The Antitumor Effects Produced By CD40 Ligand Expressed On Mouse Colon Cancer Cells Colon26 Are Linked With The Maturation Of Dendritic Cells

Objective: colonic cancer cell line colon26 was used as research agent. To study the anti-tumor activity of colon26 transfected with full-length mouse CD40L cDNA in vitro and in vivo. To analyse the anti-tumor effects of the co-culture of CD40L-expressed tumors and bone marrow-derived dendritic cells.Methods:1 The amplification of full-length mouse CD40L cDNA. The pMKITneo CD40L plasmid was transformated into the competence Escherichia coli DH5ɑ, and abundantly amplificated the Escherichia coli. The plasmid abstracted from the Escherichia coli was verified by enzyme digestion and sequencing, and the product expressed was detected by RT-PCR.2 Transfection of CD40L expression vector into tumor cells. Colon26 cells were transfected with the full-length mouse CD40L cDNA by lipofectamineTM2000 and then G418 resistant cells were selected. G418 resistant colon26 cells transfected with vector DNA pMKITneo were used as a control.3 Detection of the expression of CD40L. Transcription level of CD40LmRNA in colon26 transfected cells and the membrane binding rate of PE-labelled CD40L antibody were detected with RT-PCR and FCM respectively. The protein expressional level of CD40L in colon26 transfected cells were detected with western blot and laser scanning confocal microscope respectively.4 The proliferation of the transfected cells was determined. The cells proliferation was detected by MTT assay. To determinate proliferation response by MTS assay and draw curve of cells growth. 5 Detection of the invasion of the transfected cells. The expression level of MMP-2 mRNA in cells was detected through semi-quantitative RT-PCR assay. Using invasion experiment to detect the cells'invasive abilities.6 The preparation of dendritic cells. Bone marrow cells from tibias and femurs were depleted of erythrocytes with spallation buffer. These cells were further cultured in RPMI1640 medium supplemented with recombinant mouse GM-CSF and IL-4, and 10% fetal calf serum for 7 days. Nonadherent cells were harvested and used as DCs. CD11c+ DCs were separated by CD11c-conjugated magnetic beads. The expressions of CD11c on DCs were examined with flow cytometry.7 Test of in vitro immunity function of DC culture with tumor cells expressing CD40L. Tumor cells were cultured with DCs for 24 hours and the phenotype of DCs was examined with flow cytometry. The expression level of IL-12,IL-23,IL-18,IFN-γand IFN-(γMig)mRNA in co-cultured DCs- tumor cells were detected by RT-PCR. The level of IL-12,IL-23 and IFN-γin supernatant of co-cultured DCs- tumor cells were detected by ELISA assay.8 The BALB/C mouse was inoculated colon26 cells to establish tumor model. Logarithmic phase of colon26 cells were collected into single-cell suspension. To adjust cell concentration 1×107/ml. Colon26 cells were injected in the BALB/C mice's right department of shoulder (0.1ml, 1×106 once) to estabilish mouse xenograft model.9 Anti-tumor effects of tumor vaccine expressing CD40L in vivo. After mice were injected tumor cells 3 days, the DCs, tumor vaccine expressing CD40L, co-cultured DCs-tumor cells expressing CD40L (experiment group) or PBS (control group) was re-infused through peritoneal cavity in tumor barring mice. Every group has 5 mice. Tumor changing and treatment effect were detected. Peripheral blood of tumor barring mice, tumor tissue, liver and spleen were collected after treatmented in four groups.10 The level of IL-12, IL-23, IFN-γ, IL-10 and TGF-βin peripheral blood of tumor barring mice was detected by ELISA assay.11 After HE staining, the histophological changed and tumor cells infiltrated of liver and spleen were observed by microscope.Results:1 Identification of inserted CD40L DNA sequence in this plasmid was performed by enzyme digestion, PCR and DNA sequencing. The results showed that it fully matched the parent plasmid's nucleic acid database with right direction.2 Colon26 cells were transfected with the mouse CD40L gene and a clone expressing a large amount of CD40L on cell surface was selected. The shape of transfected cells has no change with wild cells. The cells were rhombus, full shape.3 Expression of CD40L on colon26/CD40L cells was much higher than that of colon26 cells, were detected with RT-PCR, FCM, western blot and laser scanning confocal microscope respectively. It showed the recombinant vector could play a normal role in translation of CD40L, transcription and protein synthesis.4 In vitro the proliferation rates of parent and CD40L expressed colon26 cells were not different were detected with MTS assay.5 Expression of MMP-2 mRNA on colon26/CD40L cells was gradually decreased than that of colon26 cells, were detected with RT-PCR. The results of invasion experiment shows that the invasive ability of colon26/CD40L cells decrease remarkable, compared with colon26 cells.6 The bone marrow dendritic cells were cultured by different cytokines in vitro. The cells grew nonadherently, and showed branching-like and pseudopod-like under invert microscope. DCs were separated by CD11c-conjugated magnetic beads at 7 days, and the purity of CD11c may reach above 92.88%.7 The Expression of the CD80, CD86, MHCⅠand MHCⅡof DCs that were cultured with colon26/CD40L was gradually increased than that of in control group. The Expression of the IL-23, IL-12, IL-18, IFN-γand Mig (monokine induced by IFN-γ) genes was induced in the DCs that were cultured with colon26/CD40L but not with colon26 cells. The expression level of the IL-23, IL-12 and IFN-γin co-cultured DCs-colon26/CD40L cells was gradually increased than that of in control group.8 After subcutaneous injected colon26 cells 3 days, the mouse xenograft rate was 100%. The mouse xenograft model was successful established.9 The tumor size in the injecting co-cultured DCs-tumor cells expressing CD40L group was smaller than that in the injecting DCs group, the injecting tumor vaccine expressing CD40L group and PBS. The inhibitory rates were 47.92%, 32.26%, 32.95% and 0%, respectiveily.10 The level of IL-12,IL-23 and IFN-γin the injecting co-cultured DCs-tumor cells expressing CD40L group was higher than that in the injecting DCs group, the injecting tumor vaccine expressing CD40L group and PBS. But the level of IL-10 and TGF-βwas decresed.11 The results of HE showed that the sections of tumor tissues in the injecting DCs group and tumor vaccine expressing CD40L group, and there were a small quantity inflammatory cells and necrosis. However in the injecting co-cultured DCs-tumor cells expressing CD40L group has many inflammatory cells infliltrated and mass necrosis, and tumor cells scattered and circumscribed. Cancer nodules were not found in spleen and sliver in that three group. Many tumor cells and less inflammatory cells were detected in the section of tumor tissues in the PBS group, and cancer nodules were found in spleen and sliver in this group.Conclusions: These data directly showed that the expression of CD40L in colon cancer cells facilitated the interaction between DCs and tumors, enhanced the maturation of DCs, induced secretion of cytokines, and consequently produced T-cell-dependent systemic immunity. DC-CD40L -tumor cells may be a useful strategy for cancer immunotherapy.