The number of published studies on peritoneal dissemination of scirrhous gastric carcinoma is very small as a result of the unavailability of highly reproducible animal models. Orthotopic implantation of HSC-44PE and HSC-58 (scirrhous gastric carcinoma-derived cell lines) cells into nude mice led to dissemination of the tumor cells to the greater omentum, mesenterium, peritoneum and so on, and caused ascites in a small number of animals. Cycles of isolation of the ascitic tumor cells and orthotopic inoculation of these cells were repeated in turn to animals. This was to isolate highly metastatic cell lines with a strong capability of inducing the formation of ascites (44As3 from HSC-44PE; 58As1 and 58As9 from HSC-58).

CUB-domain-containing protein 1 (CDCP1) is a type-I transmembrane protein that is highly expressed in colon, breast, and lung cancers. We recently revealed that CDCP1 is associated with and phosphorylated by Src family kinases and is involved in the regulation of anchorage independence of certain lung cancer cell lines. In this study, we examined whether CDCP1 is involved in the regulation of tumor progression of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Expression and phosphorylation levels of CDCP1 correlated with the invasive potential of scirrhous gastric cancers. Reduction of CDCP1 expression by siRNA suppressed migration, invasion, and anchorage independence without affecting the proliferation of highly invasive scirrhous gastric cancer cells. However, CDCP1 overexpression promoted gastric cancer cell migration with low potential of invasion. Loss of CDCP1 suppressed invasion and dissemination of cancer cells that were orthotopically implanted in the gastric wall of nude mice. Expression and phosphorylation of CDCP1 were also detected in cancer cells of surgically resected tissues of human scirrhous gastric cancer by immunohistochemical analysis. Our results suggest that CDCP1 promotes invasion and peritoneal dissemination of cancer cells through the regulation of cell migration and anchorage independence. Therefore, it is both a potential prognostic and therapeutic target in certain types of gastrointestinal cancers, and suppression of its phosphorylation might be a useful strategy for modulating cancer metastasis. (Am J Pathol

lthough gastric carcinoma has recently shown a gradual decrease in prevalence, it still accounts for a significant proportion of cancer-related deaths in Japan. To improve the cure rate, more attention should be directed to early detection and prevention of metastasis of this cancer. Scirrhous carcinoma of the stomach, known as diffusely infiltrative carcinoma or Borrmann's type-IV carcinoma, or linitis plastica-type carcinoma, is characterized clinically as having the worst prognosis among the various types of gastric cancer, because it is frequently associated with metastases to lymph nodes and peritoneal dissemination. However, the mechanisms underlying this propensity for metastasis are not yet clearly understood. Therefore, establishment of relevant animal models of metastasis is considered to be extremely important for the elucidation of these mechanisms and establishment of appropriate therapeutic approaches. Transplants of human tumors into nude mice have been used increasingly as experimental systems for this purpose.1) Many human tumors can proliferate when injected s.c. into nude mice, but metastasis from the site of injection is rare.2) It has been found that in most models of human cancers, including gastric carcinoma, i.v. or intrasplenic injection, or orthotopic implantation of the tumor cells is necessary to generate metastasis. [3][4][5][6] There are few reports of spontaneous metastasis from human gastric tumor xenografts in nude mice. 7,8) To date, only one experimental model of signet-ring cell or scirrhous carcinoma of the stomach has been reported. 9) In order to address this problem, we previously established and characterized four cell lines from a primary gastric carcinoma and disseminated metastatic lesions of gastric scirrhous carcinoma.10-12) These cell lines did not exhibit the potential to form experimental or spontaneous metastases when injected s.c. or i.v., or implanted orthotopically into nude mice.We now report the establishment and the biological characterization of new human signet-ring cell gastric carcinoma cell lines that exhibit the ability to metastasize spontaneously in nude mice. Materials and MethodsOrigin and establishment of the cell lines. The HSC-44PE cell line was established from the pleural fluid, obtained by thoracocentesis, of a 28-year-old female Japanese patient with scirrhous gastric carcinoma (linitis plastica-type). The previously reported 12) and the HSC-44PE cells reported in this paper, were derived from the same patient. The former was derived from the ascitic fluid in the early stage of scirrhous gastric carcinoma, while the latter was established by culture of tumor cells collected from the pleural fluid in the terminal stage of the cancer. The HSC-58 cell line was established from the ascitic fluid, obtained by peritoneocentesis, of a 57-year-old male Japanese patient with scirrhous gastric carcinoma. The HSC-60 cell line was established from the ascitic fluid, obtained by peritoneocentesis, of a 40-year-old male patient with scirrhous gastric car...

