1. Evaluation of methylene blue solution distribution in the four-point transversus abdominis plane block technique in pigs : a pilot anatomical studyJerneja Sredenšek, Jana Brankovič, Urša Lampreht Tratar, Maja Čemažar, Mihajlo Djokić, Alenka Seliškar, 2025, izvirni znanstveni članek Povzetek: Aim: This prospective pilot anatomical study aimed to develop an ultrasoundguided transversus abdominis plane (TAP) block technique that desensitises cranial and mid-abdominal wall in grower pigs. We hypothesised that a fourpoint TAP approach would be more efficient than a three-point TAP in staining relevant nerves of the cranial and mid-abdominal wall. Methods: In phase I, the ultrasound anatomy of the abdominal wall musculature was examined on three pig cadavers (two piglets and one fattening pig) and the ultrasound localization of the needle in the corresponding interfascial plane was practised. In phase II, a three-point TAP injection was performed in three freshly euthanized cadavers of grower pigs. A 1% methylene blue solution (0.3 mL/ kg per injection point) was injected between the transversus abdominis and internal oblique muscle. In phase III, methylene blue solution was injected at four points (0.2 mL/kg per injection point) in four anaesthetized grower pigs prior to euthanasia. Positive nerve staining was defined as continuous staining of at least 1 cm of the nerve length. Binary variables (positive/negative) were used for nerve staining assessment. Results: The four-point TAP technique with a lower injection volume stained more nerves than the three-point technique with a higher injection volume, i.e., 69% of the observed nerves from the eighth-last thoracic to the third lumbar nerve were stained with the four-point TAP technique. The nerves in the centre were stained with a higher success rate, while the eighth-last thoracic and the second lumbar nerve were stained with less success (1/8 and 3/8, respectively). The third lumbar nerve was not stained. Conclusion: The four-point TAP technique could be used as part of a multimodal analgesia approach for cranial and mid-abdominal surgery in pigs, but live animal studies are needed to evaluate the clinical applicability and efficacy of desensitisation.
Ključne besede: abdominal surgery, cadaveric anatomical study, pig, regional anaesthesia, transversus abdominis plane block, methylene blue
Objavljeno v DiRROS: 09.12.2025; Ogledov: 135; Prenosov: 54
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2. The role of focal adhesion kinase in bladder cancer : translation from in vitro to ex vivo human urothelial carcinomasGaja Markovič, Nataša Resnik, Aleksandar Janev, Daša Zupančič, Gašper Grubelnik, Maruša Debeljak, Maja Čemažar, Tanja Jesenko, Maša Omerzel, Tomaž Smrkolj, Mateja Erdani-Kreft, 2025, izvirni znanstveni članek Povzetek: Background: Focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, plays a crucial role in focal adhesion turnover by interfacing between the extracellular space, transmembrane integrins, and actin filaments. Its significance for the progression of several malignancies, including bladder cancer, has been well-documented. However, its precise role and the implications of its inhibition in bladder cancer tissues and urothelial in vitro models has not been fully explored. This study examined FAK expression and function in human bladder cancer biopsies and in vitro bladder cancer models. Materials and methods: Ex vivo analyses were performed using reverse transcription-quantitative PCR (qRT-PCR), western blotting, and immunohistochemistry to compare FAK expression between bladder cancer tissues and adjacent normal tissues. In vitro, FAK expression was assessed in low-grade (LG) human non-invasive papilloma urothelial cell line RT4 for NMIBC (Ta), high-grade (HG) human muscle-invasive cancer urothelial cell line T24 for MIBC (T2) and normal porcine urothelial (NPU) cells using qRT-PCR and western blotting, as well as flow cytometry for the quantification of FAK-positive RT4 and T24 cells. The role of FAK in cancer cell survival was explored in vitro using microRNA (miRNA) to silence FAK expression. Additionally, we used FAK inhibitors PND-1186, PF-573228 and defactinib to investigate the effects of FAK inhibition on normal compared to cancerous bladder urothelial cells. Results: Ex vivo analyses demonstrated significantly higher FAK expression in bladder cancer tissues compared to adjacent normal tissues. Similarly, in vitro analyses showed significantly higher FAK expression in RT4 and T24 cells than NPU cells. Silencing FAK using anti-FAK plasmids led to increased caspase-3-mediated apoptosis of RT4 and T24 cells and growth reduction of stably transfected T24 cells. Importantly, based on cell viability assays, treatment with 100 μM defactinib for 2 hours per day on 3 consecutive days was identified as a clinically relevant regimen. Under this treatment, the viability of differentiated NPU cells remained high at 108.4 ± 17.1%, while the viability of 2-day RT4 and 2-day T24 cells was drastically reduced to 4.1 ± 2.7% and 7.6 ± 2.9%, respectively. Conclusions: To our knowledge, this is the first report demonstrating the role of FAK and its inhibition across both normal and cancerous bladder urothelial models. This study highlights the critical role of FAK in the progression of human bladder cancer and establishes a foundation for exploring FAK inhibition as a potential therapeutic approach in bladder cancer treatment.
