Investigation of the effect of genotoxic stress-induced autophagy-associated secretome on natural killer cell-mediated antitumor immune response

Objective: The aim of this project was to characterize the genotoxic stress induced autophagic secretome and to investigate whether autophagic secretome affects the NK-mediated immune response to MCF-7 breast cancer cells. Material and Methods: Etoposide was used to induce genotoxic stress mediated autophagy in MCF-7 cells. Analysis of autophagy induction was tested by using autophagy markers including LC3I/II and p62 degreadation by western blot and immunfluoresence. Annexin V/7AAD analysis used to measure the rate of apoptosis in MCF-7 cells. LC/MS-MS analysis and cytokine array analyses were used to charatacterize the the chemotherapy-induced autophagic secretome content. Finally, how the chemotherapy-induced autophagic secretome affects the capacity of DNAM1-NK-92 cells to target MCF-7 cells was determined by degranulation analysis. Results: Autophagy markers such as LC3I/II and p62 showed that autophagy was induced in MCF-7 cells by 150 μM Etoposide for 24 hours and it was determined that chemotherapy-induced autophagy did not cause apoptosis. LC/MS-MS results showed that metabolic enzymes, tumor antigens, chaperones and metastasis-related proteins were secreted during chemotherapy-induced autophagy. In addition, 41 different cytokines/chemokines and growth factors were detected in the secretome by cytokine array analysis and validated by ELISA analysis for some cytokines, chemokines and growth factors such as CCL5, TGFβ and IL10. Finally, after treatment of control NK-92 and genetically modified DNAM1-NK92 cells with autophagic secretomes, differences in the capacity to target MCF-7 cells were observed between the groups. Conclusion: In this thesis, for the first time, the contents of the chemotherapy-induced autophagic secretome were fully characterized. Under in vitro conditions, important data were obtained to determine whether chemotherapy-induced autophagy induction stimulates NK cell effector functions and how they detect and respond to the types of drug-induced stresses that contribute to anticancer activity.