Mayu Ohnishi

Expression of platelet‐derived growth factor (PDGF)‐B and PDGF‐receptor β is associated with lymphatic metastasis in human gastric carcinoma

Recent study of murine fibrosarcoma has revealed that platelet‐derived growth factor (PDGF) plays a direct role in promoting lymphangiogenesis and metastatic spread to lymph nodes. Thus, we investigated the relation between PDGF and PDGF receptor (PDGF‐R) expression and lymphatic metastasis in human gastric carcinoma. We examined PDGF‐B and PDGF‐Rβ expression in four human gastric carcinoma cell lines (TMK‐1, MKN‐1, MKN‐45, and KKLS) and in 38 surgical specimens of gastric carcinoma. PDGF‐B and PDGF‐Rβ expression was examined by immunofluorescence in surgical specimens and in human gastric carcinoma cells (TMK‐1) implanted orthotopically in nude mice. Groups of mice (n = 10, each) received saline (control) or PDGF‐R tyrosine kinase inhibitor imatinib. PDGF‐B and PDGF‐Rβ mRNA expression was significantly higher in patients with lymph node metastasis than in those without and was also significantly higher in diffuse‐type carcinoma than in intestinal‐type carcinoma. In surgical specimens, tumor cells expressed PDGF‐B, but PDGF‐Rβ was expressed predominantly by stromal cells. Under culture conditions, expression of PDGF‐B mRNA was found in all of the gastric cell lines, albeit at different levels. In orthotopic TMK‐1 tumors, cancer cells expressed PDGF‐B but not PDGF‐Rβ. PDGF‐Rβ was expressed by stromal cells, including lymphatic endothelial cells. Four weeks of treatment with imatinib significantly decreased the area of lymphatic vessels. Our data indicate that secretion of PDGF‐B by gastric carcinoma cells and expression of PDGF‐Rβ by tumor‐associated stromal cells are associated with lymphatic metastasis. Blockade of PDGF‐R signaling pathways may inhibit lymph node metastasis of gastric carcinoma. (Cancer Sci 2010)

Stroma‐directed imatinib therapy impairs the tumor‐promoting effect of bone marrow‐derived mesenchymal stem cells in an orthotopic transplantation model of colon cancer

Bone marrow‐derived mesenchymal stem cells (MSCs) are reported to contribute to formation of tumor‐promoting stromal cells. We reported recently that, in an orthotopic nude mice model of colon cancer, MSCs traveled to tumor stroma, where they differentiated into carcinoma‐associated fibroblast (CAF)‐like cells. We also found that CAFs express platelet‐derived growth factor receptor (PDGFR) at a high level and that imatinib therapy targeting PDGFR in CAFs inhibits growth and metastasis of human colon cancer. These findings led us to examine whether the tumor‐promoting effect of MSCs is impaired by blockade of PDGFR signaling achieved with imatinib. Orthotopic transplantation and splenic injection of human MSCs along with KM12SM human colon cancer cells, in comparison with transplantation of KM12SM cells alone, resulted in significantly greater promotion of tumor growth and liver metastasis. The KM12SM + MSC xenograft enhanced cell proliferation and angiogenesis and inhibited tumor cell apoptosis. When tumor‐bearing animals were treated with imatinib, there was no significant increase in primary tumor volume or total volume of liver metastases, despite the KM12SM+MSC xenograft, and survival in the mixed‐cell group was prolonged by imatinib treatment. Moreover, the ability of MSCs to migrate to tumor stroma was impaired, and the number of MSCs surviving in the tumor microenvironment was significantly decreased. In in vitro experiments, treatment with imatinib inhibited migration of MSCs. Our data suggest that blockade of PDGF signaling pathways influences the interaction between bone marrow‐derived MSCs and tumor cells in the tumor microenvironment and, hence, inhibits the progressive growth of colon cancer.

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.

Hepatitis C virus infection suppresses the interferon response in the liver of the human hepatocyte chimeric mouse.

