Electrochemotherapy (ECT) exhibits high therapeutic effectiveness in the clinic, achieving up to 80% local tumor control but without a systemic (abscopal) effect. Therefore, we designed a combination therapy consisting of ECT via intratumoral application of bleomycin, oxaliplatin or cisplatin with peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (p. t. IL-12 GET). Our hypothesis was that p. t. IL-12 GET potentiates the effect of ECT on local and systemic levels and that the potentiation varies depending on tumor immune status. Therefore, the combination therapy was tested in three immunologically different murine tumor models. In poorly immunogenic B16F10 melanoma, IL-12 potentiated the antitumor effect of ECT with biologically equivalent low doses of cisplatin, oxaliplatin or bleomycin. The most pronounced potentiation was observed after ECT using cisplatin, resulting in a complete response rate of 38% and an abscopal effect. Compared to B16F10 melanoma, better responsiveness to ECT was observed in more immunogenic 4 T1 mammary carcinoma and CT26 colorectal carcinoma. In both models, p. t. IL-12 GET did not significantly improve the therapeutic outcome of ECT using any of the chemotherapeutic drugs. Collectively, the effectiveness of the combination therapy depends on tumor immune status. ECT was more effective in more immunogenic tumors, but GET exhibited greater contribution in less immunogenic tumors. Thus, the selection of the therapy, namely, either ECT alone or combination therapy with p. t. IL-12, should be predominantly based on tumor immune status.

The beneficial effects of electrochemotherapy (ECT) for superficial tumours and, more recently, deep-seated malignancies in terms of local control and quality of life are widely accepted. However, the variability in responses across histotypes needs to be explored. Currently, patient selection for ECT is based on clinical factors (tumour size, histotype, and exposure to previous oncological treatments), whereas there are no biomarkers to predict the response to treatment. In this field, two major areas of investigation can be identified, i.e., tumour cell characteristics and the tumour microenvironment (vasculature, extracellular matrix, and immune infiltrate). For each of these areas, we describe the current knowledge and discuss how to foster further investigation. This review aims to provide a summary of the currently used guiding clinical factors and delineates a research roadmap for future studies to identify putative biomarkers of response to ECT. These biomarkers may allow researchers to improve ECT practice by customising treatment parameters, manipulating the tumour and its microenvironment, and exploring novel therapeutic combinations.

Electrochemotherapy (ECT) is a clinically acknowledged method that combines the use of anticancer drugs and electrical pulses. Electrochemotherapy with bleomycin (BLM) can induce immunogenic cell death (ICD) in certain settings. However, whether this is ubiquitous over different cancer types and for other clinically relevant chemotherapeutics used with electrochemotherapy is unknown. Here, we evaluated in vitro in the B16-F10, 4T1 and CT26 murine tumor cell lines, the electrochemotherapy triggered changes in the ICD-associated damage-associated molecular patterns (DAMPs): Calreticulin (CRT), ATP, High Mobility Group Box 1 (HMGB1), and four immunologically important cellular markers: MHCI, MHC II, PD-L1 and CD40. The changes in these markers were investigated in time up to 48 h after ECT. We showed that electrochemotherapy with all three tested chemotherapeutics induced ICD-associated DAMPs, but the induced DAMP signature was cell line and chemotherapeutic concentration specific. Similarly, electrochemotherapy with CDDP, OXA or BLM modified the expression of MHC I, MHC II, PD-L1 and CD40. The potential of electrochemotherapy to change their expression was also cell line and chemotherapeutic concentration specific. Our results thus put the electrochemotherapy with clinically relevant chemotherapeutics CDDP, OXA and BLM on the map of ICD inducing therapies.

