Michiyo Kodama

Mesenchymal stem cells enhance growth and metastasis of colon cancer

Recently, mesenchymal stem cells (MSCs) were reported to migrate to tumor stroma as well as injured tissue. We examined the role of human MSCs in tumor stroma using an orthotopic nude mice model of KM12SM colon cancer. In in vivo experiments, systemically injected MSCs migrated to the stroma of orthotopic colon tumors and metastatic liver tumors. Orthotopic transplantation of KM12SM cells mixed with MSCs resulted in greater tumor weight than did transplantation of KM12SM cells alone. The survival rate was significantly lower in the mixed‐cell group, and liver metastasis was seen only in this group. Moreover, tumors resulting from transplantation of mixed cells had a significantly higher proliferating cell nuclear antigen labeling index, significantly greater microvessel area and significantly lower apoptotic index. Splenic injection of KM12SM cells mixed with MSCs, in comparison to splenic injection of KM12SM cells alone, resulted in a significantly greater number of liver metastases. MSCs incorporated into the stroma of primary and metastatic tumors expressed α‐smooth muscle actin and platelet‐derived growth factor receptor‐β as carcinoma‐associated fibroblast (CAF) markers. In in vitro experiments, KM12SM cells recruited MSCs, and MSCs stimulated migration and invasion of tumor cells through the release of soluble factors. Collectively, MSCs migrate and differentiate into CAFs in tumor stroma, and they promote growth and metastasis of colon cancer by enhancing angiogenesis, migration and invasion and by inhibiting apoptosis of tumor cells.

Vascular endothelial growth factor C stimulates progression of human gastric cancer via both autocrine and paracrine mechanisms.

Purpose: Vascular endothelial growth factor (VEGF)-C induces lymphangiogenesis by activating the VEGF receptor (VEGFR)-3, which is expressed by lymphatic endothelial cells. VEGFR-3 has also been detected on several malignant cells, but the significance of VEGFR-3 expression on malignant cells remains unclear. In this study, we examined the expression and function of VEGFR-3 in gastric carcinoma cells. Experimental Design: We examined the expression of VEGFR-3 by four human gastric carcinoma cell lines and in 36 surgical specimens of gastric carcinoma. We also used cDNA microarrays to examine the effect of VEGF-C on gene expression in VEGFR-3-expressing KKLS cells. To stimulate VEGF-C/VEGFR-3 signaling in an autocrine manner, the VEGF-C expression vector was transfected into KKLS cells, and stable transfectants were established. These cells were then transplanted into the gastric walls of nude mice. Results: Two of the four gastric carcinoma cell lines expressed VEGFR-3 mRNA. In 17 of 36 gastric carcinoma specimens, VEGFR-3-specific immunoreactivity was detected on tumor cells. In vitro treatment of KKLS cells with VEGF-C stimulated cell proliferation and increased expression of mRNAs encoding cyclin D1, placental growth factor, and autocrine motility factor. Following inoculation of VEGF-C-transfected and control cells into the gastric walls of nude mice, tumor growth of the VEGF-C-transfected cells was greatly accelerated in comparison with that of control cells. Greater angiogenesis and lymphangiogenesis were also detected in VEGF-C-transfected tumors than in control tumors. Conclusions: Gastric carcinoma cells express VEGF-C and VEGFR-3. VEGF-C may play a role in the progressive growth of human gastric carcinoma through both autocrine and paracrine mechanisms.

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)

Immune Response to CagA Protein is Associated with Improved Platelet Count After Helicobacter pylori Eradication in Patients with Idiopathic Thrombocytopenic Purpura

Background:  Improvement in platelet counts has been reported after eradication of Helicobacter pylori in patients with idiopathic thrombocytopenic purpura (ITP). We examined the levels of serum markers of gastritis and anti‐CagA (cytotoxin‐associated gene A) IgG antibody in patients with ITP to investigate whether these factors are associated with the platelet response after H. pylori eradication therapy.

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.

Antibodies to Helicobacter pylori and CagA protein are associated with the response to antibacterial therapy in patients with H. pylori‐positive API2–MALT1‐negative gastric MALT lymphoma

The aim of this study was to clarify predictive factors for response to eradication therapy in cases of Helicobacter pylori (H. pylori)‐positive API2–MALT1‐negative gastric mucosa‐associated lymphoid tissue (MALT) lymphoma. Sixty‐six patients who were examined for H. pylori infection and the presence of the API2–MALT1 chimeric transcript and who underwent H. pylori eradication therapy as first‐line therapy, were enrolled in this study. Immunohistochemical markers (p53, Ki‐67, and BCL10), microsatellite instability, loss of heterozygosity, serum levels of antibodies (anti‐H. pylori and anti‐CagA), and markers for gastritis (gastrin and pepsinogens) were examined, and the results were compared between patients whose tumors regressed completely after eradication therapy (responders) and patients whose tumors did not regress (non‐responders). Of the 66 patients with localized gastric MALT lymphoma, 47 (71.2%) showed complete remission after eradication therapy. None of the H. pylori‐negative (n = 9) and/or API2–MALT1‐positive (n = 7) patients responded to antibacterial treatment. Of 44 patients with H. pylori‐positive API2–MALT1‐negative gastric MALT lymphoma, 38 (86.4%) showed complete remission after eradication therapy. Titers of antibodies against H. pylori and CagA protein were significantly higher in the responders than in the non‐responders (P = 0.0235 and 0.0089, respectively). No significant difference between the groups was observed for the other factors. In conclusion, measurement of titers of serum antibodies to H. pylori and CagA protein may be useful for predicting the response to eradication therapy in patients with H. pylori‐positive API2–MALT1‐negative gastric MALT lymphoma. (Cancer Sci 2009; 100: 1075–1081)

Potential role for vascular endothelial growth factor-D as an autocrine factor for human gastric carcinoma cells.