Ključne besede: bladder cancer, defactinib, focal adhesion kinase, human urothelial carcinoma, urothelial cell
Objavljeno v DiRROS: 09.12.2025; Ogledov: 106; Prenosov: 52
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3. Expression of inducible damage-associated molecular patterns after interleukin-12 gene electrotransfer in mouse melanoma and colorectal cell linesAjda Medved, Maša Omerzel, Tanja Jesenko, Simon Buček, Gregor Serša, Maja Čemažar, 2025, izvirni znanstveni članek Povzetek: Interleukin-12 (IL-12) has demonstrated potent antitumor effects by activating natural killer (NK) and T cells, but its systemic administration poses challenges due to toxicity. This study investigated gene electrotransfer (GET) as a localized delivery method for IL-12 plasmids in B16F10 and CT26 tumor cells and its effect on the expression of damage-associated molecular patterns (DAMPs) and several cytokines. We observed the activation of cytosolic sensors and cytokine production, with the expression of IL-6 and TNF-α upregulated only after IL-12 GET, suggesting the involvement of inducible damage-associated molecular patterns (iDAMPs) in the immune response to IL-12 gene therapy. These findings highlight the multifaceted immune activation triggered by GET, offering insights for improving IL-12-based cancer therapies.
Ključne besede: gene electrotransfer, cytokine, interleukin-12
Objavljeno v DiRROS: 05.12.2025; Ogledov: 148; Prenosov: 44
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4. Complementary detection strategies for circulating tumor cells in breast cancer: clinical implications of combining immunofluorescence and cytopathological stainingTanja Jesenko, Cvetka Grašič-Kuhar, Živa Pišljar, Simona Miceska, Veronika Kloboves-Prevodnik, Maja Čemažar, 2025, izvirni znanstveni članek Povzetek: Background: Circulating Tumor Cells (CTCs) serve as important biomarkers for disease monitoring and treatment response in patients with metastatic breast cancer. Their detection remains challenging because of their low abundance, phenotypic diversity and non-standardized mode of detection. Cytopathological Giemsa and Immunofluorescence (IF) staining can offer complementary approaches for CTC characterization. Giemsa staining enables assessment of cellular morphology, while IF allows for marker-specific identification, together providing a more comprehensive and accurate evaluation of CTCs. Methods: We developed an IF staining protocol with antibodies against Cytokeratin (CK), vimentin (VIM), and Cluster of Differentiation 45 (CD45) to distinguish epithelial, mesenchymal, hybrid and hematopoietic cells for CTC detection and characterization and compared it with cytopathologic method of detection via Giemsa staining with regard to CTC detection rates and morphological detail. Results: Study was performed on the samples of 29 heavily pretreated patients with metastatic breast cancer (median duration of metastatic disease 19.4 months). Giemsa staining enabled the detection of a higher number of CTCs compared to our IF protocol. Lower detection rate was potentially due to the loss of fragile or loosely adherent cells during methanol fixation and IF staining. Additionally, in IF-stained samples, some CTCs presented faint nuclear signals, potentially impairing their recognition. The IF staining supported the identity of CTCs detected on Giemsa-stained slides by employing a three-color antibody panel-based approach and allowed detailed phenotypic discrimination and structural analysis of CTCs, including the identification of a distinctive CK polarization pattern suggestive of a transitional state during intravasation. Conclusion: Giemsa and IF may thus be complementary rather than mutually exclusive and relying on a single detection approach could underestimate the true CTC burden. An integrative strategy combining both techniques may offer a more comprehensive view of CTC populations in metastatic breast cancer, thereby enhancing diagnostic precision.
