Scirrhous Gastric Cancer Cells Research Articles

Dipeptidyl Peptidase-4 from Cancer-associated Fibroblasts Stimulates the Proliferation of Scirrhous-type Gastric Cancer Cells.

Cancer-associated fibroblasts (CAFs) may promote the malignancy of human scirrhous gastric cancer (SGC) cells. We conducted the present study to identify novel growth factors from CAFs. OCUM-12 and 2 CAF cell lines were used. The proliferation of cancer cells was determined by the number of cancer cells or the MTT assay. The growth factor(s) were purified and characterized by the gel filtration chromatography and protein array. The molecular weight of the growth-stimulating factor was estimated to be approximately 66-669 kDa. Protein array of conditioned medium (CM) from CAFs indicated that dipeptidyl peptidase-4 (DPP-4) was one of the growth factors. The addition of CM increased the phosphorylation of C-X-C chemokine receptor 4 (CXCR4). The DPP-4 inhibitor significantly inhibited the growth-stimulating activity of CM. DPP-4 from CAFs might be one of the growth-stimulating factors for SGC through CXCR4.

Abstract 2045: A factor (> MR 30 kDa) from cancer-associated fibroblasts may stimulate the progression of schirrous gastric cancer without FGFR2 overexpression

Abstract Purpose: Scirrhous gastric cancer (SGC), diffusely infiltrating carcinoma, proliferate rapidly with fibrosis, when the cancer cells invade into the submucosa of the stomach. It has been reported that the interactions between the cancer cells and stromal cells, such as cancer-associated fibroblasts (CAFs), surrounding them advance the proliferation and invasion of some types of solid carcinomas. We previously clarified that fibroblast growth factor-7 (FGF7) produced by CAFs promotes the proliferation and tumor progression of SGC. However, the FGF receptor type 2 (FGFR2), which is a specific receptor of FGF7, is just expressed on only 20% of SGC, suggesting that about 80% of growth-stimulating signaling by the interaction between the SGC cells and CAFs might be different from FGF7/FGFR2 signaling. Materials and Methods: In this study, we aim to clarify the interaction between SGC cells and CAFs except FGF7/FGFR2 signaling in the tumor progression of SGC. We used 4 SGC cell lines, OCUM-1, OCUM-8, OCUM-9, and OCUM-12, which do not overexpress FGFR2. Five CAF cell lines, CAF82, CAF88, CAF90, CAF91, and CAF92, were established by a primary culture from individual gastric carcinoma specimens. Serum-free conditioned medium (SF-CM) from CAFs was added to each SGC cell lines and evaluate them. The interaction of SGC and SF-CM was analyzed by CCK assay and the wound healing assay. The humoral factors in SF-CM which stimulate the proliferation of SGC is divided according to the size of the molecular weight and evaluated by the CCK assay. Results: CCK assay revealed that CAF82 SF-CM promoted the proliferation of OCUM-9 and OCUM-12 by each 27 % and 20 %. CAF88 SF-CM promoted the proliferation of OCUM-9 by 17 %. CAF90 SF-CM promoted the proliferation of OCUM-1 and OCUM-9 by each 33 % and 21 %. CAF91 SF-CM promoted the proliferation of OCUM-1 and OCUM-9 by each 29 % and 25 %. CAF92 SF-CM promoted the proliferation of OCUM-1 and OCUM-9 by each 37% and 5 %. According to the wound healing assay, the invasion activity of OCUM-12 was promoted by SF-CM from CAF82, CAF90, CAF91, and CAF92 by each 56%, 37%, 34%, and 52%. CAF82 SF-CM which contain humoral factors that molecular weight is more than 30kDa promote the proliferation of OCUM-12 by 16%. Conclusion: The factors over 30kDa from CAF might stimulate the progression of SGC cells without FGFR2 expression. Citation Format: Syuhei Kushiyama, Masakazu Yashiro, Tomohisa Okuno, Kenji Kuroda, Ryota Tanaka, Shingo Togano, Sadaaki Nishimura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. A factor (> MR 30 kDa) from cancer-associated fibroblasts may stimulate the progression of schirrous gastric cancer without FGFR2 overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2045.

