flow cytometric analysis, and the expression level of the other integrins was not different between the two cell lines. The binding ability of OCUM-2MD3 cells to matrigel, fibronectin, laminin and type I collagen was significantly increased, approximately seven times, three times, eight times, and three times greater than that of OCUM-2M cells respectively. The invasiveness of OCUM-2MD3 cells was also significantly increased 8-fold over OCUM-2M cells. The binding and invasive ability of OCUM-2MD3 cells was significantly decreased following the addition of anti-alpha 2 beta 1- and alpha 3 beta 1-integrin antibody, but not by anti-alpha 6 beta 1- and alpha 6 beta 4-integrin antibody. These results suggest that adhesiveness and invasiveness in peritoneal implantation of scirrhous gastric carcinoma might be closely associated with alpha 2 beta 1- and alpha 3 beta 1-integrin.     

Prevention of Hepatic and Peritoneal Metastases by the Angiogenesis Inhibitor FR-118487 after Removal of Growing Tumor in Mice

We established a mouse "primary tumor resection model" in which a transplanted tumor was resected after an orthotopic transplantation of colorectal cancer tissue to estimate the therapeutic effect of an angiogenesis inhibitor on metastasis. The angiogenesis inhibitor FR-118487 is a member of the fumagUlin family. Here, 1 mg/kg/day of FR-118487 was subcutaneously administered to nude mice for 1 week, 2 weeks, or 4 weeks through an osmotic pump. Liver metastasis developed in 7 of 9 control mice, 2 of 6 mice that underwent the tumor resection 2 weeks after transplantation (early resection), and in all 7 of the mice that underwent the tumor resection 4 weeks after transplantation (late resection). In the short treatment trial, the FR-118487 administration immediately after the early resection completely inhibited both hepatic and peritoneal metastases, whereas its administration after the late resection had no effect on liver metastasis. In the prolonged treatment trial, inhibitory effects of prolonged treatment with FR-118487 on both hepatic and peritoneal metastases after the late resection were clearly demonstrated. The mice of the resection-alone group all died within 106 days after tumor inoculation, due to metastases of colon carcinoma. In contrast, half of the mice that underwent resection and then received antiangiogenic therapy were alive at the end of the observation period (160 days after transplantation). In conclusion, the combination of surgery and subsequent antiangiogenic therapy may be useful to prevent the distant metastasis of colorectal cancer and to improve the prognosis of patients with colorectal cancer.     

Novel models for human scirrhous gastric carcinoma in vivo

Human scirrhous gastric carcinoma, a diffusely infiltrating type of poorly differentiated gastric carcinoma also known as linitis plastica type carcinoma, is characterized by cancer cell infiltration and proliferation accompanied with extensive stromal fibrosis. We established two new gastric cancer cell lines, designated OUCM-8 and OCUM-11, which developed the characteristic biology of scirrhous gastric carcinoma upon orthotopic implantation in mice. Involvement of lymph nodes and liver metastasis was also found in both orthotopic models. Histologically, these orthotopic models showed proliferation with extensive fibrosis, resembling human scirrhous gastric cancer. Both cell lines were derived from ascites of patients with scirrhous gastric cancer. The growth of OCUM-8 and OCUM-11 cells following the addition of KGF, FGF, and EGF was increased significantly relative to untreated cells. An increase in the number of attached and spreading cells occurred following the addition of TGF-beta 1 in both cell lines. OCUM-11 cells showed microsatellite instability. Although subcutaneous scirrhous gastric cancer cells show medullary growth, most in vivo studies of scirrhous gastric cancer have used xenografted tumors implanted subcutaneously. Only in a few cases was it confirmed that these scirrhous gastric cancer cell lines retained the original histologic characteristics. Our orthotopic models should contribute to the elucidation of disease progression in situ and to the development of therapy for scirrhous gastric cancer.