Vascular endothelial growth factor (VEGF)‐D induces lymphangiogenesis by activating VEGF receptor (VEGFR)‐3, which is expressed mainly by lymphatic endothelial cells. VEGFR‐3 has also been detected in several types of malignant cells, but the significance of VEGFR‐3 expression by malignant cells remains unclear. We examined the expression and function of VEGF‐D/VEGFR‐3 in human gastric carcinoma cells. Expression of VEGF‐D and VEGFR‐3 was analyzed in three human gastric carcinoma cell lines and 29 surgical specimens. cDNA microarray analysis was used to examine the effect of VEGF‐D on the expression of genes associated with disease progression in VEGFR‐3‐expressing KKLS cells. VEGF‐D‐transfected cells and control cells were transplanted into the gastric wall of nude mice. In 10 of the 29 (34%) gastric carcinoma specimens and two of the three cell lines, cancer cells expressed both VEGF‐D and VEGFR‐3. In vitro treatment of KKLS cells with exogenous VEGF‐D increased expression of cyclin D1 and Bcl‐2 and stimulated cell proliferation. VEGF‐D transfection into KKLS cells resulted in stimulation of angiogenesis, lymphangiogenesis, and cell proliferation, and in inhibition of apoptosis. VEGF‐D may participate in the progression of human gastric carcinoma by acting via autocrine and paracrine mechanisms. (Cancer Sci 2010)

Stroma-Directed Molecular Targeted Therapy in Gastric Cancer

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. Tumor stroma contains abundant extracellular matrix and several types of cells, including carcinoma-associated fibroblasts (CAFs), endothelial cells, pericytes and inflammatory cells including macrophages. In gastric cancer tissues, tumor cells express platelet-derived growth factor (PDGF)-B. Stromal cells, including CAFs, pericytes and lymphatic endothelial cells, express PDGF receptor (PDGFR)-β. Administration of PDGFR tyrosine kinase inhibitor significantly decreases stromal reaction, lymphatic vessel area and pericyte coverage of tumor microvessels. Administration of PDGFR tyrosine kinase inhibitor in combination with cytotoxic chemotherapeutic drug(s) impairs the progressive growth and metastasis of gastric cancer. Activated stroma might serve as a novel therapeutic target in cases of gastric cancer.

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.

Abstract 4255: Significance of mesenchymal stem cells in growth and metastasis of colon cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Recently, mesenchymal stem cells (MSCs) are reported to migrate to tumor sites as well as sites of injury and to incorporate into tumor stroma, but the effects of interactions between MSCs and tumor cells and the mechanisms underlying these effects remain unclear. We examined the role of MSCs in the tumor micro-environment using an orthotopic colon cancer model and a model of liver metastasis from colon cancer. Materials and Methods: Human MSCs labeled with fluorescent dye were injected into tail veins of nude mice bearing KM12SM human colon cancer cells. KM12SM cells alone or KM12SM cells mixed with MSCs were transplanted into the cecal wall or spleen of nude mice. Differentiation of MSCs in primary colon tumors and metastatic liver tumors were also analyzed. Migration assay and invasion assay were performed to examine attraction between KM12SM cells and MSCs in vitro. Results: In in vivo experiments, systemically injected MSCs migrated to the stroma of orthotopic colon tumors and metastatic liver tumors. Orthotopic transplantation of KM12SM cells mixed with MSCs resulted in significantly greater tumor weight than did transplantation of KM12SM cells alone (p < 0.05). The survival rate was significantly lower in the mixed-cell group, and liver metastasis was seen only in this group (p < 0.001). Moreover, tumors resulting from transplantation of mixed cells had a significantly higher proliferating cell nuclear antigen labeling index (p < 0.01), significantly greater microvessel area (p < 0.01), and significantly lower apoptotic index (p < 0.001). Splenic injection of KM12SM cells mixed with MSCs resulted in a significantly greater number of liver metastases than did splenic injection of KM12SM cells alone (p < 0.01). MSCs incorporated into the stroma of primary and metastatic tumors expressed α-smooth muscle actin and platelet-derived growth factor receptor-β as carcinoma-associated fibroblast (CAF) markers. In in vitro experiments, KM12SM cells recruited MSCs, and MSCs stimulated migration and invasion of tumor cells through the release of soluble factors (p < 0.05, respectively). Conclusion: MSCs migrate and differentiate into CAFs in tumor stroma, and they promote growth and metastasis of colon cancer by enhancing angiogenesis and by inhibiting apoptosis of tumor 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 4255.