Scientific background: In recent years, the development of new targeted therapies has led to changes in the treatment of cancer. Various immunotherapy approaches such as immune checkpoint inhibitors, cell therapy, antitumour vaccines and gene therapy with various cytotoxic and immunomodulatory molecules have come to the fore. Gene electrotransfer can be used as a delivery strategy for gene therapy in cancer, whereby electrical pulses allow the local introduction of plasmid DNA into the cells of various tissues. In this way, two plasmids with transcripts for interleukin 2 (IL -2) and interleukin 12 (IL -12) can be introduced simultaneously into tumours, which are then expressed in the transfected cells and trigger an immune response as inflammatory interleukins. The antitumour efficacy of genetic electroporation of individual plasmid IL-2 or IL-12 has been demonstrated in numerous preclinical and clinical studies. In patients who respond poorly to therapy, gene transfer of interleukins in combination with other cytokines or other immunotherapeutic approaches (e.g. immune checkpoint inhibitors) could contribute significantly to the overall efficacy of therapy. The combination of IL-2 and IL-12 is based on their synergistic mechanism of action. In this way, the immune system could be better activated, increasing antitumour efficacy compared to plasmid IL-2 or IL-12 alone. The aim of this dissertation was to determine the antitumour and antiangiogenic activity of intratumoural gene transfer plasmids transcribed for IL-2 and IL-12 in two different mouse tumour models. We investigated the effect of the gene transfer plasmids on tumour cells in vitro, the antitumour and antiangiogenic activity of the combination compared to single therapy, the effect on the expression of different cytokines after therapy and the presence of different immune cell populations in vivo. These studies were conducted to test the efficacy of this therapy and to further our understanding of the mechanisms of action of the combination of IL-2 and IL-12 in tumours.
Methods: We first investigated the effects of plasmid DNA gene transfer on survival and gene expression in two mouse tumour cell lines, melanoma B16F10 and colon cancer CT26, in vitro. Two different pulse protocols were tested. Transfection efficiency 48 hours after gene electroporation of pEGFP and pDsRed was determined using real-time fluorescence microscopy (Cytation 1 multifunctional reader). For transfection efficiency 48 hours after gene transfer of plasmids IL-2 and IL-12, RNA isolated from the cells was used to determine the expression levels of interleukins IL -2 and IL -12 by quantitative real-time PCR (qRT-PCR) and ELISA. In addition to transfection efficiency, we also determined cell survival after gene transfer using PrestoBlue〢 reagent. Next, we determined the transfection efficiency of electro-transfection of plasmid DNA transcribed for EGFP and DsRed in vivo. Tumours were analysed using a BD FacsCantoTM flow cytometer and fluorescence microscopy (Olympus BX -51 microscope). In another work, we then determined the antitumour effect of the combination treatment in the B16F10 and CT26 mouse tumour models. Tumour growth was monitored by measuring three rectangular diameters of the tumours with callipers. In mice in which a complete response was observed (100 days after treatment with no palpable tumour), the tumour was reinserted on the left flank and growth was monitored for an additional 100 days. Response to therapy or mechanisms of antitumour action after gene transfer were further investigated by determining infiltration of immune cells into the tumour by immunohistochemical staining and expression of selected cytokines by ELISA and Magpix in tumour lysates. Tumour sections were immunohistochemically stained with primary and secondary antibodies against surface markers of T helper cells (CD4+), T killer cells (CD8+), macrophages (F4/80+ and MHC II +), dendritic cells (CD11+) and endothelial cells (CD31+). Images of the sections were taken with a light microscope (Olympus BX -51 microscope) under 400x magnification. To optimise the therapy, 50 µL collagenase (250 IU/mL) was injected into the tumours 24 hours before therapy and hyaluronidase (10,000 IU/mL) was injected 2 hours before therapy. In a final step, we determined the extracellular and systemic efficacy of the
combination therapy (abscope effect) in the CT26 tumour model and assessed splenocyte activity after combination therapy. The animal experiments were performed in accordance with the instructions and approval of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (approval numbers U34401-1/2015/17 and U34401-3/2022/11). GraphPad was used to process the data and to identify differences between the groups using various statistical tests.
Results: For our experiments, we selected two mouse cell lines: melanoma B16F10 and colon cancer CT26. In an in vitro experiment, we transfected EGFP, DsRed and their combination, and IL-2, IL-12 and their combination plasmids for three days after gene electroporation and determined transfection efficiency and survival after electroporation. Transfection efficiency was higher after electroporation with the EP1 protocol and higher in B16F10 cells than in CT26 cells. This was demonstrated for both fluorescent protein expression results and for both interleukins. Genetic electroporation of pEGFP and pDsRed and their combination was also performed in both tumour models in vivo. Flow cytometry and microscopy data showed that in the B16F10 tumour model, the mean transfection efficiency was higher after electroporation with the EP1 protocol than after EP2 in all groups, but the differences were not statistically significant. In contrast, we were unable to detect transfected cells in CT26 using either method. We also investigated the antitumour effect of gene transfer of the combination of IL-2 and IL-12 plasmids in both mouse tumour models. In the B16F10 model, the group treated with the plasmid combination showed the greatest slowing of tumour growth compared to the other groups. In this group, we also observed a complete response and survival after reinjection of tumour cells. We repeated the same experiment in the CT26 tumour model, where we observed a delay in tumour growth in the group treated with the plasmid combination, but no complete responses. Therefore, we optimised the therapy in this tumour model by improving the transfection efficiency by pretreating the tumours with collagenase and hyaluronidase. After optimisation, we observed greater growth retardation in all groups treated with plasmids IL-2 and IL-12 alone and in their combination, and complete response and survival after reinjection of tumour cells in the combination group. In the CT26 tumour model, we also determined the extraventricular and systemic efficacy of the optimised combination therapy. The greatest abscopal effect and splenocyte cytotoxic activity was observed in tumours treated with the combination. We also investigated the mechanisms of antitumour action by in vivo gene transfer in the B16F10 and CT26 tumour models. To assess the efficacy of the therapy in the tumour, we performed histological analyses and determined the concentrations of 32 inflammatory cytokines in tumour and serum samples. In both models, infiltration of various immune cells was highest in the group treated with the plasmid combination. We also observed an increase in various inflammatory cytokines in all electroporation-treated groups. However, the combination plasmid group showed a significant increase in IL -12, IFNγ and TNFα, which are characterised by their pronounced anti-tumour activity.
Conclusions: Our data show successful gene transfer of a combination of IL-2 and IL-12 transporter plasmids in two tumour models with different immune status. The combination of IL-2 and IL-12 expressed in sufficient amounts in the tumour environment induces migration and stimulation of immune cells to the tumour site, which together with the anti-angiogenic effect of IL-12 leads to tumour cell killing. In addition, our combined approach also resulted in anti-tumour immune memory. In this study, we have shown that gene therapy using a combination of IL-2 and IL-12 plasmids is effective for both local and systemic treatment of tumours. Despite the good results in both tumour models, the therapy in the CT26 model could be further optimised to achieve a comparable efficacy as in the B16F10 model. The results of this PhD thesis have contributed to the understanding of the effect of IL-2 and IL-12 at the in vitro and in vivo levels. Finally, the results of the PhD thesis will also be helpful for the possible introduction of a combination therapy with these two interleukins into clinical practice.
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