Abstract 5160: Extracellular vesicles from scirrhous gastric cancer cells induce premetastatic niche formation for peritoneal metastasis

Abstract Background: Human scirrhous gastric carcinoma (SGC) is also known as diffusely infiltrating carcinoma, type 4 on Borrmann’s criteria, and linitis plastica-type carcinoma. SGC is characterized by frequent peritoneal metastasis, high proliferation and infiltration activity with extensive fibrosis in the tumor stroma. We previously reported that a soluble factor(s) from SGC cells changed the peritoneum to pre-metastatic niche such as round shape of mesothelial cells and peritoneal fibrosis, which is a favorable environment for cancer cells to metastasize. Extracellular vesicles (EVs; Exosomes) might be one of soluble factor(s) which might change the peritoneal component to pre-metastatic niche. In this study, we investigated the effect of EVs from SGC cells on the pre-metastatic niche formation of the peritoneum. Methods: Four SGC cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, 2 non-SGC cell lines, MKN74, MKN45, and peritoneal mesothelial cells (PM cells) were used. The effect of EVs from gastric cancer cells on the peritoneum was examined in vitro and in vivo, as follows. PKH26-labeled EVs from OCUM-2MD3 cells were intravenously injected into nude mice for 3 weeks, then we examined the distribution of PKH26-labeled EVs by a fluorescence microscope. Next, we injected EVs intraperitoneally into nude mice, and investigated the morphology of peritoneum. Effect of EVs on the gene expression of PM cells was examined by RT-PCR. Clinical significance of EVs markers, CD9 and CD63, was evaluated by immunohistochemistry using 63 human gastric cancer specimens. Results: PKH26-labeled EVs frequently distributed to peritoneum and the liver. The morphology of PM cells changed from round shape to spindle shape by EVs addiction in vitro and in vivo. mRNA expression level of ZEB and N-cadherin of PM cells was significantly increased following the addiction of EVs. The high expression of EVs markers, CD9 and CD63, was significantly correlated with frequent peritoneal metastasis. Conclusion: EVs from SGC cells might frequently distribute to mesothelial cells of peritoneum. EVs from SGC cells might play an important role for the formation of a pre-metastatic niche on peritoneum by the morphologic change of peritoneal mesothelial cells. Citation Format: Tomohisa Okuno, Masakazu Yashiro, Shuhei Kushiyama, Sadaaki Nishimura, Shingo Togano, Kenji Kuroda, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. Extracellular vesicles from scirrhous gastric cancer cells induce premetastatic niche formation for peritoneal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5160.

Exosomes from scirrhous gastric cancer cells might affect on the morphology of peritoneal mesothelial cells and induce peritoneal metastasis

Exosomes from scirrhous gastric cancer cells might affect on the morphology of peritoneal mesothelial cells and induce peritoneal metastasis

Abstract 4807: Novel theranostic strategy against peritoneal metastasis of scirrhous gastric cancer: Combination with fluorescence oncolytic adenovirus and chemotherapy

Abstract Background: Scirrhous gastric cancer (SGC), which is characterized by poorly differentiated tumor cells that diffusely infiltrate the gastric wall, accounts for 10% of all gastric cancers, and often causes peritoneal metastasis. The prognosis of SGC patients is very poor, because no effective treatment to overcome peritoneal metastasis of SGC has been developed yet. In recent years, intraperitoneal (i.p.) administration of paclitaxel (PTX) has been focused against various cancers with peritoneal metastasis and has been also attempted to SGC, but regrettably its effect is not satisfactory. Therefore, a novel therapeutic strategy is required for the treatment of SGC with peritoneal metastasis. Methods: We previously developed a telomerase-specific replication-competent adenovirus, OBP-401 (TelomeScan), which can replicate only within the tumor cells selectively and express green fluorescent protein (GFP). OBP-401 infection also induces tumor-specific cell death in monotherapy and combination therapy with chemotherapy. In this study, we assessed the in vitro and in vivo antitumor effects of combination therapy with OBP-401 and chemotherapeutic agent, paclitaxel (PTX), in human SGC cell lines (GCIY and KATO III) and SGC xenograft peritoneal metastasis model, respectively. The molecular mechanism underlying synergistic effect of the combination therapy was evaluated in aspects of cell death and replication efficiency of OBP-401. Results: SGC cells were visualized as GFP-positive cells selectively by the infection of OBP-401, whereas normal fibroblast cells were not. OBP-401 synergistically suppressed the viability of human SGC cells in combination with PTX. OBP-401 monotherapy dose-dependently induced apoptosis and autophagy, although PTX monotherapy induced only apoptosis. The combination therapy induced autophagy at a lower concentration of OBP-401. In the time-lapse imaging of treatment, the combination therapy enhanced the replication efficiency of OBP-401 in tumor cells. Moreover, the combination therapy with i.p. administration of OBP-401 and PTX also significantly inhibited the tumor growth of peritoneal metastasis, as compared to monotherapy. Conclusion: These results suggest that OBP-401 has a promising potential to detect peritoneal micrometastasis of SGC intraoperatively and the combination therapy with i.p. induction of OBP-401 and PTX would be a novel theranostic strategy for the treatment of SGC with peritoneal metastasis. Citation Format: Wataru Ishikawa, Satoru Kikuchi, Hiroshi Tazawa, Toshiaki Ohara, Shinji Kuroda, Kazuhiro Noma, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Novel theranostic strategy against peritoneal metastasis of scirrhous gastric cancer: Combination with fluorescence oncolytic adenovirus and chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4807.

