| Objective:Immature laminin receptor protein(iLRP)as a tumor antigen was encoded by RPSA,with high expression in various cancer,such as lymphoma,breast carcinoma,colorectal carcinoma.etc.Flagellin(FliC)is the unique ligand of TLR5 and widespread used as an adjuvant in many researches.In our study,we chose Colorectal Carcinoma(CRC)as the model for the research because the iLRP expression was much higher than para-carcinoma tissue and normal colonic epithelial cells that we checked with several methods including the analysis of reported data from TCGA database.We transfected CT26 and SW480 with lentivirus vectors to overexpress iLRP and FliC in the cell surface,and the transfected cells were then used to prepare the whole tumor cell vaccine(WTCV)after verifying the target gene expression.To test the effects of the WTCV in vitro and in vivo,we completed the transwell experiment and created a mouse CRC model for tumor killing assay.Results showed that the WTCV not only enhanced the ability of migration and polarization to M1 for macrophages(Mφ)in vitro,but also reduced tumor progression in a mouse model bearing with CRC in vivo.Methods:1.The mRNA,protein and tissue expression level of iLRP in CRC of human and mice was verified.(1)We utilized an orthotopic mouse model of CRC to detect the expression level of iLRP in murine CRC tissue and adjacent tissue by IHC.mRNA,protein expression level and subcellular localization of iLRP in FHC and SW480 was measured by Q-PCR,WB and IF assay.(2)The expression of RPSA in clinical samples of human colon and normal colon samples were analyzed by sorting out TCGA database.2.Preparation of tumor cell overexpressing iLRP/FliC.(1)Lentivirus vector that overexpression of iLRP/FliC were constructed to infect SW480 and CT26,respectively.mRNA and protein expression level of iLRP and FliC was measured by Q-PCR,WB and IF.(2)WTCV have prepared after six groups cells was inactivated by 30%ethanol and detection of inactivated by trypan blue.3.The polarization and migration ability of Mφin vitro was detected after WTCV co-cultured with Mφ.(1)Preparation of Mφ:human’s Mφwas induced from THP-1 treated with PAM at the final concentration of 100ng/ml for 24h,BALB/c female mice,6-week-old,After mice were sacrificed,the abdominal cavity was rinsed with NS,and Mφ(pMφ)was extracted from the abdominal cavity of mice in the peritoneal lavage.hMφand pMφwas identified by FACS.(2)After WTCV was co-cultured with Mφfor 24h,Q-PCR was used to detect the changes of Mφpolarization-related bio-makers and TLR5 after co-cultured.The protein expression of IL-12,TNF-αand activation of NF-κB pathway of Mφafter co-culture was detected by WB.The migration ability of Mφto tumor environment was detected by Transwell assay.4.CT26-WTCV treated with CT26 tumor-bearing mice(1)WTCV vaccination project:We were arranged for three different vaccine plans.The project with the highest IgG content was selected for subsequent treatment experiments.(2)25 BALB/c 6-week-old female mice were randomly assigned and tumor-bearing mice was established.Tumor-bearing mice was treated with WTCV after day 6 of tumor formulation.The effect of CT26-WTCV on subcutaneous tumor growth in mice was observed during the treatment.Mice were sacrificed on day 7after the last vaccination,the tumor tissue was removed.(3)Liver,lung,spleen and tumor tissue of mice was removed,and then,the expression of iLRP in tumor tissue was detected by IHC,and the percentage change of effect/memory CD4~+T cell(CD4~+,CD44~+),CD4~+T,CD8~+T,mature APCs(MHCII~+),M1 type Mφ(F4/80~+,CD86~+)was detected by FACS after preparation of spleen cell suspension,HE staining was used to detect the morphological changes of liver,lung,and tumor tissues as well as the infiltration of inflammatory cells.Cytotoxic T Lymphocyte(CTL)assay in vitro was measured by LDH release assay.Results:1.The expression of iLRP in human and mouse CRC cells and tissues is higher than that in adjacent tissues and FHC,and is expressed on the membrane surface of SW480 and CT26We utilized an orthotopic mouse model of CRC,paraffin sections were prepared from mouse CRC and adjacent tissues,human CRC tissues and adjacent tissue sections were collected.