Transfection of colorectal cancer cells with chemokine MCP-3 (monocyte chemotactic protein-3) gene retards tumor growth and inhibits tumor metastasis.

AIM: To evaluate the possibility of the induction of anti-tumor immune response by transfecting the colorectal cancer cells with chemokine MCP-3 gene. METHODS: Mouse MCP-3 gene was transduced into mouse colorectal cancer cells CMT93 by using of Liposome. G418-resistant clones were selected and the MCP-3 mRNA expression was detected by RT-PCR. The chemotactic activity of MCP-3 in the cell culture supernatant was detected by Chemotaxis assay. The tumorigenicity of wild type CMT93 and CMT93 gene transfectants were detected by in vivo experiments. The immune cell infiltrations in tumor tissue and tumor metastasis were detected histopathologically. RESULTS: MCP-3 mRNA expression was detected by RT-PCR in gene-transfected cells (CMT93/MCP-3), but not in control groups. And MCP-3 secreted in the cell culture supernatant possessed chemotatic activity. The results from in vivo experiments showed that the tumorigenicity of CMT93/MCP-3 had not decreased, but the tumors derived from CMT93/MCP-3 cells grew more slowly than those from CMT93 cells (1.021+/-0.253) cm(2) vs (1.769+/-0.371) cm(2), P<0.05) or CMT93/mock cells (1.021+/-0.253) cm(2) vs (1.680 +/-0.643)cm(2), P<0.05). Histophathological results showed few immune cells infiltrating in the tumor tissue derived from the controls. In the tumor tissue derived from CMT93/MCP-3, infiltrating immune cells increased. In addition, no tumor metastasis was found in all mice inoculated with CMT93/MCP-3 tumor cells. But all mice had tumor metastasis in CMT93 controls and 4 in 5 mice had tumor metastasis in CMT93/mock controls. CONCLUSION: The results suggested that the transfection of chemokine MCP-3 gene could promote the induction of anti-colorectal cancer immunity, but the tumor growth could not be inhibited completely by merely MCP-3 gene transfection.

[Active immunity for anti-colorectal cancer induced by chemokine MCP-3 gene transfection].

BACKGROUND & OBJECTIVE: Chemokines play an important role in the infiltration of immune cells to tumor tissues. Anti-tumor immune response had been elicited in many tumor models by the chemokine gene transfection. The aim of this study was to evaluate the possibility of inducing anti-colorectal cancer active immune response by transfection of mouse colorectal cancer CMT93 cells with chemokine MCP-3 gene. METHODS: Mouse MCP-3 gene was transduced into mouse colorectal cancer cells CMT93 by using of liposome. G418-resistant clones were selected and the MCP-3 mRNA expression was detected by RT-PCR. The chemotactic activity of MCP-3 in the cell culture supernatant was detected by chemotaxis assay. In vivo experiments were performed to observe the tumorigenicity of wild type CMT93 and MCP-3 gene modified tumor cells. The immune cell infiltration in tumor tissues and tumor metastasis were detected histopathologically. RESULTS: RT-PCR detection showed MCP-3 was expressed in MCP-3 gene-transfected G418-resistant clones(CMT93/MCP-3), but not in wild type CMT93. In chemotaxis assay, the results showed that the cell culture supernatant of CMT93/MCP-3 possess obviously chemotactic activity. The chemotactic index of the CMT93/MCP-3 supernatant was 5.57(P < 0.05). The supernatants from the control groups did not possessed the chemotactic activity. In vivo experiments showed that the tumorigenicity of CMT93/MCP-3 had not decreased significantly compared to wild type CMT93, but the tumors grew more slowly from CMT93/MCP-3 than from the controls (P < 0.05). In the tumor tissue from CMT93/MCP-3, obvious infiltrated immune cells were found, and few immune cells infiltrated in the tumor tissue from the controls. In the mice inoculated with CMT93/MCP3 tumor cells, tumor metastasis was inhibited significantly, its metastasis rate was 0(0/7), lower than that of CMT93 (100%, 4/4) and CMT93/mock (80%, 4/5) (P < 0.05). CONCLUSION: Transfection with chemokine MCP-3 gene can induce anti-colorectal cancer active immune response, but the tumor growth cannot be inhibited completely by merely MCP-3 gene transfection.

