Effect And Mechanism Research Of Attenuated Salmonella Carrying With 4-1BB Ligand And CEACAM6 In Rat Colorectal Cancer Models

Part one: Effects of recombinant attenuated Salmonella harboring CEACAM6 and 4-1BBL gene on rat experimental colorectal cancerObjective: To investigate the immune change of tumor-bearing rats and the feasibility of prevention and treatment of colorectal cancer induced by a ttenuated Salmonella typhimurium vaccine strain expressing rat 4-1BBL and CEACAM6. Methods: Rats were given subcutaneous injections of DMH once a week for 18 weeks. Eight weeks after first injection,they were divide into p IRES/SL3261, p IRES-4-1BBL/SL3261, p IRES-CEACAM6/SL3261,p IRES-CEACAM6-4-1BBL/SL3261 group, all were fed with corresponding vaccine strains, and were then sacrificed, the number of colon tumors were recorded, Dukes stage were evaluated. CMV gene expression in spleens were checked by RT-PCR.Results: Tumors were found in p IRES /SL3261 group,which was significantly higher than the other group(5.0±1.4 in p IRES-CEACAM6/SL3261 group,5.7±1.2 in p IRES-4-1BBL/SL3261 group,4.3±1.4 in p IRES-CEACAM6-4-1BBL/SL3261 group),most of the tumors in the p IRES/SL3261 group of rats were at advanced stage(stage B-C,4;Stage D,2), while most tumors in the p IRES-4-1BBL/SL3261 and p IRES-CEACAM6/SL3261 groups were at stage B-C(SL3261B: stage A, 3; stage B-C, 3; SL3261C: stage A, 1; stage B-C, 5). However, the tumors in the p IRES-CEACAM6-4-1BBL/SL3261 group of rats were at stage A(Stage A,5; Stage B-C, 1). RT-PCR test showed CMV gene in all groups. Conclusions: The bacteria could effectivly transcribe exogenous gene to rats and express related proteins. The vaccination with recombinant attenuated Salmonella harboring the CEACAM6 and 4-1BBL gene could obviously inhibite the development of colorectal cancers in rats.Part two: Anticancer immunological mechanism of recombination Attenuation Salmonella typhimurium vaccine containing p IRES-CEACAM6-4-1BBL vector----Tumor local immune responseObjective: To reveal immunology mechanism of double gene vaccine inhibiting tumor growth through TIL immunohistochemical. Methods:To test all groups’ CD3、CD4、CD8、CD56、CD45RO、Foxp3、CEACAM6、IL-4 and IL-17 expression in tumor tissues by immunohistochemical. Results: In comparison with that in the p IRES/SL3261 group, similar levels of CD3+, CD8+, CD56+, IL-4 and IL-17 expression in the p IRES-CEACAM6/SL3261 group of rats, and comparable numbers of tumors were detected in the p IRES-4-1BBL/SL3261, p IRES-CEACAM6/SL3261 groups. In contrast, higher density of CD3+CD8+, CD56+, CD45 RO and lower Foxp3+ TIL cells, IL-4、IL-17 were detected in the p IRES-CEACAM6-4-1BBL/SL3261 group of rats. Conclusions: The vaccination with recombinant attenuated Salmonella harboring the CEACAM6 and 4-1BBL gene efficiently increased the CD3+CD8+ TIL cells and NK cells, decreased FOXP3 cells, promoted the polarization of Th1 and inhibited Th2 and Th17 polarization to inhibited the development of carcinogen-induced colorectal cancers in rats.Part three: Anticancer immunological mechanism of recombination Attenuation Salmonella typhimurium vaccine containing p IRES-CEACAM6-4-1BBL vector----- Systemic immune responseObjective: To reveal immunology mechanism of double gene vaccine inhibiting tumor growth through spleens tissues immunohistochemical. Methods:To test all groups’ IFN-γ、CD3、CD4、CD8、CD56、Foxp3、IL-4 and IL-17 expression in tumor tissues by immunohistochemical. Results: In comparison with that in the p IRES/SL3261 group, similar levels of IFN-γ, CD3+, CD8+, CD56+, IL-4 and IL-17 expression in the p IRES-CEACAM6/SL3261 group of rats, and comparable numbers of tumors were detected in the p IRES-4-1BBL/SL3261, p IRES-CEACAM6/SL3261 groups. In contrast, higher density of IFN-γ, CD3+CD8+, CD56+, and lower Foxp3+ TIL cells, IL-4、IL-17 were detected in the p IRES-CEACAM6-4-1BBL/SL3261 group of rats. Conclusions: Consistent with the second part of the conclusion, the vaccination with recombinant attenuated Salmonella harboring the CEACAM6 and 4-1BBL gene could induce specificand nonspecific immune, break the immunosuppression microenvironment to restrain the CRC cancer.