Ključne besede: biomarkers, breast cancer, circulating tumor cells, cytopathological detection
Objavljeno v DiRROS: 05.12.2025; Ogledov: 200; Prenosov: 32
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5. Gene therapy of rare diseases as a milestone in medicine : overview of the field and report on initial experiences in SloveniaUrh Grošelj, Marko Kavčič, Ana Drole Torkar, Jan Kafol, Duško Lainšček, Roman Jerala, Matjaž Sever, Samo Zver, Gregor Serša, Maja Čemažar, Primož Strojan, Aleš Grošelj, Mojca Žerjav-Tanšek, Špela Miroševič, Simona Ivančan, Tomaž Prelog, David Gosar, Jasna Oražem, Matej Mlinarič, Sara Bertok, Jernej Kovač, Jana Kodrič, Saba Battelino, Marko Pokorn, Alojz Ihan, Janez Jazbec, Tadej Battelino, Damjan Osredkar, 2025, pregledni znanstveni članek Povzetek: Gene therapy has transitioned from a long-awaited promise to a clinical reality, offering transformative treatments for rare congenital diseases and certain cancers, which have a significant impact on patients’ lives. Current approaches focus on gene replacement therapy, either in vivo or ex vivo, mostly utilizing viral vectors to deliver therapeutic genes into target cells. However, refining these techniques is essential to overcome challenges and complications associated with gene therapy to ensure long-term safety and efficacy. Slovenia has witnessed significant advancements in this field since 2018, marked by successful gene therapy trials and treatments for various rare diseases. Significant strides have been made in the field of gene therapy in Slovenia, treating patients with spinal muscular atrophy and rare metabolic disorders, including the pioneering work on CTNNB1 syndrome. Additionally, immune gene therapy, exemplified by IL-12 adjuvant therapy for cancer, has been a focus of research in Slovenia. Through patient-centred initiatives and international collaborations, researchers in Slovenia are advancing preclinical research and clinical trials, paving the way for accessible gene therapies. Establishing clinical infrastructure and genomic diagnostics for rare diseases is crucial for gene therapy implementation. Efforts in this regard in Slovenia, including the establishment of a Centre for Rare Diseases, Centre for the Technologies of Gene and Cell Therapy, and rapid genomic diagnostics, demonstrate a commitment to comprehensive patient care. Despite the promises of gene therapy, challenges remain, including cost, distribution, efficacy, and long-term safety. Collaborative efforts are essential to address these challenges and ensure equitable access to innovative therapies for patients with rare diseases.
Ključne besede: gene therapy, rare genetic diseases, Slovenia, CAR-T cells, cancer, immune gene therapy
Objavljeno v DiRROS: 04.12.2025; Ogledov: 161; Prenosov: 124
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6. In vitro evaluation of electrochemotherapy combined with sotorasib in pancreatic carcinoma cell lines harboring distinct kras mutationsTanja Jesenko, Maša Omerzel, Tina Živič, Gregor Serša, Maja Čemažar, 2025, izvirni znanstveni članek Povzetek: Pancreatic cancer is among the deadliest malignancies, with limited treatment options and poor prognosis. Novel strategies are therefore urgently needed. Sotorasib, a KRAS G12C-specific inhibitor, offers targeted treatment for a small subset of patients with this mutation. Electrochemotherapy (ECT), which enhances the cytotoxicity of chemotherapeutic agents through electroporation-induced membrane permeabilization, has shown promise in various tumor types, including deep-seated malignancies such as pancreatic cancer. Combining ECT with sotorasib may potentiate antitumor effects in KRAS G12C-mutated pancreatic cancer; however, preclinical data on such combinations are lacking. This proof-of-concept study evaluated the cytotoxic effects of ECT using bleomycin (BLM) or cisplatin (CDDP) in combination with sotorasib in KRAS G12C-mutated MIA PaCa-2 and KRAS G12D-mutated PANC-1 pancreatic cancer cell lines. ECT alone significantly reduced cell viability, particularly in MIA PaCa-2 cells, where electric pulses induced approximately 75% cell death. Combining ECT with sotorasib resulted in an additive effect on KRAS G12C-mutated MIA PaCa-2 cells, though no synergy was observed, likely due to the high intrinsic sensitivity to electric pulses. These results support the potential of combining physical and molecular therapies in a subset of pancreatic cancer patients and lay the groundwork for further in vivo studies to optimize treatment parameters and explore clinical translatability.