Expression of B7 costimulation molecules by colorectal cancer cells reducestumorigenicity and induces anti-tumor immunity.

AIM:To study the tumorigenicity of colorectal cancer cells transfected with B7 gene and the anti-tumor immunity induced by B7 gene modified colorectal cancer cells.METHODS:B7 gene was transfected into mouse colon cancer cell line CMT93.The transfectants were selected in DMEM containing 800mg/L G418, and B7 molecules were detected by immunohistochemistry.Experiments in vivo include: (1)5X10(6) B7(+) CMT93 cells were inoculated into the back of C57BL/6 mice subcutanously to determine their tumorigenicity (n = 4). As control, wild type CMT93 cells were inoculated the same as the experimental group (n= 3). (2) The mice primed by B7(+) CMT93 cells whose tumors vanished were rechallenged with wild type CMT93 to observe the immune protection of these mice against the wild type CMT93 (n = 4). Non-primed 4 native mice inoculated with wild type CMT93 were used as control.With in vivo cytotoxicity assay, the mice were immunized with B7 (+) CMT93 or the wild type CMT93 by intraperitoneal injection (n = 4X2). The spleen cells and the abdominal cavity infiltrating lymphocytes were obtained and cultured for two days. Cytotoxicity of these cells against the B7 gene modified or wild type CMT93 was detected by MTT assay.RESULTS:B7 high expression clones were obtained after the transfection of the B7 gene into CMT93 cells by electroporation. Immunohistochemistry results showed mainly membrance staining and partly cytoplasm staining in B7 gene transfected CMT93 cells. in vivo experiments: (1)After the inoculation of the B7(+) CMT93 cells in the back of C57BL/6 mice, they lost their tumorigenicity greatly (P < 0.01). All the small tumors growing in the early period in the experimental group vanished in one month, and the tumors in control group grew progressively. (2) No tumors were found in all 4 mice primed by B7(+) CMT93 cells after they were rechallenged with wild type CMT93. In the control group all mice had grown tumors (P < 0.05). In vitro cytotoxicity assay, the CTLs induced by B7(+) CMT93 had a higher cytotoxity against the wild type CMT93 than that induced by wild type CMT93 (P < 0.05), and the cytotoxity of CTLs induced by B7(+) CMT93 against B7(+) CMT93 cells was higher than that against wild type CMT93 cells (P < 0.05).CONCLUSION:The results suggest that the expression of costimulation B7 molecules by colorectal cancer cells can decrease their tumorigenicity greatly, and the B7 molecule can augment the activation of the CTLs against colorectal cancer, and it plays an important role in CTL effector function as well.

Radioimmunoimaging of colorectal cancer using (99m)Tc labeled monoclonal antibody.

AIM:To determine whether Hb3 and its fragment F(ab')(2) have practical value in radioimmunoimaging of colorectal cancer.METHODS:Intact Hb3 was purified by hydroxylapatite chromatography.The fragment F(ab') (2) was prepared by cold digestion and purified as intact Hb3.Hb3 and its fragment F(ab') (2) were labeled with 99mTc by direct labeling method using SnCl(2) as reducing agent. The radioactive doses ranged from 15 to 40 mCi.The imaging was accomplished by single photon emission computered tomograph (SPECT) with imaging time ranging from 2.5 to 48 hours. In this study, 10 patients were selected. Among them, 7 were administered with intact Hb3, and 3 with F(ab') (2) fragment. All the patients were diagnosed as having colorectal adenocarcinoma.RESULTS:After purification, intact Hb3 and its fragment F(ab') (2) were fit for radioimmunoimaging. The percentage of labeling of (99m)Tc to Hb3 or F(ab') (2) was 80.6%-91.5%. Among the 10 patients, 3 of 7 patients administered with intact Hb3 had positive scans, the other 4 had negative scans, and 2 of 3 patients administered with F(ab') (2)had positive scans, the other 1 had negative scans.CONCLUSION:The results showed that both intact Hb3 and its F(ab') (2) have some practical value in radioimmunoimaging of colorectal cancer, and the effects of imaging with F(ab') (2) was better than that with intact Hb3.