The Primary Cell Culture And Identification Of The Ascites-Derived Gastric Cancer Cell Line

BackgroundInternational Agency for Research on Cancer evaluated the worldwide incidence and mortality of 27 major cancers. Overall, there were 14.1 million new cases and 8.2 million deaths in 2012. The most commonly diagnosed cancers were lung(1.82 million), breast(1.67 million), and colorectal(1.36 million); the most common causes of cancer death were lung cancer(1.6 million deaths), liver cancer(745,000 deaths), and stomach cancer(723,000 deaths) [1]. Gastric cancer is the third leading cause of the cancer deaths. With high-quality data from an additional number of populationbased registries available through the National Central Cancer Registry of China, Chen et al. estimated the cancer incidence and death of Chinese people. The results indicated that an estimated 4292,000 new cancer cases and 2814,000 cancer deaths would occur in China in 2015, with stomach cancer being the second common cancer and the second leading cause of cancer death[2]. The 5-year survival rate of gastric cancer is only about 20%[3].Gastric cancer refers to the tumours of stomach that arise from the gastric mucosa(adenocarcinoma), connective tissue of the gastric wall(gastrointestinal stromal tumours), neuroendocrine tissue(carcinoid tumours), or lymphoid tissue(lymphomas) [4]. Many gastric cancer patients after radical surgery will get recurrence and metastasis. The most common reasons for the failure of the radical surgery treatment is the peritoneal metastasis, which is the 20%-53.5% of the total recurrence rate[5-8]. The median survival period is short, almost only 3.1-5.7 months and patients always have a poor quality of life[9-12]. Gastric cancer patients with peritoneal metastasis always lead to malignant ascites. The early clinical features of the gastric cancer are not specific and always overlooked. Most patients were diagnosed at the advanced stage and would have missed the best opportunity for treatment, the treatment efficacy is poor. In recent years, the chemotherapy of the advanced gastric cancer(AGS) is still not effective [13]. The doctor may choose the palliative chemotherapy to improve the quality of life and prolong survival[14, 15]. In addition, the combination therapy with the molecular targeted drugs is the development tendency of the treatment of the gastric cancer.More than 60 years have passed since the establishment of the first human cancer cell line, HeLa, in 1951. Since then, human tumour cell lines have had an extremely important impact on cancer research and greatly facilitated development of a variety of cancer drug development that benefit human patients[16-18]. Human carcinomas that grow uncontrollably in the body are often paradoxically difficult to grow in cell culture in vitro. A robust and efficient cell line model system that predicts patient response to various drugs would greatly improve development and implementation of new drugs for personalized treatment of cancer patients[19]. The primary cell culture is the culture of the fresh tumour tissues or cells in vitro. Establshing the tumor cell lines can provide enough and effective cells for the cancer research and treatment. The in vitro survival period of primary cells are short, but with similar characteristics of primary cancer in vivo. The primary tumour cells culture will provide an useful platform for a series of cancer research, such as understanding the tumour progression and metastasis, the antitumor drug sensitivity tests, the gene mapping of metastatic gastric cancer, and even the gene therapy of cancer.Despite many decades of improvements in methods for establishing cancer cell lines [17], it remains extremely difficult to routinely establish high-quality, permanent cell lines from human primary tumours with high efficiency, limiting the number and diversity of cell lines available for research and assessment of the response of individual cancer patient to particular drugs or therapeutic regimens. So achieving the viable primary gastric cells is the key of the personalized cancer medicine.Because of most patients with gastric cancer are diagnosed at late stage and miss the time of surgery, it’s too difficult for us to doing the primary cell culture of the tumour tissues. The malignant ascites of the AGS is relatively easy to obtain and the patients suffer little, which could be a perfect source for the tumor cell culture. ObjectiveTaking the ascites-derived gastric cancer cells as the research object, we aimed to develop an optimal system for primary cell culture and identification technique, so as to establish an effective platform for carrying out a series of investigations, such as the tumour progression and metastasis, the antitumor drug sensitivity tests, the gene mapping of metastatic gastric cancer, and identification of potential target for cancer gene therapy. MethodsTo culture and purify the ascites-derived gastric cancer cells through the combination of digestion with low concentration trypsin and differential attachment. And then to observe the cell morphology by H&E and immunochemical staining, to detect cellular proliferation and invasion by RTCA xCELLigence system. The tumour growth of our established cell line in vivo was also investigated by the subcutaneous transplantation in nude mice model. ResultsWe successfully developed an ascites-derived gastric adenocarcinoma cell line(named as CFJ) that could passage steadily. The cells are consistent in morphology with nuclei showing obvious atypia and with cytoplasm expressing epithelial biomarkers. Furthermore, we succeeded in establishing the subcutaneous transplantation model in nude mice, and found the cells could metastasis to locally draining and distant lymph nodes of the mice. ConclusionMetastatic ascites of gastric cancer patients could be a good source of the primary cell culture. The purified tumor cells could act as an effective platform for the basic and clinical research of gastric cancer.