Although aberrant microRNA (miRNA) is expressed in different types of human cancer tissues, its pathophysiologic role and the relevance of tumorigenesis and metastasis are still largely unknown. Here, we defined miRNAs involved in cancer metastasis (metastamirs) using an established mouse model for peritoneal dissemination of human scirrhous gastric carcinoma cells. Highly metastatic derivatives (44As3 cells) were derived from the parental cells originally isolated from patients (HSC-44PE cells). Using microarray analysis to identify differentially expressed miRNAs in 44As3 and HSC-44PE cells, we focused on miR-516a-3p as a candidate antimetastatic miRNA (antimetastamir) whose functions in cancer had not been studied. We confirmed attenuated expression of miR-516a-3p in 44As3 cells compared with HSC-44PE cells by Northern blot analysis and quantitative reverse transcriptase PCR. Stable ectopic overexpression in 44As3-miR-516a-3p cells permitted identification of sulfatase 1 as a direct target of the miRNA, through use of the isobaric tagging reagent iTRAQ and the QSTAR Elite Hybrid LC-MS/MS system. Sulfatase 1 is known to remove 6-O-sulfates from heparan sulfate proteoglycans on the cell surface, causing release of membrane-bound Wnt ligands from cells. Consistent with this function, Western blot analyses revealed high levels of Wnt3a, Wnt5a, and nuclear b-catenin accumulation in 44As3 cells but relatively reduced levels in 44As3-miR-516a-3p cells. Notably, orthotopic inoculation of nude mice with 44As3-miR-516a-3p cells yielded significantly longer survival periods compared with mice inoculated with control 44As3 cells. Through atelocollagen-mediated delivery of an miR-516a-3p expression vector into orthotopic 44As3 tumors, we documented its feasibility as a treatment agent. Our findings define the miRNA miR-516-3p as an antimetastamir with potential therapeutic applications in blocking metastatic dissemination of gastric cancers. Cancer Res; 71(4); 1442-53. Ó2010 AACR.

We recently established a mouse model of peritoneal dissemination of human gastric carcinoma, including the formation of ascites, by orthotopic transplantation of cultured gastric carcinoma cells. To clarify the processes of expansion of the tumors in this model, nude mice were sacrificed and autopsied at different points of time after the orthotopic transplantation of the cancer cells for macroscopic and histopathologic examination of the tumors. The cancer cells grew actively in the gastric submucosa and invaded the deeper layers to reach the serosal plane. The tumor cells then underwent exfoliation and became free followed by the formation of metastatic lesions initially in the greater omentum and subsequent colonization and proliferation of the tumors on the peritoneum. Although this model allowed the detection of even minute metastases, it was not satisfactory from the viewpoint of quantitative and objective evaluation. To resolve these problems, we introduced a luciferase gene into this tumor cell line with a high metastasizing potential and carried out in vivo photon counting analysis. This photon counting technique was found to allow objective and quantitative evaluation of the progression of peritoneal dissemination on a real-time basis. This animal metastatic model is useful for monitoring the responses of tumors to anticancer agents. (Cancer Res 2006; 66(15): 7532-9)