Currently, in high-frequency electroporation, much progress has been made but limited to research groups with custom-made laboratory prototype electroporators. According to the review of electroporators and economic evaluations, there is still an area of pulse parameters that needs to be investigated. The development of an asymmetric bipolar pulse generator with a maximum voltage of 4 kV and minimum duration time of a few hundred nanoseconds, would enable in vivo evaluation of biological effects of high-frequency electroporation pulses. Herein, from a series of most commonly used drivers and optical isolations in high-voltage pulse generators the one with optimal characteristics was used. In addition, the circuit topology of the developed device is described in detail. The developed device is able to generate 4 kV pulses, with theoretical 131 A maximal current and 200 ns minimal pulse duration, the maximal pulse repetition rate is 2 MHz and the burst maximal repetition rate is 1 MHz. The device was tested in vivo. The effectiveness of electrochemotherapy of high-frequency electroporation pulses is compared to “classical” electrochemotherapy pulses. In vivo electrochemotherapy with high-frequency electroporation pulses was at least as effective as with “classical” well-established electric pulses, resulting in 86% and 50% complete responses, respectively. In contrast to previous reports, however, muscle contractions were comparable between the two protocols.

Tumor cell-based vaccines use tumor cells as a source of tumor-associated antigens. In our study, we aimed to develop and test a tumor vaccine composed of tumor cells killed by irradiation combined with in vivo interleukin-12 gene electrotransfer as an adjuvant. Vaccination was performed in the skin of B16-F10 malignant melanoma or CT26 colorectal carcinoma tumor-bearing mice, distant from the tumor site and combined with concurrent tumor irradiation. Vaccination was also performed before tumor inoculation in both tumor models and tumor outgrowth was followed. The antitumor efficacy of vaccination in combination with tumor irradiation or preventative vaccination varied between the tumor models. A synergistic effect between vaccination and irradiation was observed in the B16-F10, but not in the CT26 tumor model. In contrast, up to 56% of mice were protected from tumor outgrowth in the CT26 tumor model and none were protected in the B16-F10 tumor model. The results suggest a greater contribution of the therapeutic vaccination to tumor irradiation in a less immunogenic B16-F10 tumor model, in contrast to preventative vaccination, which has shown greater efficacy in a more immunogenic CT26 tumor model. Upon further optimization of the vaccination and irradiation regimen, our vaccine could present an alternative tumor cell-based vaccine.

The only hitherto known biological role of yeast Saccharomyces cerevisiae Tum1 protein is in the tRNA thiolation pathway. The mammalian homologue of the yeast TUM1 gene, the thiosulfate sulfurtransferase (a.k.a. rhodanese) Tst, has been proposed as an obesity-resistance and antidiabetic gene. To assess the role of Tum1 in cell metabolism and the putative functional connection between lipid metabolism and tRNA modification, we analysed evolutionary conservation of the rhodanese protein superfamily, investigated the role of Tum1 in lipid metabolism, and examined the phenotype of yeast strains expressing the mouse homologue of Tum1, TST. We analysed evolutionary relationships in the rhodanese superfamily and established that its members are widespread in bacteria, archaea and in all major eukaryotic groups. We found that the amount of sterol esters was significantly higher in the deletion strain tum1Δ than in the wild-type strain. Expression of the mouse TST protein in the deletion strain did not rescue this phenotype. Moreover, although Tum1 deficiency in the thiolation pathway was complemented by re-introducing TUM1, it was not complemented by the introduction of the mouse homologue Tst. We further showed that the tRNA thiolation pathway is not involved in the regulation of sterol ester content in S. cerevisiae, as overexpression of the tEUUC, tKUUU and tQUUG tRNAs did not rescue the lipid phenotype in the tum1Δ deletion strain, and, additionally, deletion of the key gene for the tRNA thiolation pathway, UBA4, did not affect sterol ester content. The rhodanese superfamily of proteins is widespread in all organisms, and yeast TUM1 is a bona fide orthologue of mammalian Tst thiosulfate sulfurtransferase gene. However, the mouse TST protein cannot functionally replace yeast Tum1 protein, neither in its lipid metabolism-related function, nor in the tRNA thiolation pathway. We show here that Tum1 protein is involved in lipid metabolism by decreasing the sterol ester content in yeast cells, and that this function of Tum1 is not exerted through the tRNA thiolation pathway, but through another, currently unknown pathway.