Carcinoembryonic Antigen Is A Preliminary Study On The Development Of Colon Cancer Mechanism

Objective: To explore the expression of carcinoembryonic antigen receptors (CEAR) in digestive organs.Methods: All specimens were from 20 male healthy BALB/c mice including esophagus, small intestine, stomach, colon, pancreas, and liver. Kupffer cells were obtained by density gradient centrifugation following enzymatic digesting of the fresh liver. Immunohistochemistry and Immunocytochemistry methods were used respectively to detect CEAR in those organs and Kupffer cells.Results: (1) CEAR were found both in the cytoplasm and nuclei of the digestive tract mucosa, as low-intensity in the esophageal, medium intensity in the stomach and small intestine, high intensity in the colon, and highest intensity in the epithelial cells at villus tip and near the glands opening in the colon. CEAR were also found mainly in the cytoplasm, as moderate intensity, in the smooth muscle of the esophagus, small intestine, stomach and colon; but not in the submucosal connective tissue and serosa of the digestive tract. This is consistent with the clinical phenomenon that CEA-positive incidence in digestive tract tumors from low to high is in the esophagus, stomach, and colon. (2) CEAR were found very unevenly in pancreas, as moderate intensity in almost all the pancreas islet cells, locating both in the nucleus and cytoplasm, while not in the acinar or intercalated duct. This is consistent with the clinical phenomenon that pancreatic endocrine cells tumors have higher CEA positive incidence than pancreatic cancers originated from the exocrine cells. (3) CEAR were found as medium intensity in the liver, mainly locating in the cytoplasm of the hepatocytes and Kupffer cells, while not in the outer membrane of liver or the sinusoidal endothelial cells. This is consistent with the clinical phenomenon that hepatocellular cancer has lower CEA positive incidence than colon cancer and the clinical phenomenon that colon cancer are prone to metastasize to liver. (4) CEAR expression in the main digestive tissue and cells showed significant differences both in locations (P <0.01) and intensity (P <0.01), which could be the foundation that CEA/CEAR pathway could regulate different cells to produce diverse biological effects.Conclusion: There were significantly differences in the the expression of CEAR in the main digestive organs according to the different tissue and cells, which may take an important role in the oncogenesis of digestive CEA-positive tumors, especially in the liver metastasis of the colorectal cancer. Objective: To establish a mouse colon cancer cell strain stably expressing carcinoembryonic antigen (CEA).Methods: Recombinant lentivirus conjugated with CEACAM5-eGFPcDNA or eGFPcDNA were used to transfect the wild CT26 cells. Antibiotics and fluorescence microscope were used to select the CEA-positive cells, and limited dilution methods were used to select the single CT26CEA clones. The 7th and 14th passages cells were used to detect CEACAM5 mRNA by RT-PCR, the CEACAM5 protein by western blot, immunocytochemistry and ELISA. The 14th passages CT26CEA cells were planted subcutaneously into mice by injection to creat tumors so that can be passed by in vivo. The CEACAM5 protein in the tumors was detected by immunohistochemistry and fluorescence photography. The serum concentrations of CEACAM5 were measured by ELISA after the mice were planted with tumor tissue. The serum concentrations of CEACAM5 were compared between the first generation mice and 5th generation mice. Results: (1) The monoclonal CT26CEA cell strain and CT26GFP cell strain with green fluorescence were obtained by antibiotics selection and limiting dilution methods. (2) Bright green fluorescence can be observed within the cytoplasm of the 7th and 14th generation CT26CEA cells under a fluorescence microscope, while not in CT26 cells. (3) The cytoplasm of the 7th and 14th generation CT26CEA cells were stained yellow or light brown in immunocytochemistry tests, while CT26 cells were negetive. (4) The CEACAM5 mRNA was found in the 7th and 14th generation CT26CEA cells, while not in CT26 cells. (5) The CEACAM5 protein was found in the 7th and 14th generation CT26CEA cells, while not in CT26 cells. (6) The CEA production in the 7th and 14th generation CT26CEA cells were 265±71 ng/106cell and 213±66 ng/106cell per 24 h, showing no significant difference between the different generations. (7) Both of CT26CEA and CT26 can create subcutaneous tumors within 14days in 100% mice after been planted, wether in the 1st generation or 5th generation. All CEACT26 cell subcutaneous tumors can emit green fluorescence, and show CEA-positive mainly in the cytoplasm by immunohistochemistry methods, while CT26 cell tumors not. The serum CEA concentration of the tumor-bearing mice on the 14th day show no significant difference between the first and fifth generation of mice planted by CT26CEA cell tumor (48±16.8ng/mL vs 37±18.7ng/mL), but both are significantly higher than that by CT26 (0.13±0.05 ng/mL, P<0.01).Conclusion: The monoclonal colon cancer cell strain stably expressing CEA has been successfully established, providing a new tool to facilitate research in the impact of CEA on biological characteristics of the colon cancer cells in vivo, especially the research in the impact of CEA on the process of liver metastasis in the mice with normal immunology. Objective: To investigate the effect of endogenous carcinoembryonic antigen (CEA) on the adhesion, migration, invasion, proliferation, apoptosis and other biological characteristics of the colon cancer cells and effects on tumor growth and metastasis in mice.Methods: The CT26CEA cell strain can stably expressing CEA and the control CT26GFP cell strain transfected with only eGFP- recombinant lentivirus were obtained in the previous stady. The wild CT26 cell strain was also used as a control group. All the three cells strains were respectively cultured to draw cell growth curve, calculate cell population doubling time (DT), detected adhesion by MTT assays, movement and invasion by transwell methods. The distribution in cell cycle was detected by flow cytometry to calculate proliferation index (PI) and apoptosis index (AI).Then the three cell strains were respectively planted subcutaneously into mice to study the growth of the subcutaneous tumors. After 14 days, the tumor weight was measured; the apoptosis index in tumor was detected by TUNEL methods. The mouse model of liver metastasis was also developed by injecting cancer cells into spleen and the incidence of liver metastasis was compared.Results: (1) The CT26CEA cell strain show significant increase by 14.7% in adhesion(P<0.01), 21.8% increase in movement(P<0.01), 39.5% in invasion(P<0.01), comparing to the CT26GFP cell strain. But there was no significant difference between the CT26GFP cell strain and CT26 cell strain. (2) There was no significant difference in cell cycle distribution, proliferation index and apoptosis index among the three cell strains. (3) There was no significant difference in growth curve or cell population doubling time. (4) All the three cell strains can develop subcutaneous tumors in mice on 14th day by 100%, but the formation of CT26CEA tumors were significant earlier (P<0.05) and heavier (P<0.01) comparing to CT26GFP tumors, while no significant difference between CT26GFP and CT26. All the apoptosis indices of the three cell strains in subcutaneous tumors were less than 4%, showing no significant difference. (5) The three cell strains showed no significant difference in liver metastases incidence, but CT26CEA cells can make significantly more metastasis'lesions comparing to CT26GPF cells (P<0.01). While there was no significant difference between the CT26GFP cell strain and CT26 cell strain.Conclusion: Endogenous carcinoembryonic antigen can enhance the adhesion, migration and invasion of the colon cancer cells, also can promote tumor growth and metastasis in mice, but have no significant effect on the cell cycle regulation, proliferation or apoptosis in vitro.