Background and Aims Recent studies indicate that hepatitis C virus (HCV) can modulate the expression of various genes including those involved in interferon signaling, and up-regulation of interferon-stimulated genes by HCV was reported to be strongly associated with treatment outcome. To expand our understanding of the molecular mechanism underlying treatment resistance, we analyzed the direct effects of interferon and/or HCV infection under immunodeficient conditions using cDNA microarray analysis of human hepatocyte chimeric mice. Methods Human serum containing HCV genotype 1b was injected into human hepatocyte chimeric mice. IFN-α was administered 8 weeks after inoculation, and 6 hours later human hepatocytes in the mouse livers were collected for microarray analysis. Results HCV infection induced a more than 3-fold change in the expression of 181 genes, especially genes related to Organismal Injury and Abnormalities, such as fibrosis or injury of the liver (P = 5.90E-16 ∼ 3.66E-03). IFN administration induced more than 3-fold up-regulation in the expression of 152 genes. Marked induction was observed in the anti-fibrotic chemokines such as CXCL9, suggesting that IFN treatment might lead not only to HCV eradication but also prevention and repair of liver fibrosis. HCV infection appeared to suppress interferon signaling via significant reduction in interferon-induced gene expression in several genes of the IFN signaling pathway, including Mx1, STAT1, and several members of the CXCL and IFI families (P = 6.0E-12). Genes associated with Antimicrobial Response and Inflammatory Response were also significantly repressed (P = 5.22×10−10 ∼ 1.95×10−2). Conclusions These results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy.

Abstract 2074: Blockade mTOR and PDGF pathways by combination of molecular-targeted drugs inhibited cancer-stromal cell interaction in colon cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells. We have shown previously that platelet-derived growth factor receptors (PDGF-Rs) are overexpressed by various stromal cell populations, including carcinoma-associated fibroblasts and pericytes, but not by cancer cells themselves in human colon cancer tissues. Blockade of PDGF-R signaling pathways in tumor-associated stromal cells has been shown to inhibit the stromal reaction. Activation of phosphoinositide 3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling is frequently observed in many cancer types and has been implicated in the pathogenesis of various diseases. mTOR is a central regulatory kinase that increases the production of proteins that stimulate key cellular processes such as cell proliferation, metabolism, survival, and angiogenesis. However, the effects of mTOR inhibitors on the stromal component of tumor tissues has not been studied. In the present study, the effects of PDGF-R tyrosine kinase inhibitors (nilotinib) and mTOR inhibitor (everolimus) on tumor development and spread, were examined using an orthotopic model of human colon cancer, whereby KM12SM cells were implanted into the cecum of nude mice. Groups of mice (n=7 each) received saline (control), nilotinib (100mg/kg/day) or everolimus (1mg/kg/day), or a combination of nilotinib (100mg/kg/day) and everolimus (1mg/kg/day) by daily oral gavage. After 4 weeks of treatment, tumors and lymph nodes were resected, and specimens were analyzed histologically and immunohistochemically for comparison between the treatment group and control group. After treatment with nilotinib(vs. saline), the stromal reaction was significantly decreased. After treatment with everolimus, the stromal reaction was not decreased, but tumor cell proliferation and microvessel density were decreased, and the proportion of apoptotic tumor cells was increased. With the combination therapy, both tumor cell proliferation and the stromal reaction were decreased and apoptosis of tumor cells was increased, resulting in a remarkable decrease in tumor size and the number of metastatic lymph nodes. Therefore, concurrent inhibition of tumor cells and activated stromal cells by combination therapy using PDGF-R tyrosine kinase inhibitor and mTOR inhibitor may represent a novel molecular-targeted approach for patients with colon cancer. Citation Format: Ryo Yuge, Yasuhiko Kitadai, Kei Shinagawa, Mieko Onoyama, Mayu Ohnishi, Shinji Tanaka, Wataru Yasui, Kazuhisa Chayama. Blockade mTOR and PDGF pathways by combination of molecular-targeted drugs inhibited cancer-stromal cell interaction in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2074. doi:10.1158/1538-7445.AM2013-2074

Abstract 4004: Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces metallothionein gene expression in human colon cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Bone marrow-derived mesenchymal stem cells (MSCs) are reported to contribute to the formation of tumor stroma. We reported recently that in an orthotopic nude mouse model of human colon cancer, MSCs migrated to tumor stroma, where they differentiated into carcinoma-associated fibroblast (CAF)-like cells and promoted tumor growth and metastasis. Furthermore, the migratory and invasive abilities of KM12SM colon cancer cells were shown by Transwell invasion assay to be enhanced by indirect co-culture with MSCs. However, the molecular mechanisms of this phenomenon have not been elucidated. We performed microarray analysis of gene expression in KM12SM cells and found that expression of metallothionein (MT) mRNA increased markedly by indirect co-culture of KM12SM cells with MSCs. Additionally, in an orthotopic nude mouse model of colon cancer, MT mRNA expression levels were shown to be higher in the tumors produced by KM12SM cells mixed with MSCs than in the tumors produced by KM12SM cells alone. MTs comprise a family of low-molecular-weight, cysteine-rich intracellular proteins that are associated with protection against DNA damage, oxidative stress, and apoptosis. The potential role of MTs in carcinogenesis remains unknown. We examined MT expression in surgical specimens of human colon cancer and colon adenoma by immunohistochemistry. A total of 101 tumors were analyzed. MT immunoreactivity was detected in tumor cells and in atypical stromal cells. Atypical stromal cells with strong MT expression were found in invasive cancers, at the invasive edge, but not in mucosal cancers or adenomas. Levels of MT expression in tumor cells correlated inversely with disease stage. These findings suggest that the interaction between MSCs recruited into tumor stroma and the tumor cells of invasive colon cancers induces and depends on MT expression. Further studies are needed to clarify the specific effects of MTs on tumor cells and stromal cells. Citation Format: Kei Shinagawa, Yasuhiko Kitadai, Ryo Yuge, Mieko Onoyama, Mayu Ohnishi, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces metallothionein gene expression in human colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4004. doi:10.1158/1538-7445.AM2013-4004

