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 EVsmarkers, 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.
The sentinel node (SN) technique has been established for the treatment of some types of solid cancers to avoid unnecessary lymphadenectomy. If node disease were diagnosed before surgery, minimal surgery with omission of lymph node dissection would be an option for patients with early gastric cancer. Although SN biopsy has been well ascertained in the treatment of breast cancer and melanoma, SN navigation surgery (SNNS) in gastric cancer has not been yet universal due to the complicated lymphatic flow from the stomach. Satisfactory establishment of SNNS will result in the possible indication of minimally invasive surgery of gastric cancer. However, the results reported in the literature on SN biopsy in gastric cancer are widely divergent and many issues are still to be resolved, such as the collection method of SN, detection of micrometastasis in SN, and clinical benefit. The difference in the procedural technique and learning phase of surgeons is also varied the accuracy of SN mapping. In this review, we outline the current status of application for SNNS in gastric cancer.
Abstract Background. A microRNA (miRNA) is non-coding RNA molecules of 21-25nt. miRNAs are post-transcriptional regulators that bind to messenger RNA transcripts, resulting in translational repression of target degradation and gene silencing. miRNAs are wrapped in exosome and secreted stably in body fluid, which can prevent RNase from degrading the miRNAs. Therefore several methods for miRNA-based early cancer detection using serum, plasma, and urine are reported. However there is no report to detect peritoneal dissemination of gastric cancer using malignant ascitis. Aim. To detect predictive factors for peritoneal dissemination of gastric cancer, global analysis of exosomal miRNAs in gastric malignant ascites, intraoperative peritoneal lavage fluid and cell culture supernatant were performed. Materials and Methods. A total of 6 gastric malignant ascites, 20 peritoneal lavage fluids and 2 human gastric carcinoma cell culture supernatants (OCUM-2M and its sub line with high potential for metastasis to the peritoneal cavity named OCUM-2MD3) were included in this study. The amount of 10 ml samples underwent a final ultracentrifugation at 120,000g twice to pellet the exosomes. miRNA was extracted and analyzed using Bioanalyzer. Exosomal miRNA expression profiling was performed using the Agilent Human miRNA Microarrays. To quantitatively detect mature miRNAs, TaqMan MicroRNA Assays was used. Statistical tests by spearman's rank-correlation coefficient were performed to investigate correlation of exosomal miRNAs that were expressed between malignant ascites and cell culture supernatant. Results. miRNAs were detected in each gastric malignant ascites at concentrations ranging from 61.7 ng to 220 ng, however no or very low amounts of ribosomal RNA (18S and 28S). Microarray analysis detected 327 miRNAs in malignant ascites, 389 in OCUM-2M and 414 in OCUM-2MD3. miRNA expression of Malignant ascites correlated that of cell culture supernatants; OCUM-2M(r=0.774, p<0.01) and OCUM-2MD3(r=0.776, r<0.001). Total of 140 miRNAs in OCUM-2MD3 showed a higher expression level (>2-fold change) than in OCUM-2M. Among these 140 miRNAs, 7 miRNAs detected at high expression in malignant ascites were selected for confirmation by qRT-PCR. All 7 miRNA was detectable both malignant ascites and intraoperative peritoneal lavage fluids by qRT-PCR. Conclusions. Exosomal miRNAs were highly detectable in malignant ascites and peritoneal lavage fluids. In this study 7 miRNAs related to peritoneal dissemination were identified. This result may provide a new diagnostic approach of peritoneal dissemination. Citation Format: Motohiko Tokuhisa, Yasushi Ichikawa, Takashi Kosaka, Satoshi Hasegawa, Hirotoshi Akiyama, Takashi Ishikawa, Nobuyoshi Kosaka, Takahiro Ochiya, Masakazu Yashiro, Kousei Hirakawa, Chikara Kunisaki, Ayumu Goto, Noritoshi Kobayashi, Itaru Endo. Expression of exosomal microRNAs in malignant ascites, peritoneal lavage fluid and cell culture supernatant of gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2013-5279 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Purpose: It has been reported that the interaction between cancer cells and cancer-associated fibroblasts (CAFs) is closely associated with the progression of various types of cancer. The aim of this study is to clarify the role of bone marrow-derived stromal cells (BM-SCs), which are supposed to be the origin of CAFs in the tumor microenvironment, on the development of gastric cancer.Methods: The effect of human BM-SCs which frequently express CD271 on the proliferation and motility of six gastric cancer cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, NUGC-3, and MKN-74, was examined. CD271 expression levels in BM-SCs were analyzed by flow cytometry. We also generated a gastric tumor model by orthotopic inoculation of OCUM-2MD3 cells in mice that had received transplantation of bone marrow from CAG-EGFP mice. The correlation between the clinicopathological features of 279 primary gastric carcinomas and CD271 expression in tumor stroma was examined by immunohistochemistry.Results: Numerous BM-SCs infiltrated the gastric tumor microenvironment; CD271 expression was found approximately 25% of BM-SCs. Conditioned medium from BM-SCs significantly increased the proliferation of gastric cancer cell lines. Furthermore, conditioned medium from gastric cancer cells significantly increased the number of BM-SCs, while migration of OCUM-12 and NUGC-3 cells was significantly increased by conditioned medium from BM-SCs. CD271 expression in stromal cells was significantly associated with macroscopic type-4 cancers, diffuse-type tumors, and tumor invasion depth. The overall survival of patients (n=279) with CD271-positive stromal cells was significantly worse than that of patients with CD271-negative stromal cells. This is the first report of the significance of BM-SCs in gastric cancer progression.Conclusions: BM-SCs were abundant in the tumor microenvironment of gastric cancer in vivo. In vitro study also indicated that the crosstalk between BM-SCs and gastric cancer cells might play an important role for the development of gastric cancer. CD271-positive tumor stromal cells might be a useful predictive prognostic factor for patients with gastric cancer. BM-SCs, therefore, might be one of therapeutic targets for cancer treatment.Citation Format: Masakazu Yashiro, Hiroaki Kasashima, Syuhei Kushiyama, Sadaaki Nishimura, Kenji Kuroda, Shingo Togano, Tomohisa Okuno, Yuichiro Miki, Tatsunari Fukuoka, Hirohisa Nakamae, Masayuki Hino, Masaichi Ohira. Bone marrow-derived stromal cells in the tumor microenvironment might be associated with the progression of gastric cancer [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 2742.
