Genska terapija postaja čedalje bolj zanimiva tudi v onkologiji. Med aplikacijami je morda najzanimivejša imunostimulacija. Pripravimo lahko plazmidno DNA, ki nosi zapis za različne imunostimulatorne ...molekule, ki jih vnesemo v celice tumorjev ali normalnih tkiv. Ta tkiva postanejo proizvajalci teh molekul, ki lahko delujejo lokalno ali pa se izločajo tudi sistemsko v krvni obtok. Ker plazmidna DNA ne prehaja celične membrane, so potrebni dostavni sistemi, virusni ali nevirusni. V naših študijah uporabljamo predvsem nevirusni dostavni sistem – elektroporacijo. Interlevkin 12 (IL-12) je eden od zanimivih citokinov, za katerega je znano protitumorsko delovanje s spodbujanjem imunskega odziva in antiangiogenim delovanjem. Namen projekta SmartGene.si je bil pripraviti plazmid z zapisom za interlevkin 12 (plazmid phIL12) in pripraviti vse potrebno za njegovo klinično testiranje za zdravljenje kožnih tumorjev. V konzorciju smo združili moči s partnerji z akademskega in industrijskega področja. Treba je bilo pripraviti plazmid za uporabo v humani onkologiji po zahtevah Evropske agencije za zdravila (EMA). Za prijavo klinične študije na Javno agencijo za zdravila in medicinske pripomočke (JAZMP) smo morali izvesti tudi vse neklinične raziskave o varnosti in učinkovitosti zdravila. Nato je bilo treba razviti postopek priprave zdravila, zagotoviti primerne prostore za pripravo in izvedbo postopka priprave zdravila. V treh letih smo dosegli vse te zastavljene cilje in dobili dovoljenje za izvajanje klinične študije na kožnih tumorjih, ki ga je izdala JAZMP na osnovi pozitivnega mnenja Komisije Republike Slovenije za medicinsko etiko. Zdaj poteka klinična študija faze I preizkušanja plazmida phIL12 na kožnih tumorjih glave in vratu z namenom preveriti varnost in sprejemljivost genskega elektroprenosa plazmida v tumorje. Cilj študije je prav tako določiti primeren odmerek zdravila, ki bi ga v nadaljnji klinični študiji uporabili kot adjuvantno zdravljenje k ablativnim terapijam, kot sta radioterapija ali elektrokemoterapija.
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.
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•Combination of electrochemotherapy and gene electrotransfer of IL-12 was tested.•Electrochemotherapy is more effective in more immunogenic tumors.•Gene electrotransfer exhibits greater contribution in less immunogenic tumors.•Therapy selection should be predominantly based on tumor immune status.
Calcium electroporation (CaEP) is a novel anti-tumour treatment that induces cell death by internalization of large quantities of calcium. The anti-tumour effectiveness of CaEP has been demonstrated ...in vitro, in vivo, and in preliminary clinical trials; however, its effects on the vasculature have not been previously investigated. Using a dorsal window chamber tumour model, we observed that CaEP affected to the same degree normal and tumour blood vessels in vivo, as it disrupted the vessels and caused tumour eradication by necrosis. In all cases, the effect was more pronounced in small vessels, similar to electrochemotherapy (ECT) with bleomycin. In vitro studies in four different cell lines (the B16F1 melanoma, HUVEC endothelial, FADU squamous cell carcinoma, and CHO cell lines) confirmed that CaEP causes necrosis associated with acute and severe ATP depletion, a picture different from bleomycin with electroporation. Furthermore, CaEP considerably inhibited cell migratory capabilities of endothelial cells and their potential to form capillary-like structures. The finding that CaEP has anti-vascular effects and inhibits cell migration capabilities may contribute to the explanation of the high efficacy observed in preclinical and clinical studies.
Interest in platinum-based chemotherapeutics such as oxaliplatin (OXA) and cisplatin (CDDP) has been reinvigorated by their newly described impacts on tumor-specific immune responses. In addition to ...CDDP, OXA is frequently used to treat cancers. Based on the characteristics of OXA, which are similar to those of CDDP, and the presumably more pronounced immunomodulatory effect of OXA, OXA is a candidate for electrochemotherapy (ECT). We compared the effectiveness of intratumoral ECT with OXA to that of ECT with CDDP in murine B16F10 melanoma to determine the equieffective dose. Special attention was given to the elicitation of immunogenic cell death and local immune response. Based on the in vitro and in vivo results pertaining to effectiveness and drug uptake in cells and tumors, ECT with OXA is as effective as ECT with CDDP when the OXA dose is increased 1.6-fold. Exposure of melanoma cells to ECT induces immunogenic cell death when either OXA or CDDP is used, which correlates with a comparable increase in lymphocyte infiltration into tumors after ECT with either OXA or CDDP. Based on these results, OXA is a valid platinum-based drug for use with ECT, and the effectiveness of ECT with OXA is comparable to that of the well-established ECT with CDDP. Furthermore, both drugs display equal and specific immune responses following ECT.
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•Our findings indicate the potential clinical application of OXA in ECT.•OXA is highly cytotoxic when its uptake is facilitated by electroporation of cells.•ECT with OXA is equally effective as ECT with CDDP when OXA dose is increased 1.6-fold.•Both OXA and CDDP induce immunogenic cell death in electroporated cells.•ECT using either OXA or CDDP induces significant lymphocyte infiltration in tumors.
In this article, the reversible electroporation induced by rectangular long unipolar and short bipolar voltage pulses on 3D cells is studied. The cell geometry was reconstructed from 3D images of ...real cells obtained using the confocal microscopy technique. A numerical model based on the Maxwell and the asymptotic Smoluchowski equations has been developed to calculate the induced transmembrane voltage and pore density on the plasma membrane of real cells exposed to the pulsed electric field. Moreover, in the case of the high-frequency pulses, the dielectric dispersion of plasma membranes has been taken into account using the second-order Debye-based relationship. Several numerical simulations were performed and we obtained suitable agreement between the numerical and experimental results.
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