Epirubicin is widely used to treat various human tumors. However, it is difficult to achieve a sufficient antitumor effect because of dosage limitation to prevent cardiotoxicity. We hypothesized that epirubicin-incorporating micelle would reduce cardiotoxicity and improve the antitumor effect. NC-6300 comprises epirubicin covalently bound to PEG polyaspartate block copolymer through an acid-labile hydrazone bond. The conjugate forms a micellar structure of 40-80 nm in diameter in an aqueous milieu. NC-6300 (10, 15 mg ⁄ kg) and epirubicin (10 mg ⁄ kg) were given i.v. three times to mice bearing s.c. or liver xenograft of human hepatocellular carcinoma Hep3B cells. Cardiotoxicity was evaluated by echocardiography in C57BL ⁄ 6 mice that were given NC-6300 (10 mg ⁄ kg) or epirubicin (10 mg ⁄ kg) in nine doses over 12 weeks. NC-6300 showed a significantly potent antitumor effect against Hep3B s.c. tumors compared with epirubicin. Moreover, NC-6300 also produced a significantly longer survival rate than epirubicin against the liver orthotopic tumor of Hep3B. With respect to cardiotoxicity, epirubicin-treated mice showed significant deteriorations in fractional shortening and ejection fraction. In contrast, cardiac functions of NC-6300 treated mice were no less well maintained than in control mice. This study warrants a clinical evaluation of NC-6300 in patients with hepatocellular carcinoma or other cancers. (Cancer Sci 2013; 104: 920-925) H epatocellular carcinoma (HCC) is the fifth most common cancer and the third largest cause of cancer mortality worldwide.(1,2) The range of available oncological treatment for HCC is sometimes limited due to poor liver function caused by concomitant chronic liver disease, especially liver cirrhosis, which is mainly the result of hepatitis virus infection. Surgical resection is widely considered the mainstay for curative treatment and yields a certain survival rate. However, <20% of patients with HCC can undergo surgical resection. (3,4) With the exception of patients at an early stage and with adequate liver function, recurrence rates after surgical resection are unfortunately high. High recurrence rates are also seen in patients treated by other local treatment options, such as ablation, percutaneous ethanol injection, and trans-arterial chemoembolization.(5) For advanced HCC, the only available option is sorafenib, a tyrosine kinase inhibitor, which was recently approved; however, the survival rate associated with its use is far from satisfactory.

T-cell-mediated immunotherapy with a chimeric antigen receptor (CAR) is expected to become a powerful treatment for cancer. CD38, highly expressed in B-cell nonHodgkin lymphoma (B-NHL) cells, is an attractive target in immunotherapy for B-NHL. We retrovirally transduced a T-cell line, Hut78, expressing little CD38, with an anti-CD38-CAR. Hut78 cells with the anti-CD38-CAR were cocultured with B-NHL cell lines bearing CD38 and also B-NHL cells from patients. Four days later most of the lymphoma cells were killed (the level of cytotoxicity was >95%). By contrast, there was undetectable cytotoxicity against CD38-negative cell lines. Then, we introduced the anti-CD38-CAR into human peripheral T cells. However, the recovery of viable cells was very low, presumably because of an autolytic reaction caused by the association of the anti-CD38-CAR with CD38 on the cell surface. The addition of an anti-CD38 antibody increased the yield of viable transduced T cell probably by blocking the autolytic reaction. We cocultured human peripheral T cells bearing anti-CD38-CAR with B-NHL cells. The median specific cytotoxicity was greater than 90%. These cells were injected 4 times into NOD/SCID mice, which were inoculated with B-NHL cells luciferase. Luciferase activity was not detectable even 30 days after the inoculation in 5 of 6 mice injected. By contrast, it increased in all of the mice injected with the mock vectortransduced T cell. In conclusion, T cell with the anti-CD38-CAR showed powerful cytotoxicity against B-NHL cells in vitro and in vivo. These findings may provide an important clue for improving the methodology of T-cell-mediated immunotherapy.

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