The significance of scirrhous gastric cancer cell lines: the molecular characterization using cell lines and mouse models.

Scirrhous gastric cancer (SGC) exhibits aggressiveness of the rapid infiltrating tumor cells with abundant fibroblasts. Experimental studies using SGC cell lines have obtained useful information about this cancer. Our literature search divulged a total of 18 SGC cell lines; two cell lines were established from primary SGC and the other lines were established from a metastatic lesion of SGC. Fibroblast growth factor receptor 2 (FGFR2) and transforming growth factor-beta receptor (TβR) are linked to the rapid development of SGC. Cross-talk between the cancer cells and cancer-associated fibroblasts (CAFs) has been shown to contribute to the progression of SGC. Chemokine (C-X-C motif) receptor 1 (CXCR1) from SGC cells might be associated with the abundant CAFs in cancer microenvironments. The in vivo models established using SGC cell lines are expected to serve as a useful tool for the development of drugs such as FGFR2 inhibitors, TβR inhibitors, and CXCR1 inhibitors, which might be promising as SGC treatments. However, the number of available SGC cell lines is insufficient for the clarification of the entire biologic behavior of SGC. Since the mechanisms responsible for the characteristic aggressiveness of SGC are not fully elucidated, the establishment of new SGC cell lines could help clarify the biological behavior of SGC and contribute to its treatment.

Hypoxia-induced ANGPTL4 sustains tumour growth and anoikis resistance through different mechanisms in scirrhous gastric cancer cell lines

Patients with scirrhous gastric cancer (SGC) frequently develop peritoneal dissemination, which leads to poor prognosis. The secreted protein angiopoietin-like-4 (ANGPTL4), which is induced by hypoxia, exerts diverse effects on cancer progression. Here, we aimed to determine the biological function of ANGPTL4 in SGC cells under hypoxia. ANGPTL4 levels were higher in SGC cells under hypoxia than in other types of gastric cancer cells. Hypoxia-induced ANGPTL4 mRNA expression was regulated by hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, monolayer cultures of ANGPTL4 knockdown (KD) 58As9 SGC (58As9-KD) cells were arrested in the G1 phase of the cell cycle through downregulation of c-Myc and upregulation of p27, in contrast to control 58As9-SC cells. Moreover, the ability of 58As9-KD xenografts to form tumours in nude mice was strongly suppressed. When 58As9-KD cells were cultured in suspension, hypoxia strongly increased their susceptibility to anoikis through suppression of the FAK/Src/PI3K-Akt/ERK pro-survival pathway, followed by activation of the apoptotic factors caspases-3, -8 and -9. The development of peritoneal dissemination by 58As9-KD cells was completely inhibited compared with that by 58As9-SC cells. In conclusion, ANGPTL4 is uniquely induced by hypoxia in cultured SGC cells and is essential for tumour growth and resistance to anoikis through different mechanisms.

Abstract 1103: Novel theranostic strategy against scirrhous gastric cancer; combination of chemotherapy and fluorescence oncolytic adenovirus

