201. Contrast-enhanced ultrasound for evaluation of tumor perfusion and outcome following treatment in a murine melanoma modelMaja Brložnik, Nina Boc, Maja Čemažar, Maša Omerzel, Monika Savarin, Nataša Kejžar, Gregor Serša, Darja Pavlin, Simona Kranjc Brezar, 2021, original scientific article Published in DiRROS: 23.09.2022; Views: 528; Downloads: 320
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202. Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune statusKatja Uršič Valentinuzzi, Špela Kos, Urška Kamenšek, Maja Čemažar, Simona Miceska, Boštjan Markelc, Simon Buček, Barbara Starešinič, Veronika Kloboves-Prevodnik, Richard Heller, Gregor Serša, 2021, original scientific article Abstract: 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.
Keywords: electrochemotherapy, gene electrotransfer, interleukin-12
Published in DiRROS: 21.09.2022; Views: 621; Downloads: 213
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203. Evaluation of a novel plasmid for simultaneous gene electrotransfer-mediated silencing of CD105 and CD146 in combination with irradiationMonika Savarin, Urška Kamenšek, Katarina Žnidar, Vesna Todorović, Gregor Serša, Maja Čemažar, 2021, original scientific article Abstract: Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.
Keywords: CD105, CD146, plasmid, gene electrotransfer
Published in DiRROS: 21.09.2022; Views: 578; Downloads: 330
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205. PARP inhibitor olaparib has a potential to increase the effectiveness of electrochemotherapy in BRCA1 mutated breast cancer in miceMaša Omerzel, Tanja Jesenko, Boštjan Markelc, Larisa Janžič, Maja Čemažar, Gregor Serša, 2021, original scientific article Abstract: Electrochemotherapy (ECT), a local therapy, has different effectiveness among tumor types. In breast can-cer, its effectiveness is low; therefore, combined therapies are needed. The aim of our study was to com-bine ECT with PARP inhibitor olaparib, which could inhibit the repair of bleomycin or cisplatin inducedDNA damage and potentiate the effectiveness of ECT. The effects of combined therapy were studied inBRCA1mutated (HCC1937) and non-mutated (HCC1143) triple negative breast cancer cell lines.Therapeutic effectiveness was studied in 2D and 3D cell cultures andin vivoon subcutaneousHCC1937 tumor model in mice. The underlying mechanism of combined therapy was determined withthe evaluation ofcH2AX foci. Combined therapy of ECT with bleomycin and olaparib potentiated theeffectiveness of ECT inBRCA1mutated HCC1937, but not in non-mutated HCC1143 cells. The combinedtherapy had a synergistic effect, which was due to the increased number of DNA double strand breaks.Addition of olaparib to ECT with bleomycinin vivoin HCC1937 tumor model had only minimal effect,indicating repetitive olaparib treatment would be needed. This study demonstrates that DNA repar inhibiting drugs, like olaparib, have the potential to increase the effectiveness of ECT with bleomycin.
Keywords: electrochemotherapy, breast cancer, olaparib, bleomycin
Published in DiRROS: 21.09.2022; Views: 560; Downloads: 192
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206. Mutational burden, MHC-I expression and immune infiltration as limiting factors for in situ vaccination by TNF[alfa] and IL-12 gene electrotransferUrška Kamenšek, Katja Uršič Valentinuzzi, Boštjan Markelc, Maja Čemažar, Vita Šetrajčič Dragoš, Gregor Serša, 2021, original scientific article Abstract: In situ vaccination is a promising immunotherapeutic approach, where various local ablative therapies are used to induce an immune response against tumor antigens that are released from the therapy-killed tumor cells. We recently proposed using intratumoral gene electrotransfer for concomitant transfection of a cytotoxic cytokine tumor necrosis factor-% (TNF%) to induce in situ vaccination, and an immunostimulatory cytokine interleukin 12 (IL-12) to boost the primed immune response. Here, our aim was to test the local and systemic effectiveness of the approach in tree syngeneic mouse tumor models and associate it with tumor immune profiles, characterized by tumor mutational burden, immune infiltration and expression of PD-L1 and MHC-I on tumor cells. While none of the tested characteristic proved predictive for local effectiveness, high tumor mutational burden, immune infiltration and MHC-I expression were associated with higher abscopal effectiveness. Hence, we have confirmed that both the abundance and presentation of tumor antigens as well as the absence of immunosuppressive mechanisms are important for effective in situ vaccination. These findings provide important indications for future development of in situ vaccination based treatments, and for the selection of tumor types that will most likely benefit from it.
