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2. Razvoj raziskav cirkulirajočih tumorskih celic pri raku dojk na Onkološkem inštitutu LjubljanaTanja Jesenko, Cvetka Grašič-Kuhar, Živa Pišljar, Simona Miceska, Veronika Kloboves-Prevodnik, Maja Čemažar, 2024, review article Abstract: Cirkulirajoče tumorske celice (CTC) so postale pomemben biološki označevalec pri raku dojk, saj omogočajo vpogled v razvoj in napredovanje razsejane bolezni ter spremljanje odziva na zdravljenje. Zaradi njihove izjemne redkosti in kompleksnosti sestave krvi, v kateri se nahajajo, sta njihova izolacija in karakterizacija velik izziv. Posebne metode izolacije omogočajo obogatitev CTC iz vzorca krvi in olajšajo nadaljnjo analizo. Na Onkološkem inštitutu Ljubljana smo leta 2018 začeli s prvimi koraki v smeri razvoja preproste metode za izolacijo in karakterizacijo CTC, ki bi omogočala prepoznavanje teh celic s citopatološkimi analizami. Ocenili smo dve različni metodi izolacije CTC pri bolnicah z rakom dojk, ki temeljita na različnih pristopih. Prva metoda temelji na bioloških lastnostih celic, kot je izražanje epitelijskega označevalca celične adhezije (EpCAM), medtem ko druga metoda temelji na fizikalnih lastnostih CTC, kot sta večja velikost in stisljivost v primerjavi z drugimi krvnimi celicami. Ugotovili smo, da je fizikalna metoda primernejša, saj omogoča izolacijo večjega števila morfološko ohranjenih CTC in tudi skupkov CTC. Po izolaciji pripravimo citološke preparate, ki jih nato opredelimo s citopatološko analizo in dodatnimi imunocitokemičnimi ter imunofluorescenčnimi barvanji. Na ta način lahko trenutno določimo število CTC in skupkov CTC v krvi, ocenimo njihovo morfološko ohranjenost ter prepoznamo njihov fenotip. Poleg preučevanja vzorcev posamičnih CTC in skupkov CTC v okviru trenutno potekajočih kliničnih raziskav in načrtovane vzpostavitve translacijske platforme na mišjih modelih, pa v prihodnosti želimo nabor raziskav CTC še razširiti na genomsko in transkriptomsko analizo.
Keywords: cirkulirajoče tumorske celice, rak dojk, eksperimentalna onkologija
Published in DiRROS: 21.05.2025; Views: 122; Downloads: 42
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3. Muscle gene electrotransfer is increased by the antioxidant tempol in miceBoštjan Markelc, Gregor Tevž, Maja Čemažar, Simona Kranjc Brezar, Jaka Lavrenčak, Bojana Žegura, Justin Teissié, Gregor Serša, 2012, original scientific article Abstract: Electropermeabilization (EP) is an effective method of gene transfer into different tissues. During EP, reactive oxygen species (ROS) are formed, which could affect transfection efficiency. The role of generated ROS and the role of antioxidants in electrotransfer in myoblasts in vitro and in Musculus tibialis cranialis in mice were, therefore, investigated. We demonstrate in the study that during EP of C2C12 myoblasts, ROS are generated on the surface of the cells, which do not induce long-term genomic DNA damage. Plasmid DNA for transfection (pEGFP-N1), which is present outside the cells during EP, neutralizes the generated ROS. The ROS generation is proportional to the amplitude of the electric pulses and can be scavenged by antioxidants, such as vitamin C or tempol. When antioxidants were used during gene electrotransfer, the transfection efficiency of C2C12 myoblasts was statistically significantly increased 1.6-fold with tempol. Also in vivo, the transfection efficiency of M. tibialis cranialis in mice was statistically significantly increased 1.4-fold by tempol. The study indicates that ROS are generated on cells during EP and can be scavenged by antioxidants. Specifically, tempol can be used to improve gene electrotransfer into the muscle and possibly also to other tissues.