Abstract Background: Gastric cancer is one of the most common cancers worldwide, especially in Eastern Asia. Scirrhous gastric cancer (SGC), which is characterized by poorly differentiated tumor cells that diffusely infiltrate the gastric wall, accounts for 10% of all gastric cancers, and often causes peritoneal metastasis. Peritoneal carcinomatosis shows the worst prognosis among various metastatic patterns. Since it is difficult to detect peritoneal micrometastasis preoperatively or intraoperatively, SGC patients with peritoneal micrometastasis show the recurrence and refractory to conventional systemic chemotherapy after surgery. In recent year, intraperitoneal (i.p.) administration of paclitaxel (PTX) has been shown to have therapeutic effects against ovarian cancer with peritoneal metastasis. However, the effective treatment option to overcome peritoneal metastasis of SGC has not been developed yet, although i.p. administration of some chemotherapeutic agents has been attempted to SGC. Therefore, a novel therapeutic strategy is required for the treatment of peritoneal metastasis of SGC. Methods: We previously developed a telomerase-dependent replication-selective adenovirus OBP-401 (TelomeScan), which can replicate within the tumor cells selectively and express green fluorescent protein (GFP). Moreover, OBP-401 infection also induces tumor-specific cell death in monotherapy or combination therapy with chemotherapy. In this study, we assessed synergistic effects in combination with novel theranostic agent OBP-401 and chemotherapeutic agent paclitaxel (PTX) using human SGC cell lines (GCIY and KATO III). In vitro experiment, SGC cells and normal human lung fibroblast (NHLF) cells were infected with OBP-401 at various doses. PTX was administrated after 48 hours of virus infection. Twenty-four hours after PTX administration, we evaluated the anti-tumor effect by XTT assay, and analyzed the synergistic effect by CalcuSyn Software. Results: SGC cells were visualized as GFP-positive cells selectively by the infection of OBP-401, whereas NHLF cells were not visualized with OBP-401. In both SGC cell lines, the suppression of cell viability dose-dependently by administration of OBP-401 or PTX alone was confirmed. OBP-401 synergistically suppressed the viability of SGC cells in combination with PTX as compared to monotherapy. Conclusions: These results suggest that OBP-401 has a promising potential to detect peritoneal micrometastasis of SGC intraoperatively and combination of OBP-401 and PTX would be a novel theranostic strategy for the treatment of peritoneal metastasis of SGC. Citation Format: Wataru Ishikawa, Satoru Kikuchi, Hiroshi Tazawa, Shinji Kuroda, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Toshiyoshi Fujiwara. Novel theranostic strategy against scirrhous gastric cancer; combination of chemotherapy and fluorescence oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1103. doi:10.1158/1538-7445.AM2017-1103

Aminopeptidase N (APN/CD13) as a target molecule for scirrhous gastric cancer

SummaryBackgroundScirrhous gastric cancer is associated with peritoneal dissemination and advanced lymph node metastasis from an early stage, and the prognosis is still poor. In this study, we aimed to analyze candidate molecules for targeted therapy of scirrhous gastric cancer. We searched for molecules/metabolic activity that might be predominantly expressed in a subpopulation of scirrhous gastric cancer cells and might function as cancer stem cell markers.ResultsFor this purpose, we investigated the expression of various cell surface markers and of aldehyde dehydrogenase (ALDH) activity. These analyses showed that the scirrhous gastric cancer cell lines HSC-58 and HSC-44PE heterogeneously expressed CD13, while CD44, CDCP1, EpCAM and ABCG2 were expressed uniformly. Moreover, 10% of the total HSC-58 cell population expressed ALDH enzyme activity. A subpopulation of cells strongly positive for ALDH also expressed high levels of CD13, both of which are known as cancer stem cell markers. HSC-58 cells expressing high levels of CD13 showed lower sensitivity to a cancer drug cisplatin than cells with low levels of CD13. In contrast, CD13-high subpopulation of HSC-58 was more sensitive to an aminopeptidase N inhibitor bestatin. In terms of antibody-drug therapy, anti-CD13-immunotoxin was highly cytotoxic towards HSC-58 cells and was more cytotoxic than anti-EpCAM-immunotoxin.ConclusionThese data suggest that CD13 is a suitable cell surface candidate for targeted antibody-drug therapy of scirrhous gastric cancer.

Impacts of Cancer-Associated Stromal Cells on Growth of Human Gastric Cancer Cell Lines

Scirrhous gastric cancer (SGC) is highly resistant to treatment and has a poor prognosis. Tumor stromal cells are considered to play vital roles in the development of SGC. However, interactions between cancer cells and stromal cells are poorly understood. We evaluated the importance of direct contact between cancer cells and stromal cells in vitro . We obtained gastric cancer-associated stromal cells (GCSCs) and normal gastric stromal cells (GSCs) from six patients. We co-cultured these cells with MKN45 solid-type gastric cancer and HSC43 SGC cells, respectively, for 7 days. Both gastric cancer cell lines were labeled with fluorescent protein to distinguish them from stromal cells in culture. We used two co-culture models: direct contact (GCSCs and GSCs), in which cancer cells and stromal cells were mixed, and indirect contact (GCSCs only), in which cells were separated by cell culture inserts. We also compared the gene expression profiles of GCSCs and GSCs by microarray assays. The cell growth rate was significantly higher in the direct-contact GCSC model compared with that in monocultured cells in MKN45, but not in HSC43 cells. For either cell line, there was no significant difference in growth rates between indirect GCSC co-cultures and monocultures, and no difference between direct or indirect GSC co-cultures and monocultures. In terms of gene expression, fibroblast growth factor 9, which has an anti-apoptotic effect on gastric cancer cells, was more up-regulated in GCSCs than in GSCs. Flow cytometry revealed no difference between GCSCs and GSCs in terms of marker expression. In conclusion, although stromal cells can influence the growth of gastric cancer cells, our results suggest that the impacts of interactions with stromal cells might vary according to the specific characteristics of the cancer cells.

