Study On Anti-tumor Immune Response Induced By Dendritic Cells Loaded With Colon Cancer Stem Cells-derived Antigens

Objective Colon cancer is one of the most common malignant tumors in the digestivetract. So far, surgical resection with or without adjuvant chemo-and/or radiationtherapy remains the key modality for colon cancer, but unfortunately shows limitedclinical benefits due to high rate of tumor metastasis. Although current adjuvantchemo-radiation therapy has been shown to extend patient survival in the presence ofrecurrent lesions, severe side effects usually limit the efficacy of this anti-cancermodality. Therefore, it is urgently required to develop more effective treatmentstrategies for colon cancer. In recent years, preclinical data indicate that cancer stemcells (CSC) drive tumor growth and are thought to be responsible for tumorself-renewal/maintenance, recurrence, distant metastasis, angiogenesis, anddrug/radiation resistance, the CSC hypothesis proposes that these cells must beeradicated to cure the cancer. Thus,in this study we isolated CD44+CT-26cells as thesource of colon cancer stem cells and investigate whether colon cancer stemcell-derived lysate or total RNA-pulsed dendritic cells (DC) could induce cytotoxic Tlymphocyte(CTL)activity against colon cancer stem cells in vivo.Methods CD44+CT-26cells were magnetically isolated using CD44MicroBeads andcultivated in serum-free DMEM/F12medium with the20ng/ml EGF,20ng/ml bFGFand2%B27. The bone marrow-derived DC pulsed with mouse colon cancer stemcells(CT-26SC) lysate (Pro-DC) or RNA (RNA-DC) were collected to immunizesyngeneic na ve BALB/c mice as vaccination. And then CT-26colon tumor stem cellswere implanted subcutaneously(s.c.)in BALB/c mice. In order to determine whetheror not vaccination induce the therapeutic potential in the established colon tumormodel, tumor-bearing mice were injected with vaccination. Tumor growth wasassessed every2-3days. Finally, the spleen of immunized and tumor-bearing micewere collected and then CTL activity and IFN-γ secretion were evaluated.Results The tumor volume of Pro-DC and RNA-DC group was significantly smallerthan that of unpulsed-DC group, and the median survival time of mice immunizedwith Pro-DC and RNA-DC was significantly longer than that of those immunized with unpulsed-DC (P<0.05). The results of the LDH assay show that splenocytes frommice that received Pro-DC vaccination displayed significantly stronger cytolyticactivity against CT-26SC at ratios10:1,20:1, and40:1of effector cells to targetcells(P<0.05).We further examined the secretion of IFN-γ produced from splenocytesderived from immunized and control mice by ELISA assay. Splenic T cells of Pro-DCor RNA-DC group with CT-26SC at an E:T ratio of20:1produced higher levels ofIFN-γ in vitro; however, the splenic T cells from unpulsed DC or PBS group did notproduce higher levels of IFN-γ when stimulated with CT26SC. Vaccination withPro-DC and RNA-DC could also induce anti-tumor immunity against CT-26SC inmurine therapeutic models.Conclusion (1) Immune protection group: The tumor volume of Pro-DC andRNA-DC group was significantly smaller than that of unpulsed-DC group, and themedian survival time of mice immunized with Pro-DC and RNA-DC wassignificantly longer than that of those immunized with unpulsed-DC. T cells ofPro-DC and RNA-DC group were able to induce effective CTL activity against CT-26SC at a serial E/T ratio of10:1,20:1, and40:1, but not unpulsed-DC group, andproduced significantly higher levels of IFN-γ when stimulated with CT-26SC.(2)Immunotherapy group: vaccination with Pro-DC and RNA-DC could also induceanti-tumor immunity against CT-26SC in murine therapeutic models. Inconclusion,CT-26SC lysate or RNA-pulsed DC can induce tumor-specific CTLactivity against colon cancer stem cells, suppress the growth of colon cancer andretarded tumor growth in vivo and serve as a novel DC-based tumor vaccine targetingcolon cancer stem cells.