Abstract 1509: Modulation of stromal reaction by PDGF-R tyrosine kinase inhibitor in an orthotopic mice model of gastric carcinoma

Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells. Many human tumors secrete platelet-derived growth factor (PDGF) ligands, and PDGF receptors (PDGF-Rs) are expressed by various stromal cell populations, including carcinoma-associated fibroblasts (CAFs). We reported recently that, in a mice model of colon cancer, bone marrow-derived mesenchymal stem cells (MSCs) traveled to tumor stroma, where they differentiated into CAF-like cells. In the present study, we examined PDGF-B and PDGF-Rβ expression in relation to clinicopathologic features of human gastric carcinomas. We also examined the effects of PDGF-R tyrosine kinase inhibitors (imatinib, nilotinib) on tumor stroma in an orthotopic nude mouse model of human gastric carcinoma. Five gastric carcinoma cell lines and 38 surgical specimens of gastric carcinoma were used. Under culture conditions, gastric cancer cell lines expressed PDGF-B at various levels but not PDGFR-β. When cells from these lines were implanted orthotopically into the gastric wall of nude mice, cells with high PDGF-B expression resulted in tumors with abundant stroma. In contrast, cells with low PDGF-B expression resulted in medullary tumors with little stromal reaction. In surgical specimens, we found PDGF-B expression to be associated with stromal reaction. PDGF-B and PDGF-Rβ mRNA expression was significantly higher in patients with lymph node metastasis than in those without and also significantly higher in diffuse-type carcinoma than in intestinal-type carcinoma. In surgical specimens and orthotopic tumors, tumor cells expressed PDGF-B, but PDGF-Rβ was expressed predominantly by stromal cells, including CAFs, pericytes, and lymphatic endothelial cells. Treatment with PDGF-R tyrosine kinase inhibitors did not inhibit tumor growth but did significantly decrease the stromal reaction, lymphatic invasion, lymphatic vessel area, and pericyte coverage of tumor microvessels. In addition, tumor tropism of MSCs was also inhibited by treatment with PDGF-R inhibitors in vivo and in vitro. The PDGF/PDGF-R signaling pathway appears to regulate cancer-stromal cell interaction, and target molecule-based inhibition of the stromal reaction may hold promise as an effective anti-tumor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1509. doi:1538-7445.AM2012-1509

Abstract 519: Blockade of PDGFR signaling impairs the tumor promoting effect of bone marrow-derived mesenchymal stem cells in colon cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Bone marrow-derived mesenchymal stem cells (MSCs) are reported to migrate to tumor stroma as well as injured tissue. We reported recently that, in an orthotopic nude mice model of colon cancer, MSCs traveled to tumor stroma, where they differentiated into carcinoma-associated fibroblasts (CAF)-like cells. We have also found that CAFs express platelet-derived growth factor receptor (PDGFR) at a high level and that imatinib therapy targeting PDGFR in CAFs combined with administration of a cytotoxic drug significantly inhibits growth and metastasis of human colon cancer. These findings led us to examine whether the tumor promoting effect of MSCs is impaired by blockade of PDGFR signaling achieved with imatinib. KM12SM colon cancer cells alone or KM12SM cells mixed with MSCs in a 1:2 ratio were transplanted into the cecal wall of nude mice. Orthotopic transplantation of KM12SM cells mixed with MSCs, in comparison to transplantation of KM12SM cells alone, resulted in tumors of greater weight. The survival rate was significantly lower in the mixed-cell group. Co-injection of MSCs with tumor cells promotes tumor growth and metastasis of colon cancer by enhancing angiogenesis and tumor migration and invasion and by inhibiting tumor cell apoptosis. When tumor bearing animals were treated with imatinib (by gavage once daily at 50 mg/kg for 35 days), there was no significant increase in primary tumor volume or number of metastases with the KM12SM+MSC xenograft and the lower survival rate in the mixed-cell group was prolonged by the treatment. Moreover, migratory ability of MSCs to tumor stroma was impaired, and MSCs surviving in the tumor microenvironment were significantly decreased. In in vitro experiments, treatment with imatinib inhibited migration and proliferation of MSCs. Our data suggest that blockade of PDGFR signaling pathways influences the interaction between bone marrow-derived MSCs and tumor cells in the tumor microenvironment and, hence, inhibits the progressive growth of colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 519. doi:10.1158/1538-7445.AM2011-519