Abstract 5338: Preclinical study of telomerase-specific p53 tumor suppressor gene overexpression in human scirrhous gastric cancer cells with different p53 status

Abstract Background: Despite multimodal therapy for scirrhous gastric cancer, many patients show refractory to conventional therapy and rapid progression, leading to poor prognosis. Therefore, the development of novel therapeutic strategy is required to improve the clinical outcome in patients with scirrhous gastric cancers. As novel therapeutic strategies, Ad-p53, a replication-defective adenovirus expressing a tumor suppressor p53 gene, is currently under clinical evaluation for various types of cancers. We recently developed a novel p53-expressing telomerase-specific replication-competent oncolytic adenovirus OBP-702, which induces the p53 gene expression in a telomerase-dependent manner. In this study, we investigated the antitumor effects of Ad-p53 and OBP-702 in human scirrhous gastric cancer cells with different p53 status. Methods: We used 3 human scirrhous gastric cancer cell lines with different p53 status, NUGC4 (p53 wild-type), GCIY (p53 mutant-type), and KATOIII (p53 null). The antitumor effects of Ad-p53 and OBP-702 were evaluated using XTT assay. The 50% inhibiting dose (ID50) value of Ad-p53 and OBP-702 for each cell was calculated using cell viability data obtained 3 days after virus infection. We evaluated the virus-mediated cell death in the sphere-forming cells by LIVE/DEAD assay. The apoptosis- and autophagy-related cell death was assessed by western blot analysis for cleaved poly (ADP-ribose) polymerase (PARP) and p62. Results: Ad-p53 suppressed the cell viability in p53-inactivated scirrhous gastric cancer cells (KATO III, GCIY), whereas p53-intact NUGC4 cells were resistant to Ad-p53. OBP-702 efficiently suppressed the cell viability in all scirrhous gastric cancer cells with different p53 function. The ID50 value of OBP-702 was lower than that of Ad-p53 in all cell lines. LIVE/DEAD assay showed that OBP-702 induced cell death more efficiently than Ad-p53 in the sphere-forming cells. OBP-702 induced more profound p53 expression and apoptotic cell death than Ad-p53. Moreover, only OBP-702 induced not only apoptosis but also autophagy. Conclusions: These results suggest that p53-expressing tumor-specific oncolytic adenovirus OBP-702 is a promising antitumor agent to induce profound cell death in scirrhous gastric cancers with different p53 status. Now, in vivo experiments are under way to investigate the antitumor effect of OBP-702 in the peritoneal dissemination of scirrhous gastric cancer cells. Citation Format: Naoto Hori, Hiroshi Tazawa, Masahiko Nishizaki, Satoru Kikuchi, Shuya Yano, Michihiro Ishida, Megumi Watanabe, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Preclinical study of telomerase-specific p53 tumor suppressor gene overexpression in human scirrhous gastric cancer cells with different p53 status. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5338. doi:10.1158/1538-7445.AM2015-5338

Abstract 2368: Differentiated gastric cancer cells have a potential to induce cancer-associated fibroblasts