Keywords: in situ vaccination, gene electrotransfer, interleukin 12, tumor necrosis factor [alfa]
Published in DiRROS: 19.09.2022; Views: 591; Downloads: 192
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207. Gene electrotransfer of proinflammatory chemokines CCL5 and CCL17 as a novel approach of modifying cytokine expression profile in the tumor microenvironmentTim Božič, Gregor Serša, Simona Kranjc Brezar, Maja Čemažar, Boštjan Markelc, 2021, original scientific article Abstract: The effectiveness of immunotherapy highly correlates with the degree and the type of infiltrated immune cells in the tumor tissue. Treatments based on modifying the immune cell infiltrate of the tumor microenvironment are thus gaining momentum. Therefore, the aim of our study was to investigate the effects of gene therapy with two proinflammatory chemokines CCL5 and CCL17 on inflammatory cytokine expression profile and immune cell infiltrate in two murine breast tumor models, 4T1 and E0771, and two murine colon tumor models, CT26 and MC38. In vitro, lipofection of plasmid DNA encoding CCL5 or CCL17 resulted in changes in the cytokine expression profile similar to control plasmid DNA, implying that the main driver of these changes was the entry of foreign DNA into the cell%s cytosol. In vivo, gene electrotransfer resulted in high expression levels of both Ccl5 and Ccl17 transgenes in the 4T1 and CT26 tumor models. Besides a minor increase in the survival of the treated mice, the therapy also resulted in increased expression of Cxcl9 and Ifn%, potent activators of the immune system, in CT26 tumors. However, this was not recapitulated in changes of TME, implying that a further refinement of the dosing schedule is needed.
Keywords: chemokines, cytokine expression, gene electrotransfer, CCL5
Published in DiRROS: 19.09.2022; Views: 585; Downloads: 165
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208. Poročilo 2021 o izvajanju letnega programa dela javnih služb v oljkarstvuMaja Podgornik, Viljanka Vesel, Dunja Bandelj, Bojan Butinar, Elizabeta Bonin, Janko Brajnik, Erika Bešter, Jakob Fantinič, Katja Fičur, Vasja Juretič, Matjaž Prinčič, Vasilij Valenčič, Saša Volk, Milena Bučar-Miklavčič, 2022, other monographs and other completed works Abstract: V okviru javne službe za izvajanje strokovnih nalog v oljkarstvu, so bile v letu 2021 izvedene naloge: selekcija, introdukcija, razmnoževanje sadilnega material, ugotavljanje ustreznih tehnologije za pridelavo oljk ter ugotavljanje vrednosti oljk za predelavo. V poročilu so zbrane morfološke in agronomske lastnosti ter značilnosti oljčnega olja sort 'Mata', 'Coratina', 'Frantoio', 'Oblica', 'Pendolino', 'Picholine' in 'Grignan' za opazovanja v letu 2021. Predstavljeni so tudi rezultati genotipizacije izbranih sort ter agronomskega vrednotenja in fenofaze različnih sort, ki so zasajene v introdukcijsko-kolekcijskem nasadu Purissima in Šempeter. V poročilu so zbrani podatki tehnoloških poskusov pridelave in predelave; parametri, ki karakterizirajo posamezna sortna olja ter parametri kakovosti oljčnega olja letnika 2021.
Ključne besede: oljkarstvo, oljka, oljčno olje, tehnologija pridelave, ocena letnika, introdukcija, selekcija
Keywords: elektronske knjige
Published in DiRROS: 14.09.2022; Views: 680; Downloads: 330
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210. Non-clinical in vitro evaluation of antibiotic resistance gene-free plasmids encoding human or murine IL-12 intended for first-in-human clinical studyŠpela Kos, Maša Omerzel, Tanja Jesenko, Boštjan Markelc, Urška Kamenšek, Katarina Žnidar, Urška Matkovič, Andrej Renčelj, Gregor Serša, Rosana Hudej, Aneja Tuljak, Matjaž Peterka, Maja Čemažar, 2021, original scientific article Abstract: Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of expression and plasmid copy number were determined in vitro in the human squamous cell carcinoma cell line FaDu and the murine colon carcinoma cell line CT26. The results of the non-clinical evaluation in vitro set the basis for further in vivo testing and evaluation of antitumor activity of therapeutic molecules in murine models as well as provide crucial data for further clinical trials of the constructed antibiotic resistance gene-free plasmid in humans.
Keywords: interleukin 12, gene electrotransfer, antibiotic resistance, plasmids
Published in DiRROS: 07.09.2022; Views: 541; Downloads: 309
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