Ključne besede: bleomycin, cisplatin, electrochemotherapy, pancreatic cancer
Objavljeno v DiRROS: 26.11.2025; Ogledov: 113; Prenosov: 51
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7. Long-term outcomes of reduced-dose bleomycin in electrochemotherapy for basal cell carcinoma in elderly patientsAleš Grošelj, Črt Jamšek, Simona Kranjc Brezar, Maja Čemažar, Maša Omerzel, Luka Pušnik, Gregor Serša, 2025, izvirni znanstveni članek Povzetek: Electrochemotherapy (ECT) is a minimally invasive treatment option for basal cell carcinoma (BCC), which is particularly advantageous in the elderly population. This study evaluated the long-term effects of treating BCC in older patients using ECT with a reduced dose of bleomycin (10,000 IU/m2) and compared the results to patients who received the standard dose of bleomycin (15,000 IU/m2). The retrospective analysis included 116 patients aged over 65 years with 257 histologically confirmed BCCs. Tumors were treated with either the standard dose (n = 82) or the reduced dose (n = 175) of bleomycin. The results showed that the recurrence rate was comparable between the groups, particularly in the first year after treatment. The reduced-dose group exhibited a greater recurrence rate after the first year, which may be attributed to a weaker local immune response due to the de-escalated dose of bleomycin. Nonetheless, administering a standard bleomycin dosage as a salvage treatment in the event of recurrence proved highly effective. These findings suggest that ECT with a reduced bleomycin dose is a viable option for treating BCC in elderly patients, particularly those with shorter life expectancy.
Ključne besede: bleomycin, electrochemotherapy, head and neck, nonmelanoma skin cancer
Objavljeno v DiRROS: 25.11.2025; Ogledov: 153; Prenosov: 119
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8. Electrochemotherapy with bleomycin, oxaliplatin, or cisplatin in mouse tumor models, from tumor ablation to in situ vaccinationKatja Uršič Valentinuzzi, Urška Kamenšek, Simona Kranjc Brezar, Chloe Heranney, Tilen Komel, Simon Buček, Maja Čemažar, Gregor Serša, 2025, izvirni znanstveni članek Povzetek: Introduction: In addition to its direct cytotoxic effects, ablative therapies as electrochemotherapy (ECT) can elicit indirect antitumor effects by triggering immune system responses. Here, we comprehensively analyzed this dual effectiveness of intratumoral ECT with chemotherapeutic drugs bleomycin (BLM), oxaliplatin (OXA), and cisplatin (CDDP). Our aim was to determine if ECT can act as in situ vaccination and thereby induce an abscopal effect. By evaluating ECT’s potential for in situ vaccination, our goal was to pave the way for future advancements for its combination with emerging (immuno)therapies, leading to enhanced responses and outcomes. Methods: We employed two mouse tumor models, the immunologically cold B16F10 melanoma and 4T1 mammary carcinoma, to explore both local and systemic (i.e., abscopal) antitumor effects following equieffective intratumoral ECT with BLM, OXA, and CDDP. Through histological analyses and the use of immunodeficient and metastatic (for abscopal effect) mouse models, we identified and compared both the cytotoxic and immunological components of ECT’s antitumor efficiency, such as immunologically recognizable cell deaths (immunogenic cell death and necrosis) and immune infiltrate (CD11+, CD4+, CD8+, GrB+). Results: Differences in immunological involvement after equieffective intratumoral ECT were highlighted by variable kinetics of immunologically recognizable cell deaths and immune infiltrate across the studied tumor models. Particularly, the 4T1 tumor model exhibited a more pronounced involvement of the immune component compared to the B16F10 tumor model. Variances in the antitumor (immune) response were also detected based on the chemotherapeutic drug used in ECT. Collectively, ECT demonstrated effectiveness in inducing in situ vaccination in both tumor models; however, an abscopal effect was observed in the 4T1 tumor model only. Conclusions: This is the first preclinical study systematically comparing the immune involvement in intratumoral ECT’s efficiency using three distinct chemotherapeutic drugs in mouse tumor models. The demonstrated variability in immune response to ECT across different tumor models and chemotherapeutic drugs provides a basis for future investigations aimed at enhancing the effectiveness of combined treatments.
Ključne besede: electroporation, chemotherapeutic drugs, mouse tumor models
Objavljeno v DiRROS: 19.11.2025; Ogledov: 195; Prenosov: 87
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9. Klinične raziskave na področju onkologijeErika Matos, Neža Gros, Barbara Bonač, Maja Čemažar, 2024, druge monografije in druga zaključena dela Ključne besede: klinične raziskave, onkologija, raziskovalna dejavnost, zgibanke, drobni tisk
Objavljeno v DiRROS: 08.08.2025; Ogledov: 418; Prenosov: 143
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