Keywords: electropermeabilization, gene electrotransfer, muscle, tempol, reactive oxygen species
Published in DiRROS: 26.02.2025; Views: 287; Downloads: 145
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6. Addition of 2-(ethylamino)acetonitrile group to nitroxoline results in significantly improved anti-tumor activity in vitro and in vivoAna Mitrović, Izidor Sosič, Špela Kos, Urša Lampreht Tratar, Barbara Breznik, Simona Kranjc Brezar, Bojana Mirković, Stanislav Gobec, Tamara Lah Turnšek, Maja Čemažar, Gregor Serša, Janko Kos, 2017, original scientific article Abstract: Lysosomal cysteine peptidase cathepsin B, involved in multiple processes associated with tumor progression, is validated as a target for anti-cancer therapy. Nitroxoline, a known antimicrobial agent, is a potent and selective inhibitor of cathepsin B, hence reducing tumor progression in vitro and in vivo. In order to further improve its anti-cancer properties we developed a number of derivatives using structure-based chemical synthesis. Of these, the 7-aminomethylated derivative (compound 17) exhibited significantly improved kinetic properties over nitroxoline, inhibiting cathepsin B endopeptidase activity selectively. In the present study, we have evaluated its anti-cancer properties. It was more effective than nitroxoline in reducing tumor cell invasion and migration, as determined in vitro on two-dimensional cell models and tumor spheroids, under either endpoint or real time conditions. Moreover, it exhibited improved action over nitroxoline in impairing tumor growth in vivo in LPB mouse fibrosarcoma tumors in C57Bl/6 mice. Taken together, the addition of a 2-(ethylamino)acetonitrile group to nitroxoline at position 7 significantly improves its pharmacological characteristics and its potential for use as an anti-cancer drug.
Keywords: nitroxoline derivative, cathepsin B, inhibition, tumor invasion, cell migration
Published in DiRROS: 26.07.2024; Views: 596; Downloads: 425
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7. Bleomycin electrosclerotherapy (BEST) for the treatment of vascular malformations : an International Network for Sharing Practices on Electrochemotherapy (InspECT) study group reportTobian Muir, Giulia Bertino, Aleš Grošelj, Lakshmi Ratnam, Erika Kis, Joy Odili, Ian McCafferty, Walter A Wohlgemuth, Maja Čemažar, Aljoša Krt, Maša Omerzel, Alessandro Zanasi, Michela Battista, Francesca De Terlizzi, Luca Giovanni Campana, Gregor Serša, 2023, review article Abstract: Biomedical applications of electroporation are expanding out of the field of oncology into vaccination, treatment of arrhythmias and now in the treatment of vascular malformations. Bleomycin is a widely used sclerosing agent in the treatment of various vascular malformations. The application of electric pulses in addition to bleomycin enhances the effectiveness of the drug, as demonstrated by electrochemotherapy, which utilizes bleomycin in the treatment of tumors. The same principle is used in bleomycin electrosclerotherapy (BEST). The approach seems to be effective in the treatment of low-flow (venous and lymphatic) and, potentially, even high-flow (arteriovenous) malformations. Although there are only a few published reports to date, the surgical community is interested, and an increasing number of centers are applying BEST in the treatment of vascular malformations. Within the International Network for Sharing Practices on Electrochemotherapy (InspECT) consortium, a dedicated working group has been constituted to develop standard operating procedures for BEST and foster clinical trials. By treatment standardization and successful completion of clinical trials demonstrating the effectiveness and safety of the approach, higher quality data and better clinical outcomes may be achieved.
Keywords: vascular malformations, electrosclerotherapy, bleomycin
Published in DiRROS: 25.07.2024; Views: 683; Downloads: 204
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8. Treatment of vulvar cancer recurrences with electrochemotherapy : a detailed analysis of possible causes for unsuccessful treatmentGregor Vivod, Tanja Jesenko, Gorana Gašljević, Nina Kovačević, Maša Omerzel, Gregor Serša, Sebastjan Merlo, Maja Čemažar, 2023, original scientific article Abstract: Background. Electrochemotherapy has good local effectiveness in the treatment of vulvar cancer. Most studies have reported the safety and effectiveness of electrochemotherapy for palliative treatment of gynecological cancers and mostly vulvar squamous cell carcinoma. Some tumors, however, fail to respond to electrochemotherapy. The biological features/determinants for the nonresponsiveness are not determined yet. Patient and methods. A recurrence of vulvar squamous cell carcinoma was treated by electrochemotherapy using intravenous administration of bleomycin. The treatment was performed by hexagonal electrodes according to standard operating procedures. We analyzed the factors that could determine nonresponsiveness to electrochemotherapy. Results. Based on the presented case of nonresponsive vulvar recurrence to electrochemotherapy, we hypothesize that the vasculature of the tumors prior to treatment may predict the response to electrochemotherapy. The histological analysis showed minimal presence of blood vessels in the tumor. Thus, low perfusion may reduce drug delivery and lead to a lower response rate because of the minor antitumor effectiveness of vascular disruption. In this case, no immune response in the tumor was elicited by electrochemotherapy. Conclusions. In this case, of nonresponsive vulvar recurrence treated by electrochemotherapy, we analyzed possible factors that could predict treatment failure. Based on histological analysis, low vascularization of the tumor was observed, which hampered drug delivery and distribution and resulted in no vascular disrupting action of electrochemotherapy. All these factors could contribute to ineffective treatment with electrochemotherapy.
