Investigation of antitumor immune responses of dendritic cells loaded with cancer stem cell lysate

The use of the body's own defensive cells to eliminate the "cancer stem cells" as the root of cancer is one of the most important revolutions of cancer immunotherapy. Cancer stem cells (CSCs) represent a unique small subpopulation that possess capacity for self-renewal and tumor initiation. The main purpose of cancer immunotherapy is to stimulate a strong immune response to CSCs. The immune system is powerful and also complex and consists of an army that reflects the relationship between cells, tissues and organs. Dendritic cells (DCs), one of the most important members of the army, are professional cells that serve as an important bridge between innate and adaptive immunity. Following the maturation process, DCs play an essential role role in the induction and maintenance of anti-tumor responses. However, the therapeutic efficacy of DC-based therapy in cancer patients is not satisfactory and new strategies need to be developed. Sialic acids on the surface of DCs are very critical in uptake, processing, and presentation to T-cell of antigen and formation of immunological synapses. The goal of this study is twofold: i) to investigate changes in DCs treated with CSCs antigens after removal of sialic acids in various aspects, ii) to evaluate of the immune response to CSCs. Breast cancer stem cells (CD44+/CD24-/low) were isolated by fluorescence activated cell sorting (FACS) and the lysates containing the antigens of CSCs were obtained. DCs isolated from bone-marrow were characterized using CD11c. After removal of the sialic acids from the surface of the DCs by neuraminidase, the DCs were treated with the CSCs lysate. The effects of CSCs lysate on dendritic cells were evaluated by cell viability, apoptosis and cell cycle assays. Changes in co-stimulatory molecules such as CD40, CD80 and CD86 in immature (imDC) and mature (mDC) cells were analyzed using flow cytometry. The genes associated with various signaling pathways were evaluated by PCR. Biochemical (glycogen, lipid, protein, nucleic acid) changes in the dendritic cells were analyzed using ATR-FTIR. Morphological changes in cells were examined visually by SPM and histochemical staining. Changes in IL-12 production in the cells were analyzed by ELISA assay. In the in vivo experiments, the immune response effects of imDC and mDCs were evaluated by using a variety of parameters. The data obtained from the study show that mDCs exhibit a more effective immune response compared to imDCs. It has been shown that DCs treated with CSCs antigens form podosome structures and cell skeleton elements such as actin, gelsoline, talline, Arp/2, WASp play an important role in antigen uptake. After the maturation, there was an increase in the expression of co-stimulatory molecules such as CD86, CD80 and CD40. Significant changes in Notch, MAPK and interleukin signaling pathways, which are known to be associated with the differentiation and function of DCs, have been identified. There were significant differences in nucleic acid, lipid, carbohydrate and protein structures, and a decrease in lipid droplets and an increase in glycogen after maturation were determined. Morphological analyzes showed significant changes in the size, surface area and roughness properties of the cells. After maturation, there was an increase in IL-12 production. In vivo tumor model for the investigation of anti-tumor immune responses, it was found that the mice vaccinated with mDC had a significant decrease in both tumor volume and weight. However, a decrease in proliferation-related markers and an increase in apoptotic index were determined. Significant increases in CD4 and CD8 ratios were found in mDCs compared to imDCs. Vaccination with mDCs has been shown to inhibit metastasis. A reduction in the expression of markers such as Snail, Slug and Twist associated with epithelial mesenchymal transition has been identified. The data obtained from this study showed that sialic acid removal and loading with CSCs antigens provide important molecular, biochemical, morphological and functional changes. These changes contributed to the increase of anti-cancer immune responses of DCs. Keywords; cancer stem cell; dendritic cell vaccine; immunotherapy; sialic acid; maturation; immune response