| Background: Colon cancer is a common clinical tumor, liver metastases has become amajor factor in colon cancer death. The main treatments for colon cancer livermetastasis include surgical resection (embolization, radiofrequency ablation),radiotherapy and chemotherapy, but a single method can not effectively reverse thepatients’ tumor-bearing state, so the treatment of colon cancer liver metastasis should bean integrated strategy. At present, as one of the main treatments of colon cancer livermetastasis, adoptive cellular imunotherapy (ACI) has got growing concern by thepeople. Domestic and foreign studies have shown that agonistic CD40monoclonalantibody (CD40mAb) can reverse tumor immune suppression and enhance DCsmediated T-cell anti-tumor effect. However, there is no report about CD40monoclonalantibody used in adoptive cellular imunotherapy for colon cancer liver metastases athome and abroad so far.Objective: Inducing dendritic cells (DCs) by agonistic CD40monoclonal antibody(CD40mAb) to study its act on enhancing the effect of tumor antigen-specific cytotoxicT lymphocytes (CTL) to kill the CT-26colon cancer cells of mice in vitro, and theimmunetherapeutic effect on murine colon carcinoma liver metastases.Method:1) Liver metastases model were established in BALB/c mice by intrasplenicinjection of colonic carcinoma cells (CT-26).2) Remove normal mouse spleens,separate and extract mouse lymphocytes by using mouse lymphocyte separation liquid,the adherent cells and adherent cells were cultured respectively; adherent of DCs wereloaded with apoptotic tumor cells CT-26and pulsed with or without agonistic CD40monoclonal antibody (CD40mAb) in order to activate DCs maturely.3) Mature DCs and cognate suspension cells co-cultured, adding cytokines and respectively to getCTLCD40Aband CTL.4) Cell morphology and activity was observed in culturing process,the proliferation of CTLs in each group were detected by cell counts and CTLs inkilling of murine colon carcinoma CT-26cells in vitro were detected by LDH assay,analyse the killing effects.5) Continuously inject saline, CD40mAb, CTLCD40AbandCTL (1times/week,3times in all) through tail vein in order to treat the colon cancerliver metastasis model in mice and analyse the antitumor effects.Results:1) Successfully constructed the mouse colon cancer liver metastasis model andthe tumor formation rate is86.7%(13/15), the pathological findings in line with thecharacteristics of colon cancer liver metastasis.2) Observed by light microscopy, thenumber of cells and colony of CTLCD40Abare more than the CTL cells and theCTLCD40Abgroup has larger cell density and single cell volume. Cells count showed thatCTLCD40Abcells proliferate more obviously than CTL cells and the most obviously isCTLCD40Ab1ug/ml group.3) At the10:1anti-target ratio, the killing rates ofCTLCD40Ab1ug/ml, CTLCD40Ab5ug/ml and CTL group against murine coloncarcinoma CT-26cells were65%,53%and29%respectively. And the killing rate ofCTLCD40Ab1ug/ml and CTLCD40Ab5ug/ml group compared with the CTL group wassignificantly higher (P <0.05).4) Tumor inhibition in vivo: Tumor suppression rate ofthe CD40Ab group, the CTL group and CTLCD40Aggroup was (23.1±12.5)%,(53.8±9.8)%and (64.6±10.2)%respectively; Compared with control group, the mice tumorweight of CTL and CTLCD40Aggroups reduced dramaticlly (P <0.05), and CTLCD40Aggroup was also significantly lower than the CD40Ab group (P <0.05). There was nosignificant difference between CTL group and CTLCD40Aggroup, as well as the CTLgroup and CD40Ab group in tumor weight (P=0.089and P=0.077, respectively).Histopathology show a large number of lymphocytes infiltrating in tumor region in micetreated with CTL and CTLCD40Ag.Conclusion:1) Tumorigenicity of the mouse colon cancer liver metastasis model of thisstudy is high, security and stability, and a realistic simulation of human colon cancerliver metastases pathological process. It is a simple and operable preparation technique and has a better value in basic and clinicalthe study for colon cancer liver metastases.2)Agonistic CD40monoclonal antibody (CD40mAb) can activate tumor antigen loadedDCs in vitro and then act on T cells, thereby significantly enhance the proliferativeactivity of tumor-specific cytotoxic T lymphocytes (CTLs) and the cytotoxic activityagainst tumor cells in vitro.3) In vivo, activation of tumor antigen loaded DCs by theagonistic CD40monoclonal antibody (CD40mAb) and thus the tumor-specific cytotoxicT lymphocytes (CTLs) can significantly inhibit the growth of metastatic tumors of theliver in mice, and agonistic CD40monoclonal antibody (CD40mAb) has possible effectof enhancing antitumor effect of tumor-specific cytotoxic T lymphocytes (CTLs) inmice. |
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