(provided by the Laboratory of Gastroenterology,Zunyi Medical University),IHC,Q-PCR and WB results showed that the expression level of iLRP in tumor tissues was significantly higher than that in adjacent(**p<0.01).The expression of iLRP in SW480 was also significantly higher than that of FHC(**p<0.01).Cellular immunofluorescence results showed that iLRP was expressed on the surface of SW480 and CT26 cell membranes;we analyzed the expression of RPSA gene in clinical samples of human colon cancer and normal colon tissue using TCGA database(*p<0.05).2.Preparation of SW480 and CT26 expressing iLRP/FliC respectivelyWe constructed tumor cell that overexpressed iLRP and FliC.The results of Q-PCR and WB showed that iLRP and FliC were overexpressed in SW480 and CT26 tumor cells and expressed on tumor cell membranes.IF showed that FliC was expressed on SW480 and CT26 tumor cell membranes.3.Co-culture of WTCV and induce the Mφto M1 type polarization and the ability of Mφto migrate to tumor cells by inducing activation of the MφNF-κB signaling pathwayIn vitro:The results of Q-PCR showed that the co-cultivation group in which FliC-WTCV participated not only promoted the expression of TLR5 in Mφ,but also the expression of polarization-related molecules IL-12,TNF-α,and INOS in M1 type Mφ,and the expression of TNF-αsecreted by Mφincreased significantly,while the expression of IL-12 in the co-culture of the FliC and iLRP group and the combined group increased significantly;The results of transwell experiments showed that the migration ability of Mφto tumor cells after co-culture was enhanced;The WB results showed that the expression of phosphorylated p65(S536)in the Mφof the co-culture group with FliC-WTCV was increased(*p<0.05).The results shows that WTCV may promote the migration of Mφto tumor cells and the polarization of Mφto M1 type by inducing the activation of the MφNF-κB signaling pathway,and forming a beneficial conditions to fight with the tumor.4.iLRP-WTCV and FlliC-CT26 inhibit the growth of tumors in mouse by inducing the activation and maturation of APC and CTLIn vivo:The WTCV treatment results shows that iLRP-CT26,FliC-CT26,iLRP-CT26+FliC-CT26 groups can effectively inhibit the growth of subcutaneous xenograft tumors in mice(*p<0.05);The results of FACS indicates that iLRP-CT26have increased the proportion of effector/memory T cells,CD4~+T cells and CD8~+T cells(*p<0.05).The proportion of M1 type Mφand mature APCs have increased in the treatment group of FliC-CT26.The CTL experiments results shows that the killing ability of CTL cells in mice against iLRP~+CT26 was enhanced in iLRP-CT26group(*p<0.05).We concluded that iLRP-CT26 have increased that effect/memory T cells,CD4~+T cells,CD8~+T cells,and promotion of CTL effects against iLRP~+tumors in mice to achieve antitumor effects,while FliC-CT26 can promote M1 type of Mφpolarization,APC maturation and activation.The combination treatment group resulting both a significant antitumor effect with CT26 tumor growth of tumor-bearing mice.Conclusion:1.The expression of iLRP in human and mouse CRC cells and tissues is higher than that in adjacent tissues and FHC,and is expressed on the membrane surface of SW480 and CT26.2.Preparation of SW480 and CT26 expressing iLRP/FliC respectively.3.WTCV and Mφco-culture promote the expression of TLR5 of Mφby inducing the activation of MφNF-κB pathway,and promote the Mφpolarization to M1 type and the ability of Mφto migrate to tumor cells.4.iLRP-WTCV and FlliC-CT26 inhibit the growth of tumors in mouse by inducing the activation and maturation of APC and CTL. |
PDF Full Text Request