Abstract LB-296: Involvement of T-cell immunity in changes in the plasma amino acid profiles of patients with colorectal cancer

Metabolic changes in patients with various disorders, including liver disease and cancer, lead to alterations in amino acid balance, and plasma amino acid profiles can be used to discriminate between non-malnourished patients with cancer and healthy individuals. Not with standing, the mechanisms of amino acid imbalance are not well understood. In patients with cancer, various factors including metabolic changes in the tumor itself and immune responses may affect plasma amino acid levels. To investigate involvement of the immune system, we measured plasma amino acid levels in an orthotopic transplant model of colon cancer established in both conventional BALB/c mice and athymic BALB/c nude mice (T-lymphocyte-deficient mice). CT26 colon cancer cells (murine colon cancer cell line) were implanted into the cecal wall of the BALB/c mice (n=10) and nude mice (n=10). Plasma samples were collected from the tumor-bearing mice at 7 and 14 days after transplantation, and changes in plasma amino acid profiles were compared. Plasma was also collected from mice that underwent sham operation (control mice, n=10). Plasma amino acid concentrations were measured by LC/MS/MS. The plasma concentrations of several amino acids changed significantly in tumor-bearing mice compared to concentrations in control mice. Although cells from the same line were injected into the cecal wall of BALB/c mice and nude mice, a difference was noted in the change in plasma amino acid levels. Plasma Val, Ile, Leu, and Met levels decreased in BALB/c mice but increased in nude mice. There was no difference in the amino acid profile of CT26 tumor tissues between BALB/c mice and nude mice. We then injected antiCD3-antibody into the peritoneal cavity of tumor-bearing BALB/c mice (n=5) to eliminate T-cell function. We also injected IgG into tumor-bearing BALB/c mice (n=5) for control. The pattern of change in amino-acid profiles differed between the two groups of mice. Our findings suggest that T-lymphocyte-mediated immunoreactions are involved in changes in the plasma amino acid profile in patients with colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-296. doi:10.1158/1538-7445.AM2011-LB-296

Abstract 1295: Inhibition of platelet derived growth factor receptor tyrosine kinase reduces lymphangiogenesis of human gastrointestinal cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Objective: Vascular endothelial growth factor (VEGF)-C/D induces lymphangiogenesis by activating VEGF receptor (VEGFR)-3, which is expressed on lymphatic endothelial cells. Platelet derived growth factor receptor (PDGF-R) has also been detected on lymphatic endothelial cells and PDGF-BB acts as a lymphangiogenic factor in murine fibrosarcoma. We have previously reported that PDGF-R is expressed by lymphatic endothelial cells in human gastric cancer and its expression levels in tumor tissue correlate with lymph node metastasis. In this study, we examined whether inhibition of PDGF-R tyrosine kinase signaling by imatinib affects lymphangiogenesis and lymph node metastasis of human gastrointestinal cancer cells growing in the orthotopic site of nude mice. Methods: TMK-1 human gastric cancer cells or KM12SM human colon cancer cells were injected into the orthotopic site of nude mice. Groups of mice (n=10) received saline (control), imatinib, the cancer chemotherapeutic irinotecan, or a combination of imatinib and irinotecan. The lymphatic vessels were then stained with antibodies against Lyve 1. Results: Lymphatic endothelial cells as well as activated fibroblasts in the tumor stroma express PDGF-R. Four weeks of treatment with imatinib and irinotecan significantly inhibited lymph node metastasis (relative to control or single-agent therapy). Imatinib alone or in combination with irinotecan significantly decreased number and area of lymphatic vessels in the tumor stroma. Conclusion: Administration of a PDGF-R tyrosine kinase inhibitor in combination with irinotecan inhibits the lymph node metastasis of orthotopically implanted gastrointestinal cancer cells in nude mice by blocking PDGF-R signaling in tumor-associated lymphatic endothelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1295.