Pre-clinical Study On The Treatment Of Malignant Solid Tumors With Cytokine-Induced Killer Cells

Part Ⅰ: CIK cells preparation and biological charactersPurpose: CIK cells are new immunological competent cells that are employed in adoptive cellular immunotherapy. In this study, we established a multi-cytokine culture system to prepare CIK cells by amplifying peripheral blood mononuclear cells in vitro. We evaluated the phenotype, multiple cytokines secretion of CIK cells and anti-tumor activities of CIK cells against the gastric carcinoma cell line and the colon cancer cell line. Based on these experiment results, we shed lights on the possibility of the application of CIK cells in clinical treatment against cancer.Material and Method:1. Preparation of CIK cells in vitro:Peripheral blood mononuclear cells (PBMC) from donor were incubated in vitro with various cytokines, such as CD3 monoclonal antibody (CD3McAb), interleukin-2 (IL-2), interferon-gamma (IFN-Y) and interleukin-1α (IL-1α), in a certain sequence. Cells were incubated in a culture system that is renewed every 3 days with fresh complete medium and various types of cytokines such as IL-2 and IFN-γ. After 12 to 14 days, CIK cells were harvested and tested for proliferation profiles and survival rates.2. The phenotype and cytokine secretion of CIK cells:Flow cytometry analysis was used to analyze the phenotype of CIK cells. ELISA was used to test cytokines, such as IL-2, IFN-γ and IL-10, that are secreted fromCIK cells.3. Anti-tumor cytotoxicity of CIK cells:The gastric carcinoma cell line (823 cells) and the colon cancer cell line (HCT-8 cells) were used as target cells. The LDH method was employed to measure the cytotoxicity of CIK cells against cancer cells.Results:1. Cells were amplified to a total population of 49.8 times of initial cellular population after in vitro culture and incubation. The survival rate of CIK cells is above 95%.2. After the induction of various cytokins, the phenotypes of CIK cells, such as CD3, CD3/CD8> CD3/CD4> CD3/CD56, have more than that of peripheral blood mononuclear cells. Cytokines secreted from CIK cells , such as IL-2> IFN-y, are more than that from peripheral blood mononuclear cells. In the contrast, cytokine of IL-10 from CIK cells is less than that from peripheral blood mononuclear cells.3. Results from in vitro cytotoxicity experiment show that CIK cells have potent killing activity to 823 cells and HCT-8 cells.Conclusion: The multi-cytokine culture system is capable of amplifying peripheral blood mononuclear cells into CIK cells in vitro. The CD3+/CD56+ cell, which is the major effect cell of CIK cells, responds with a consequence of prompt proliferation and productive secretion of cytokines. These downstream events have potent killing activity to cancer cells that can be applied towards adoptive immunotherapy against malignant solid tumors.Part II: Impacts of cancer cells on CIK cells and Joint abilities of CIK cells and 5FU to kill cancer cells in vitroPurpose: To discuss the impact of malignant cancer cells on the preparation, proliferation inhibition and induced apoptosis of CIK cells. To discuss the possibility of combinational application of chemotherapy and CIK cells in clinical treatment against cancer.Material and Method:1. Supernatant from 823 cells and HCT- cells culture system and the chemotherapy drug (5FU) were added separately into the culture system of CIK cells. Experiment data, such as CIK cells proliferation rate, percentage of the major effect cell (CD3+/CD56+ cells) and killing activities was taken individually to evaluate the impact of each experiment condition on CIK cells.2. CIK cells and cancer cells are co-cultured in medium. Flow cytometry analysis was used to measure the influence of cancer cells to CIK cells in respect of the proliferation inhibition and induced apoptosis.3. ELISA was used to measure the content of immunoinhibitory cytokines in the culture supernatant from CIK cells. Ultracentrifugation was used to separate exosome from culture system. Those experiment data were analyzed to evaluate their inhibitory effect on CIK cells.4. The LDH assay and the MTT assay were used separately to test killing activities of either CIK cells to cancer cells that have been pre-incubated with 5FU or mixture of CIK cells and 5FU to cancer cells without pre-treatment.Results:1. The addition of 5FU and the supernatant from cancer cell culture system decreases the proliferation rate of CIK cells and the purity of CD3+/CD56+ cells. The addition of the supernatant from cancer cell culture system suppresses thecyiotoxiciiy 01 l,ijs. ceiis 10 omer similar cancer cen lines.2. Co-culture of cancer cells and CIK cells increases the apoptosis of CIK cells, while decreases the proliferation rate of CIK cells.3. Immunoinhibitory cytokines were found to exist in the supernatant of 823 cells and HCT-8 cells. Exosomes which are small(40-100 run in diameter) single-membrane bound vesicles are extracted by ultra-speed centrifugation from the supernatant of 823 cells, and identified by electron microscopy. Depletion of the exosomes from the supernatants of 823 cells lowers their suppression to the proliferation rate of CIK cells.4. Pre-incubating cancer cells with 5FU increases their sensitivity to CIK cells. Applying 5FU and CIK cells in a certain sequence increases the cytotoxicity of CIK cells to cancer cells. After cancer cells are pre-incubated with 5FU, the killing activity of CIK cells to cancer cells is better than that if both CIK cells and 5FU are applied to cancer cells at the same time.Conclusion: Cancer cells have inhibitory effects to CIK cells on their biological activities and preparation. The mechanism could be immunoinhibitory cytokines and exosomes secreted from cancer cells. 5FU increases the sensitivity of cancer cells to CIK cells. The combinational application of CIK cells and chemotherapy drugs increases effects on anti-cancer treatment.Part III: Organ distribution and tumor restraint function ofCIK cells in vivoPurpose: To discuss the distribution pattern of CIK cells and their anti-tumor...