Keywords: electrochemotherapy, bleomycin, vulvar cancer
Published in DiRROS: 25.07.2024; Views: 593; Downloads: 255
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9. Treatment of skin tumors with intratumoral interleukin 12 gene electrotransfer in the head and neck region : a first-in-human clinical trial protocolAleš Grošelj, Maša Omerzel, Tanja Jesenko, Maja Čemažar, Boštjan Markelc, Primož Strojan, Gregor Serša, 2022, original scientific article Abstract: Immune therapies are currently under intensive investigation providing in many cases excellent re-sponses in different tumors. Other possible approach for immunotherapy is a targeted intratumoral delivery of inter-leukin 12 (IL-12), a cytokine with anti-tumor effectiveness. Due to its immunomodulatory action, it can be used as an imunostimulating component to in situ vaccinating effect of local ablative therapies. We have developed a phIL12 plasmid devoid of antibiotic resistance marker with a transgene for human IL-12 p70 protein. The plasmid can be delivered intratumorally by gene electrotransfer (GET). Patients and methods. Here we present a first-in-human clinical trial protocol for phIL12 GET (ISRCTN15479959, ClinicalTrials NCT05077033). The study is aimed at evaluating the safety and tolerability of phIL12 GET in treatment of basal cell carcinomas in patients with operable tumors in the head and neck region. The study is designed as an ex-ploratory, dose escalating study with the aim to determine the safety and tolerability of the treatment and to identify the dose of plasmid phIL12 that is safe and elicits its biological activity. Conclusions. The results of this trail protocol will therefore provide the basis for the use of phIL12 GET as an adjuvant treatment to local ablative therapies, to potentially increase their local and elicit a systemic response.
Keywords: skin tumors, gene electrotransfer, interleukin 12, clinical trial
Published in DiRROS: 24.07.2024; Views: 567; Downloads: 205
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10. Sunitinib potentiates the cytotoxic effect of electrochemotherapy in pancreatic carcinoma cellsMaša Omerzel, Tanja Jesenko, Boštjan Markelc, Anja Cerovšek, Gregor Serša, Maja Čemažar, 2022, original scientific article Abstract: One of the new treatment options for unresectable locally advanced pancreatic cancer is electro-chemotherapy (ECT), a local ablative therapy that potentiates the entry of chemotherapeutic drugs into the cells, by the application of an electric field to the tumor. Its feasibility and safety were demonstrated in preclinical and clinical studies; however, there is a lack of preclinical studies assessing the actions of different drugs used in ECT, their mechanisms and interactions with other target drugs that are used in clinical practice. Materials and methods. The aim of the study was to determine the cytotoxicity of two chemotherapeutic drugs usually used in ECT (bleomycin and cisplatin) in the BxPC-3 human pancreatic carcinoma cell line and evaluate the interactions of ECT with the targeted drug sunitinib. First, the cytotoxicity of ECT using both chemotherapeutics was determined. In the next part, the interactions of ECT and sunitinib were evaluated through determination of combined cytotoxicity, sunitinib targets and kinetics of cell death.Results. The results demonstrate that ECT is effective in pancreatic cancer cell line, especially when bleomycin is used, with the onset of cell death in the first hours after the treatment, reaching a plateau at 20 hours after the treat-ment. Furthermore, we provide the rationale for combining ECT with bleomycin and the targeted drug sunitinib to potentiate cytotoxicity. The combined treatment of sunitinib and ECT was synergistic for bleomycin only at the high-est used concentration of bleomycin 0.14 μM, whereas with lower doses of bleomycin, this effect was not observed. The interaction of ECT and treatment with sunitinib was confirmed by course of the cell death, also indicating on synergism
Keywords: electrochemotherapy, pancreas, sunitinib, pancreatic cancer
Published in DiRROS: 24.07.2024; Views: 571; Downloads: 318
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