Abstract Gastric cancer is one of the most common causes of cancer death. Stromal fibroblasts in the tumor microenvironment have recently been implicated in tumor growth, invasion and metastasis. Cancer-associated fibroblasts (CAFs) have been suggested to be responsible for progression of undifferentiated scirrhous gastric cancers with vast fibrous stroma. However, the relationship between differentiated gastric cancer cells and CAFs in tumor progression remains unclear. In this study, we investigated whether differentiated and/or undifferentiated human gastric cancer cells induce CAFs. We used two differentiated human gastric cancer cells (MKN-7, MKN-74) and two undifferentiated human scirrhous gastric cancer cells (KATO-III, NUGC-4). Two types of human normal fibroblasts (WI-38, FEF-3) were used for induction of CAFs. The conditioned media (CM) from all cancer cells were obtained 48 hours after serum starvation. The morphological change and expression of CAF-specific markers (α-smooth muscle actin (α-SMA), fibroblast activation protein-α (FAP)) were analyzed by microscopy and Western blot analysis, respectively, in fibroblasts treated with CM for 120 hours. Moreover, the effect of CM from CAFs in the migration ability of cancer cells was evaluated using in vitro Migration assay. The CM from differentiated MKN-7 and MKN-74 cells induced the morphological change like CAFs in normal human fibroblasts, whereas CM from undifferentiated KATO-III and NUGU-4 cells did not induce. In fact, morphologically changed fibroblasts like CAFs showed increased α-SMA expression, whereas FAP expression was not increased. The CM from morphologically changed fibroblasts, but not normal fibroblasts, significantly enhanced the migration ability of MKN-7 cells. These results suggest that differentiated gastric cancer cells have a potential to induce CAFs in the invasion process of gastric cancers. CAF-targeting therapy may be a promising antitumor strategy in differentiated gastric cancers as well as undifferentiated scirrhous gastric cancers with vast fibrous stroma. Citation Format: Yuncheng Li, Hiroshi Tazawa, Nishizaki Masahiko, Yuuri Hashimoto, Naoto Hori, Ryoichi Katsube, Shinji Kuroda, Kazuhiro Noma, Shunsuke Kagawa, Toshiyoshi Fujiwara. Differentiated gastric cancer cells have a potential to induce cancer-associated fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2368. doi:10.1158/1538-7445.AM2015-2368

Abstract 3612: Effect of cancer associated fibroblast on gastric cancer cells under hypoxia

Abstract Background: Cancer-associated fibroblasts (CAF) have been suggested to be associated with the scirrhous gastric cancer (SGC) progression. SGC have a heterogeneously hypoxic environment which has been currently considered to be associated with aggressive tumor phenotypes cancer. Stromal cell-derived factor (SDF)-1 and C-X-C chemokine receptor type 4 (CXCR4) form an important chemokine/receptor pair, and are supposed to be up-regulated by hypoxia inducible factor-1 (HIF-1). The aim of our study was to clarify the effect of hypoxic environment on the CAF and CXCR4/SDF-1 axis in SGC. Experimental Design: Proliferation of SGC cells grown in monoculture and co-culture with CAF under hypoxia. SGC and CAF expression level of CXCR4 and SDF-1 under hypoxia were examined by RT-PCR and ELISA. Proliferation and migration of SGC cells with SDF-1 or hepatocyte growth factor (HGF) or conditioned medium from CAF under hypoxia, furthermore, added c-Met inhibitor or CXCR4 inhibitor. Results: The proliferation that co-culture of SGC cells with CAF was significantly increased under hypoxia. The expression level of CXCR4 mRNA was significantly increased under hypoxia in all of SGC cells. SDF-1 concentration level was significantly increased under hypoxia. The proliferation of SGC cells with SDF-1 and CAF was significantly increased. The proliferation of SGC cells with CAF was inhibited by CXCR4 inhibitor under hypoxia. The migration of SGC cells with HGF and CAF was significantly increased. The migration of SGC cells with CAF was inhibited by c-Met inhibitor. Conclusion: CAF might up-regulate the proliferation and migration of SGC cells under hypoxia, in comparison with that under normoxia. CXCR4/SDF-1 axis might play an important role for the proliferation of SGC cells under hypoxia. Citation Format: Haruhito Kinoshita, Masakazu Yashiro, Go Masuda, Hiroaki Kasashima, Tamami Morisaki, Tatsunari Fukuoka, Masatune Shibutani, Sadaaki Yamazoe, Katsunobu Sakurai, Hisashi Nagahara, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Hiroshi Otani, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. Effect of cancer associated fibroblast on gastric cancer cells under hypoxia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3612. doi:10.1158/1538-7445.AM2014-3612

Cancer-associated fibroblasts might sustain the stemness of scirrhous gastric cancer cells via transforming growth factor-β signaling

