The incidence of cancer has been steadily increasing over the past hundred years, and standard treatment approaches often prove insufficient. In recent years, immunotherapy has emerged as one of the key new approaches to treatment, utilizing the host’s immune system to recognize and destroy cancer cells and to prevent tumor growth and development. One type of immunotherapy is inducible immunotherapy, which allows external control of treatment through the use of small molecules, light, ultrasound, or radiation. Pyroptosis is a form of immunogenic cell death that is triggered by various external or internal stress stimuli and leads to a strong immune response. The effectors of pyroptosis are gasdermins, whose active N-terminal domains bind to the cell membrane, oligomerize, and form pores. During pyroptosis of tumor cells, tumor antigens are released into the surrounding environment, potentially leading to a long-term antitumor immune response. It has been shown that pyroptosis of even a small number of tumor cells is sufficient to promote infiltration of immune cells into the tumor immune microenvironment.
The aim of this master's thesis was to develop a synthetic biology system that induces immunogenic cell death by pyroptosis in mammalian HEK293 or HEK293T cells in response to a selected stressor. In the first part, we focused on selecting a promoter and a suitable stressor that would activate that promoter. We tested two stress-responsive promoters: p21 and a minimal promoter with NF-κB response elements (minNF-κB). We attempted to induce these promoters with reactive oxygen species (hydrogen peroxide) and chemotherapeutic agents. We measured promoter activation levels using a luciferase assay. We found that the best activation was achieved with the combination of the minNF-κB promoter and the chemotherapeutic doxorubicin. To determine whether promoter control could influence the induction of cell death, we fused the minNF-κB promoter to the sequence encoding the N-terminal domain of gasdermin D. To assess pyroptosis activation, we measured the proportion of cell death using propidium iodide (PI) and confocal microscopy, and found that the system combining minNF-κB and doxorubicin successfully triggered pyroptosis. To determine whether this system could also be used to induce the expression of other proteins, we fused the minNF-κB promoter to the sequence encoding interleukin-15 and demonstrated that the addition of doxorubicin triggered protein expression. Thus, we developed a system for controlling cellular processes that can be used in research and also holds potential for clinical therapy.
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