Misato Takigahira

Development and biological analysis of peritoneal metastasis mouse models for human scirrhous stomach cancer

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). All three cell lines induced tumor formation at the site of orthotopic injection, and caused fatal cancerous peritonitis and bloody ascites in 90–100% of the animals approximately 3–5 weeks after the inoculation. When the parent cells were implanted, the animals became moribund in approximately 12–18 weeks, however, none of the animals developed ascites. Complementary DNA microarray and immunohistochemical analyses revealed differences in the expression levels of genes coding for the matrix proteinase, cell adhesion, motility, angiogenesis and proliferation between the highly metastatic‐ and parent‐cell lines. The usefulness of this model for the evaluation of drugs was assessed by analyzing the stability of the metastatic potential of the cells and the reproducibility. Animals intravenously treated with CPT‐11 and GEM showed suppressed tumor growth and significantly prolonged survival. The metastatic cell lines and the in vivo model established in the present study are expected to serve as a model of cancerous peritonitis developing from primary lesions, and as a useful means of clarifying the pathophysiology of peritoneal dissemination of scirrhous gastric carcinoma and the development of drugs for its treatment. (Cancer Sci 2005; 96: 323–332)

Establishment of two cell lines from human gastric scirrhous carcinoma that possess the potential to metastasize spontaneously in nude mice

Few experimental studies have been conducted to clarify the mechanism of development of metastasis in scirrhous carcinoma of the stomach. In the present study, we attempted to establish gastric carcinoma cell lines by incubation of cancer cells collected from the body fluids of patients with gastric cancer. At the same time, xenografting of these cells to nude mice was performed. It was found that, of the gastric carcinoma cell lines thus established, two cell lines, designated as HSC‐44PE and HSC‐58, formed s.c. tumors with a high infiltrative potential (often invading the lymphatics around the cancer tissue) when implanted. Metastasis to the lymph nodes and lungs was observed in 20–40% of all the animals, indicating that the two cell lines are also capable of metastasizing spontaneously. Through repeated selection, i.e., repeated cycles of removal, culture, and implantation of the HSC cancer cells from metastatic lesions, we obtained 5 subclones of HSC‐44PE and HSC‐58 (designated as m2509, m2615, m2792, m2917, and m2691), which, when implanted orthotopically, exhibited the following characteristics as compared to the parent cells: (1) a higher percentage take (survival), similar frequency of metastasis, shorter time to metastasis (less than 100 days), and consistent metastasizing potential; (2) a relatively high frequency of metastasis to lymph nodes, including distant metastasis to axillary lymph nodes; (3) the potential to cause occasional bloody ascites; (4) enhanced expression of dysadherin, CD44, and other molecules. This is the first report of cultured scirrhous gastric carcinoma cells showing the potential for spontaneous metastasis.

CUB-Domain-Containing Protein 1 Regulates Peritoneal Dissemination of Gastric Scirrhous Carcinoma

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.

The Metastasis-Associated microRNA miR-516a-3p Is a Novel Therapeutic Target for Inhibiting Peritoneal Dissemination of Human Scirrhous Gastric Cancer

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 β-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.

NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin

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.

Antitumor Effect of SN-38–Releasing Polymeric Micelles, NK012, on Spontaneous Peritoneal Metastases from Orthotopic Gastric Cancer in Mice Compared with Irinotecan

7-Ethyl-10-hydroxy-camptothecin (SN-38), an active metabolite of irinotecan hydrochloride (CPT-11), has potent antitumor activity. Moreover, we have reported the strong antitumor activity of NK012 (i.e., SN-38-releasing polymeric micelles) against human cancer xenografts compared with CPT-11. Here, we investigated the advantages of NK012 over CPT-11 treatment in mouse models of gastric cancer with peritoneal dissemination. NK012 or CPT-11 was i.v. administered thrice every 4 days at their respective maximum tolerable doses (NK012, 30 mg/kg/day; CPT-11, 67 mg/kg/day) to mice receiving orthotopic transplants of gastric cancer cell lines (44As3Luc and 58As1mLuc) transfected with the luciferase gene (n = 5). Antitumor effect was evaluated using the photon counting technique. SN-38 concentration in gastric tumors and peritoneal nodules was examined by high-performance liquid chromatography (HPLC) 1, 24, and 72 hours after each drug injection. NK012 or CPT-11 distribution in these tumors was evaluated using a fluorescence microscope on the same schedule. In both models, the antitumor activity of NK012 was superior to that of CPT-11. High concentrations of SN-38 released from NK012 were detected in gastric tumors and peritoneal nodules up to 72 hours by HPLC. Only a slight conversion from CPT-11 to SN-38 was observed from 1 to 24 hours. Fluorescence originating from NK012 was detected up to 72 hours, whereas that from CPT-11 disappeared until 24 hours. NK012 also showed antitumor activity against peritoneal nodules. Thus, NK012 showing enhanced distribution with prolonged SN-38 release may be ideal for cancer treatment because the antitumor activity of SN-38 is time dependent.