Cancer-associated fibroblasts (CAFs) have recently been implicated in tumor growth and metastasis in gastric cancer. Cancer stem cells (CSCs) have been proposed to have an important role in cancer progression. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric carcinoma. Scirrhous gastric cancer cell lines, OCUM-12 and OCUM-2MD3, and non-scirrhous gastric cancer cell lines, MKN-45 and MKN-74, were used. OCUM-12/side population (SP) cells and OCUM-2MD3/SP cells were sorted by flow cytometry as CSC-rich cells from the parent cells. CaF-37 was established from the tumoral gastric specimens as CAFs. Flow cytometric analysis of SP fraction, spheroid colony assay, and RT-PCR analysis of CSC markers were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM-12/SP cells was examined using nude mice. CAF CM significantly increased the percentages of the SP fraction of OCUM-12/SP and OCUM-2MD3/SP cells, but not that of MKN-45/SP and MKN-74/SP cells. Taken together, CM from CaF-37 significantly increased the number of spheroid colonies and the expression level of CSC markers of OCUM-12/SP and OCUM-2MD3/SP cells. These stimulating-activities by CM were significantly decreased by TGFβ inhibitors, but not FGFR and cMet inhibitor. Tumorigenicity by subcutaneous coinoculation of OCUM-12/SP cells with CAFs was significantly high in comparison with that by OCUM-12/SP cells alone. Phospho-Smad2 expression level was significantly increased by co-inoculation with CAFs. These findings suggested that CAFs might regulate the stemness of CSCs in scirrhous gastric cancer by TGFβ signaling.

N-myc Downstream Regulated Gene 1 (NDRG1) Promotes Metastasis of Human Scirrhous Gastric Cancer Cells through Epithelial Mesenchymal Transition

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.

Nanomedicine for Cancer: Lipid-Based Nanostructures for Drug Delivery and Monitoring

Recent advances in nanotechnology, materials science, and biotechnology have led to innovations in the field of nanomedicine. Improvements in the diagnosis and treatment of cancer are urgently needed, and it may now be possible to achieve marked improvements in both of these areas using nanomedicine. Lipid-coated nanoparticles containing diagnostic or therapeutic agents have been developed and studied for biomedical applications and provide a nanomedicine strategy with great potential. Lipid nanoparticles have cationic headgroups on their surfaces that bind anionic nucleic acids and contain hydrophobic drugs at the lipid membrane and hydrophilic drugs inside the hollow space in the interior. Moreover, researchers can design nanoparticles to work in combination with external stimuli such as magnetic field, light, and ionizing radiation, which adds further utility in biomedical applications. In this Account, we review several examples of lipid-based nanoparticles and describe their potential for cancer treatment and diagnosis. (1) The development of a lipid-based nanoparticle that included a promoter-enhancer and transcriptional activator greatly improved gene therapy. (2) The addition of a radiosensitive promoter to lipid nanoparticles was sufficient to confer radioisotope-activated expression of the genes delivered by the nanoparticles. (3) We successfully tailored lipid nanoparticle composition to increase gene transduction in scirrhous gastric cancer cells. (4) When lipophilic photosensitizing molecules were incorporated into lipid nanoparticles, those particles showed an increased photodynamic cytotoxic effect on the target cancer. (5) Coating an Fe(3)O(4) nanocrystal with lipids proved to be an efficient strategy for magnetically guided gene-silencing in tumor tissues. (6) An Fe(16)N(2)/lipid nanocomposite displayed effective magnetism and gene delivery in cancer cells. (7) Lipid-coated magnetic hollow capsules carried aqueous anticancer drugs and delivered them in response to a magnetic field. (8) Fluorescent lipid-coated and antibody-conjugated magnetic nanoparticles detected cancer-associated antigen in a microfluidic channel. We believe that the continuing development of lipid-based nanomedicine will lead to the sensitive minimally invasive treatment of cancer. Moreover, the fusion of different scientific fields is accelerating these developments, and we expect these interdisciplinary efforts to have considerable ripple effects on various fields of research.

Inhibitory effect of a TGFβ receptor type-I inhibitor, Ki26894, on invasiveness of scirrhous gastric cancer cells

Background:Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-β (TGF-β) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-β receptor (TβR) phosphorylation inhibitor on the invasiveness of gastric cancer cells.Methods:Four gastric cancer cell lines, including two scirrhous-type cell lines and two non-scirrhous-type cell lines, were used. A TβR type I (TβR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TβR-I. We investigated the expression levels of TβR and phospho-Smad2, and the effects of TGF-β in the presence or absence of Ki26894 on Smad2 phosphorylation, invasion, migration, epithelial-to-mesenchymal transition (EMT), Ras homologue gene family member A (RhoA), ZO-2, myosin, and E-cadherin expression of gastric cancer cells.Results:TβR-I, TβR-II, and phospho-Smad2 expressions were found in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. Ki26894 decreased Smad2 phosphorylation induced by TGF-β1 in scirrhous gastric cancer cells. Transforming growth factor-β1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-β1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-β1 decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cells. Interestingly, Ki26894 inhibited these characteristics in scirrhous gastric cancer cells. In contrast, non-scirrhous gastric cancer cells were not affected by TGF-β1 or Ki26894 treatment.Conclusion:A TβR-I kinase inhibitor decreases the invasiveness and EMT of scirrhous gastric cancer cells. Ki26894 is therefore considered to be a promising therapeutic compound for the metastasis of scirrhous gastric carcinoma.

