Study On The Treatment Of Anti-colon Cancer Mediated By DC Vaccine Loaded With Cancer Stem Cells-derived DRibbles

Objective:Colon cancer is a common malignant tumor of the digestive tract,and its incidence and mortality are increasing year by year.Because colon cancer is easy to relapse after surgery,and easy to produce radiotherapy and chemotherapy tolerance,current clinical treatment methods are not satisfactory.Cancer stem cells(CSCs)are a small number of subpopulation of tumors,with self-renewal and infinity,capable of multi-directional differentiation and stay in a long-term“dormant”state.CSCs can not easy to be killed by traditional radiotherapy and chemotherapy,which due to CSCs becomes the key factor in tumore metastasis and recurrence.The strategy of treating cancer with CSCs as a target becomes a new breakthrough in cancer therapy.Dendritic cells(DCs)are the most powerful professional antigen presenting cells found so far,which can activate T cells,induce specific immune responses,and enhance the ability of T cells to kill tumors.Numerous studies have shown that preparation of anti-tumor vaccine using DCs can effectively activate the anti-tumor responses of immune cells.The efficacy of DC vaccines is mainly limited by DC isolation and culture techniques,immunogenicity of loaded antigens,vaccine application methods,and so on.At present,the most widely used load antigen is the lysates of tumor cells,but its loading effect still needs to be improved.Studies have shown that autophagosomes produced by cell autophagy contain defective ribosomal products(DRiPs),misfolded proteins,which have the potential to act as novel carrier antigens,named DRibbles(DRiPs in belbs).Therefore,in this study,we used DRibbles derived CSCs as an antigen to prepare DC vaccines and measure their effects on colon cancer by examing a serie of assay on mouse colon cancer xenograft model.Methods:(1)CD44~+CT-26 cancer stem cells were selected from mouse colon cancer cell line CT-26 by immunomagnetic beads method;(2)Mouse bone marrow mononuclear cells were isolated and induced differentiated to dendritic cells by cytokines;(3)Autophagy formation induced by addition of 100 nM rapamycin,100 nM bortezomib,10 mM ammonium chloride to cancer stem cell culture system,and antophagosomes were accumulated in cells;(4)DRibbles and lysates derived from CT-26 tumor cells and CD44~+CT-26 cancer stem cells stimulated spleen lymphocytes in vitro respectively,the effects of different antigens on the proliferation of mouse lymphocytes were examed;(5)DRibbles and lysates derived from CT-26 tumor cells and CD44~+CT-26 cancer stem cells were loaded on DCs as vaccines applied to the mouse colon cancer xenograft model respectively,PBS-loaded DCs as treatment control,PBS as negative control.The tumor growth,survival status,tumor wight were measured,the killing ability of mouse effector lymphocytes in vitro were detected by LDH assay.Results:(1)The purity of CD44~+CT-26 obtained by immunomagnetic beads method was98%.It was able to form single-cell cloned spheres suspend in serum free medium,which couled induced to differentiate into adherent tumor cells by serum;(2)After 7days of induction and differentiation,the expression of CD11c on cells surface reached 97.2%,indicating the mouse bone marrow mononuclear cells can be induced into DCs with high purity by this method.(3)The induced autophagosomes from tumor cells and cancer stem cells were collected and identified by transmission electronmicroscopy.ComparedwithCT26-derivedDRibbles(TD),CD44~+CT-26-derived DRibbles(SD)can significantly promote the proliferation of mouse splenic lymphocytes in vitro(P<0.001);compared with CD44~+CT-26-derived lysate(SL),SD can significantly promote the proliferation of mouse spleen lymphocytes in vitro(P<0.001);(4)Compared with Control group,all treatment groups show the significantly inhibited on tumor growth of mice(P<0.01).At the end of experiment,the tumor volume of each group was ascending to large:SD-DC group<TD-DC group<SL-DC group<TL-DC Group<PBS-DC group<Control group,each group had significant difference(P<0.01);the order of tumor weight of each group was ranked same as tumor volume,and the differences were significant(P<0.05).The survival period of mice showed that,on the 30~thh day after inoculation of tumor cell,the survival rate of SD-DC group and SL-DC group was87.5%,which was significantely higher than that of Control group(P<0.01);the survival rate of TD-DC group was 75%,which was significantly higher than that of Control group(P<0.05);(5)After vaccined,the ability of cell killing of spleen lymphocytes in vitro was ranked from high to low as:SD-DC group>TD-DC group>SL-DC group>TL-DC group>PBS-DC group>Control group,SD-DC group had a significantly stongest killing effect among other groups(P<0.0001).Conclusion:CD44~+CT-26 stem cell-derived DRibbles can effectively stimulate the proliferation of T lymphocytes.In mouse colon cancer xenograft models,DC vaccine loaded with CD44~+CT-26 cell-derived DRibbles have a better treatment effect than that of CD44~+CT-26 cell-derived lysate,and also superior to CT-26 cell-derived DRibbles.