Scientific background: Various ablative techniques are used to treat tumors, including radiotherapy (RT) and electrochemotherapy (ECT). With RT, the tumor cells die due to the effect of ionizing radiation (IR), while with ECT, as a result of a combination of chemotherapeutic agents and the electroporation of tissues, the tumor cells die at the site where the electrical pulse is applied due to the cytotoxic effect of the drug. Both therapies damage the genetic material of the tumor cells (DNA) and thus prevent their further proliferation, trigger the activation of the immune system and cause various types of cell death. These include immunogenic cell death (ICD), apoptosis, necrosis, and autophagy. ICD is a form of cell death in which the adaptive immune system is stimulated against tumor neoantigens. The molecules involved in ICD are: calreticulin exposed on the cell membrane (CRT), extracellularly released HMGB1, and adenosine triphosphate (ATP). In addition to these molecules, the immune (non-)response is influenced by other immunologically important changes in tumor cells, including the molecules MHC I and II, PD-L1, and CD40. RT has been shown to stimulate activation of the host immune system and trigger ICD, resulting in immune memory and sometimes systemic antitumor effects. However, it is not yet known which doses and procedures activate the immune system most effectively in different tumor models. The efficacy of ECT depends on the immunogenicity of the tumors and their intrinsic sensitivity to chemotherapeutic agents. Therefore, the type of chemotherapeutic agent, its concentration, and the time frame after therapy that induces the most immunological changes in tumor cells must be determined to optimize the activation of the immune response by ECT. The aim of this dissertation was to determine which chemotherapeutic agent used in ECT induces the most important immunological changes in tumor cells in vitro, at which concentration, and in which time frame after therapy, and to compare these with the changes after IR.
Methods: In the first part of the dissertation, we investigated the sensitivity of the murine tumor cell lines B16-F10 (melanoma), 4T1 (mammary carcinoma), and CT26 (colorectal carcinoma) in vitro to ECT with different chemotherapeutic agents and to IR. First, we determined the electropermeability of the cell lines by adding propidium iodide to the suspension cells. The suspension was pipetted between two stainless steel plate electrodes (2.4 mm between the electrodes) and electroporated with pulses (8 pulses, 100 µs duration, 1 Hz) with different amplitude–to–distance ratios. In the next part of the study, we determined the survival of the cell lines after ECT using cisplatin (CDDP), oxaliplatin (OXA), and bleomycin (BLM), and after IR with a clonogenic assay. The Jouan GHT beta electrical pulse generator was used to deliver electrical pulses as used in clinical ECT (8 pulses, 1300 V/cm, 100 µs duration, 1 Hz), and cells were seeded in plates at different densities. To determine the radiosensitivity, the cells were seeded in plates at different densities and irradiated with different doses after three hours. The inhibitory concentrations were determined for each chemotherapeutic agent using the survival curves, and the inhibitory doses IC30, IC50, IC70 were calculated using a linear-quadratic model. In the second part, cells were treated in vitro with inhibitory doses, and then the exposure of CRT was determined by flow cytometry. HMGB1 release was determined by ELISA assay, and ATP release by luminescence assay. Two methods were used to determine apoptosis and necrosis. In the first method, the cell lines were treated in vitro with inhibitory doses, and then flow cytometry was used for determination. In the second method, cells were seeded on plates, treated with inhibitory doses, then fluorescent reagents were added and cells were monitored with time-lapse imaging. To determine autophagy, the cell lines were treated in vitro with IC50, then seeded on plates and incubated for 24 hours. The assay to determine autophagy was used for analysis, and images of the samples were taken with a microscope. In the last part, we determined the changes in the presence of immunologically important membrane markers after both therapies. The expression of the cell markers MHC I, MHC II, PD-L1, and CD40 was determined in vitro at different time points after treatment with inhibitory doses.
Results: In the first part, we found that all three cell lines (B16-F10, 4T1, CT26) were comparably permeable at all tested voltage-to-distance ratios and equally sensitive to CDDP, BLM, and IR. In the second part, we showed that cells usually die immediately after ECT, whereas after IR they try to repair the DNA damage caused, and that cell death is actually the result of unsuccessful repair. In general, ECT had more favorable effects on the development of ICD compared to IR, even in less immunogenic cell lines, as it caused ICD in more lines and at different concentrations. In contrast, IR caused ICD only in CT26. Furthermore, we determined the development of apoptosis and necrosis after both therapies and showed that two separate peaks of cell death occur after ECT. The first occurs immediately after ECT and the second 24 to 48 hours after ECT, with necrosis being the main modality of death, regardless of the type of chemotherapeutic agent or cell line. In contrast, cells died at later time points after IR, but again necrosis was the main modality. The number of dying cells increased in proportion to both the dose and the duration following IR. In further experiments, we found the increase in autophagy only in the 4T1 after ECT with CDDP and BLM. In contrast, we found a decrease in autophagy in B16-F10 after IR. When examining the changes in the expression of MHC I, MHC II, PD-L1, and CD40, we were able to show that all chemotherapeutic agents caused an increase in the expression of MHC I and PD-L1 in all cell lines after ECT. The expression of MHC II decreased with all chemotherapeutics except for the B16-F10 cell line, where expression increased. Next, we showed that the CD40 molecule is expressed only by the 4T1 cell line and that its expression increased after ECT with all chemotherapeutics, except IC30 and IC50 of BLM. Furthermore, the expression of MHC I increased in all cell lines and after all doses used after IR, except in 4T1 at dose IC30. The expression of PD-L1 also increased in all cell lines and doses, and the expression of CD40 also increased in 4T1. Most changes in the MHC II molecule were observed in the B16-F10 cell line.
Conclusions: With this study, we showed that ECT, together with the clinically relevant chemotherapeutics CDDP, OXA, and BLM, induces different immunologically important changes in tumor cells in vitro, depending on the type of chemotherapeutic agent, its concentration, and the tumor model. In addition, IR induces comparable changes in the expression of the cell surface markers MHC I, MHC II, PD-L1, and CD40, which, however, are not correlated in time with the changes after ECT. ECT caused the most changes in the expression of the DAMP molecules (CRT, HMGB1, ATP), which correlated with ICD in the less immunogenic cell line 4T1. IR caused the most changes in the expression of DAMP in the most immunogenic cell line CT26. ECT, together with the clinically relevant chemotherapeutics CDDP, OXA, and BLM, can therefore be included in the list of therapies that induce ICD in vitro. Our results provide mechanistic insights into how the potential of ICDs to trigger a systemic anti-tumor immune response can be exploited, especially when ECT is combined with immunotherapy.
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