Cancer–Stromal Interactions in Scirrhous Gastric Carcinoma

Fibroblasts play an important role in the progression, growth and spread of gastric cancers. Cancer-stroma interactions have been especially evident in the scirrhous type of gastric carcinoma. Fibroblasts are associated with the cancer progression at the primary and metastatic site. The proliferative and invasive ability of scirrhous gastric cancer cells are closely associated with the growth factors produced by organ-specific fibroblasts. Fibroblasts are therefore a key determinant in the malignant progression of gastric cancer and represent an important target for cancer therapies.

Suppression of gastric cancer dissemination by ephrin‐B1‐derived peptide

Interaction of the Eph family of receptor protein tyrosine kinases and their ligands, ephrin family members, induces bidirectional signaling through cell-cell contacts. High expression of B-type ephrin is associated with high invasion potential of tumors, and we previously observed that signaling through the C-terminus of ephrin-B1 mediates the migration and invasion of cells, and is involved in the promotion of carcinomatous peritonitis in vivo. Here we show that the intracellular introduction of a synthetic peptide derived from ephrin-B1 C-terminus blocks ephrin-B1 mediated signaling in scirrhous gastric cancer cells. Treatment of cancer cells with a fusion peptide consisting of HIV-TAT and amino acids 331-346 of ephrin-B1 (PTD-EFNB1-C) suppressed the activation of RhoA, mediated by the association of ephrin-B1 with an adaptor protein Dishevelled, and also inhibited extracellular secretion of metalloproteinase. Moreover, injection of PTD-EFNB1-C peptide into the peritoneal cavity of nude mice suppressed carcinomatous peritonitis of intraperitoneally transplanted scirrhous gastric cancer cells. These results indicate the possible application of ephrin-B1 C-terminal peptide to develop novel protein therapy for scirrhous gastric carcinoma, especially in the stage of tumor progression, including peritoneal dissemination.

Synergistic antiproliferative effect of mTOR inhibitors in combination with 5‐fluorouracil in scirrhous gastric cancer

The aim of this study is to clarify the benefit of combination chemotherapy in gastric cancer based on a cell-signal inhibitor and an anticancer drug. Two scirrhous gastric cancer cell lines and two non-scirrhous gastric cancer cell lines were used. Five anticancer drugs (5-fluorouracil [5FU], paclitaxel, oxaliplatin, irinotecan, and gemcitabine) and four cell-signal inhibitors, mammalian target of rapamycin (mTOR) inhibitor, glycogen synthase kinase 3beta, p38alphabetaMAPK, and cyclin-dependent kinase, were used. The proliferation of cancer cells was examined by MTT assay and in vivo study. The apoptosis of cancer cells and the expression of apoptosis-related molecules were examined by flow cytometry, real-time PCR, and immunostaining. mTOR inhibitors with 5FU showed a synergistic antiproliferative effect in scirrhous gastric cancer, whereas the other signal inhibitors showed no synergistic effect with any anticancer drugs. mTOR inhibitor decreased the IC(50) of 5FU and increased the apoptosis rate in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. The pan-caspase inhibitor, zVAD-fmk, inhibits apoptosis induced in combination with 5FU and mTOR inhibitor. mTOR inhibitor decreased dihydropyrimidine dehydrogenase, thymidylatesynthase, and bcl-2 expression, and increased caspase-3 and p21 expression of scirrhous gastric cancer cells, but did not affect those of non-scirrhous gastric cancer cells. In an in vivo study, mTOR inhibitor significantly enhanced the therapeutic efficacy of S1, an analog of 5FU. These findings suggest that mTOR inhibitor interacts with 5FU in a synergistic manner in scirrhous gastric cancer cells by the activation of the apoptosis signal. Therefore, mTOR inhibitor is a promising therapeutic agent in combination with 5FU in scirrhous gastric cancer.