ZD6474 inhibits tumor growth and intraperitoneal dissemination in a highly metastatic orthotopic gastric cancer model

Angiogenesis inhibitors have been used to treat some cancers, but the therapeutic potential of these agents for gastric cancer has remained unclear. To investigate their therapeutic potential, we examined the effect of ZD6474, an agent that selectively targets vascular endothelial growth factor receptor‐2 (VEGFR‐2; KDR) tyrosine kinase and epidermal growth factor receptor (EGFR) tyrosine kinase, in a highly metastatic orthotopic model using an undifferentiated gastric cancer cell line, 58As1. ZD6474 (100 mg/kg/day, p.o., 2 weeks) significantly inhibited tumor growth (p < 0.05 vs. control) and reduced tumor dissemination into the peritoneal cavity (p < 0.05 vs. control). In addition, to identify putative tumor biomarkers that would reflect the effects of ZD6474 treatment in clinical settings, we examined the gene expression profiles of implanted gastric tumors treated with ZD6474 in vivo. Twenty‐eight candidate genes were identified, including IGFBP‐3, ADM, ANGPTL4, PLOD2, DSIPI, NDRG1, ENO2, HIG2 and BNIP3L, which are known to be hypoxia‐inducible genes. These genes and gene products may be useful biomarkers for monitoring the effects of ZD6474 treatment. ZD6474 also improved the survival of mice with implanted another undifferentiated gastric cancer cell line, 44As3. In conclusion, our results suggest that ZD6474 may have clinical activity against gastric cancer, particularly undifferentiated gastric cancer with peritoneal dissemination. We also identified putative biomarkers for monitoring the pharmacodynamic effects of ZD6474 by gene expression profiling.

A Photon Counting Technique for Quantitatively Evaluating Progression of Peritoneal Tumor Dissemination

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.

The significance of microscopic mass spectrometry with high resolution in the visualisation of drug distribution

The visualisation and quantitative analysis of the native drug distribution in a pre-clinical or clinical setting are desirable for evaluating drug effects and optimising drug design. Here, using matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-IMS) with enhanced resolution and sensitivity, we compared the distribution of a paclitaxel (PTX)-incorporating micelle (NK105) with that of PTX alone after injection into tumour-bearing mice. We demonstrated optically and quantitatively that NK105 delivered more PTX to the tumour, including the centre of the tumour, while delivering less PTX to normal neural tissue, compared with injection with PTX alone. NK105 treatment yielded a greater antitumour effect and less neural toxicity in mice than did PTX treatment. The use of high-resolution MALDI-IMS may be an innovative approach for pharmacological evaluation and drug design support.

Synergistic and persistent effect of T-cell immunotherapy with anti-CD19 or anti-CD38 chimeric receptor in conjunction with rituximab on B-cell non-Hodgkin lymphoma

Using artificial receptors, it is possible to redirect the specificity of immune cells to tumour‐associated antigens, which is expected to provide a useful strategy for cancer immunotherapy. Given that B‐cell non‐Hodgkin lymphoma (B‐NHL) cells invariably express CD19 and CD38, these antigens may be suitable molecular candidates for such immunotherapy. We transduced human peripheral T cells or a T‐cell line with either anti‐CD19‐chimeric receptor (CAR) or anti‐CD38‐CAR, which contained an anti‐CD19 or anti‐CD38 antibody‐derived single‐chain variable domain respectively. Retroviral transduction led to anti‐CD19‐CAR or anti‐CD38‐CAR expression in T cells with high efficiency (>60%). The T cell line, Hut78, when transduced with anti‐CD19‐CAR or anti‐CD38‐CAR, exerted strong cytotoxicity against the B‐NHL cell lines, HT and RL, and lymphoma cells isolated from patients. Interestingly, use of both CARs had an additive cytotoxic effect on HT cells in vitro. In conjunction with rituximab, human peripheral T cells expressing either anti‐CD19‐CAR or anti‐CD38‐CAR enhanced cytotoxicity against HT‐luciferase cells in xenografted mice. Moreover, the synergistic tumour‐suppressing activity was persistent in vivo for over 2 months. These results provide a powerful rationale for clinical testing of the combination of rituximab with autologous T cells carrying either CAR on aggressive or relapsed B‐NHLs.