Kei Shinagawa

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.

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.

mTOR and PDGF pathway blockade inhibits liver metastasis of colorectal cancer by modulating the tumor microenvironment.

Tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells, and platelet-derived growth factor receptors (PDGF-Rs) are overexpressed by various stromal cell populations. Activation of PI3K-AKT-mTOR signaling is frequently observed in many cancer types. We investigated whether the mTOR inhibitor everolimus, alone or in combination with the PDGF-R tyrosine kinase inhibitor nilotinib, can inhibit growth and metastasis of human colon cancer. The effects of nilotinib and everolimus on tumor growth and metastasis were examined in an orthotopic mouse model of human colon cancer and a model of liver metastasis. After treatment with nilotinib (versus distilled water), the stromal reaction of xenografts growing in the cecal wall and liver was significantly decreased. After treatment with everolimus, the stromal reaction did not decrease, but tumor cell proliferation and microvessel density decreased. With the two drugs in combination, both stromal reaction and tumor cell proliferation decreased and apoptosis of tumor cells increased, resulting in remarkable inhibition of tumor growth at both the orthotopic and the metastatic site. Concurrent inhibition of tumor cells and activated stromal cells by a PDGF-R tyrosine kinase inhibitor and an mTOR inhibitor used in combination may represent a novel therapeutic approach for colorectal cancer.

Crucial involvement of the CCL3‐CCR5 axis‐mediated fibroblast accumulation in colitis‐associated carcinogenesis in mice

Patients with inflammatory bowel diseases often develop colon carcinoma. Combined treatment of azoxymethane (AOM) and dextran sulfate sodium (DSS) recapitulates colitis‐associated cancer in mice. AOM/DSS‐induced tumor formation was reduced in CCL3‐ or its specific receptor, CCR5‐deficient mice despite the presence of a massive infiltration of inflammatory cells. However, AOM/DSS‐induced type I collagen‐positive fibroblast accumulation in the colon was reduced in CCL3‐ or CCR5‐deficient mice. This was associated with depressed expression of heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), which is expressed mainly by fibroblasts. Moreover in vitro, CCL3 induced fibroblasts to proliferate and to enhance HB‐EGF expression. Furthermore, CCR5 blockade reduced tumor formation together with reduced fibroblast accumulation and HB‐EGF expression, even when administered after the development of multiple colon tumors. Thus, CCL3‐CCR5‐mediated fibroblast accumulation may be required, in addition to leukocyte infiltration, to induce full‐blown colitis‐associated carcinogenesis. Our studies shed light on a therapeutic potential of CCR5 antagonist for patients with colitis‐associated cancer.

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)

Multikinase inhibitor regorafenib inhibits the growth and metastasis of colon cancer with abundant stroma.

Interaction between tumor cells and stromal cells plays an important role in the growth and metastasis of colon cancer. We previously found that carcinoma‐associated fibroblasts (CAFs) expressed platelet‐derived growth factor receptor‐β (PDGFR‐β) and that PDGFR targeted therapy using imatinib or nilotinib inhibited stromal reaction. Bone marrow‐derived mesenchymal stem cells (MSCs) migrate to tumor stroma and differentiate into CAFs. A novel oral multikinase inhibitor regorafenib inhibits receptor tyrosine kinases expressed on stromal cells (vascular endothelial growth factor receptor 1–3, TIE2, PDGFR‐β, and fibroblast growth factors) and tumor cells (c‐KIT, RET, and BRAF). These molecules are involved in tumor growth, angiogenesis, lymphangiogenesis, and stromal activation. Therefore, we examined whether regorafenib impaired the tumor‐promoting effect of CAFs/MSCs. KM12SM human colon cancer cells alone or KM12SM cells with MSCs were transplanted into the cecal wall of nude mice. Co‐implantation of KM12SM cells with MSCs into the cecal wall of nude mice produced tumors with abundant stromal component and promoted tumor growth and lymph node metastasis. Single treatment with regorafenib inhibited tumor growth and metastasis by inhibiting both tumor cells and stromal reaction. This tumor‐inhibitory effect of regorafenib was more obvious in tumors developed by co‐implanting KM12SM cells with MSCs. Our data suggested that targeting of the tumor microenvironment with regorafenib affected tumor cell–MSC interaction, which in turn inhibited the growth and metastasis of colon cancer.

Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Transition in Colon Cancer Cells through Direct Cell-to-Cell Contact

We previously reported that in an orthotopic nude mouse model of human colon cancer, bone marrow–derived mesenchymal stem cells (MSCs) migrated to the tumor stroma and promoted tumor growth and metastasis. Here, we evaluated the proliferation and migration ability of cancer cells cocultured with MSCs to elucidate the mechanism of interaction between cancer cells and MSCs. Proliferation and migration of cancer cells increased following direct coculture with MSCs but not following indirect coculture. Thus, we hypothesized that direct contact between cancer cells and MSCs was important. We performed a microarray analysis of gene expression in KM12SM colon cancer cells directly cocultured with MSCs. Expression of epithelial-mesenchymal transition (EMT)–related genes such as fibronectin (FN), SPARC, and galectin 1 was increased by direct coculture with MSCs. We also confirmed the upregulation of these genes with real-time polymerase chain reaction. Gene expression was not elevated in cancer cells indirectly cocultured with MSCs. Among the EMT-related genes upregulated by direct coculture with MSCs, we examined the immune localization of FN, a well-known EMT marker. In coculture assay in chamber slides, expression of FN was seen only at the edges of cancer clusters where cancer cells directly contacted MSCs. FN expression in cancer cells increased at the tumor periphery and invasive edge in orthotopic nude mouse tumors and human colon cancer tissues. These results suggest that MSCs induce EMT in colon cancer cells via direct cell-to-cell contact and may play an important role in colon cancer metastasis.