1. The lysosome–cathepsin axis in pancreatic cancer : mechanisms of stromal remodeling, immune evasion, and therapy resistanceNika Mazej Jeram, Emanuela Senjor, Janko Kos, Milica Perišić, 2026, review article Abstract: Pancreatic cancer remains one of the most lethal malignancies worldwide, with pancreatic ductal adenocarcinoma accounting for the vast majority of cases and characterized by extensive desmoplasia, immune exclusion, and resistance to systemic therapies. Increasing evidence implicates lysosomal cathepsins as important regulators of these defining features of pancreatic tumor biology. Cathepsin-dependent proteolysis and lysosome-associated signaling pathways contribute to extracellular matrix remodeling, regulate immune cell trafficking, and influence antigen processing and presentation. Beyond their classical degradative functions, cathepsins participate in stress-adaptive cellular programs linked to autophagy, metabolic regulation, and proteostasis, supporting tumor cell survival under hypoxic, nutrient-limited, and therapy-induced stress conditions. Within the tumor microenvironment, dysregulated cathepsin activity promotes immune evasion by reshaping cytokine networks, impairing effective antigen presentation, and reinforcing physical and functional barriers to cytotoxic T-cell infiltration. Collectively, these mechanisms position the lysosome–cathepsin system as a central regulator of proteolytic remodeling, immune exclusion, and adaptive therapy resistance in pancreatic cancer, highlighting its potential relevance for emerging combinatorial therapeutic strategies.
Keywords: tumor microenvironment, autophagy, therapeutic resistance, immune evasion, extracellular matrix remodelling, cytotoxic cells, antigen presentation, lysosome
Published in DiRROS: 05.06.2026; Views: 244; Downloads: 226
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2. Amyloid-▫$\beta$▫ 1-42 fibrils regulate SH-SY5Y cell adhesion in a delayed mannerUrša Pečar Fonović, Slavko Kralj, Janko Kos, 2026, original scientific article Abstract: Amyloid-β peptide (Aβ), a hallmark peptide in the pathology of Alzheimer's disease, together with the amyloid-β protein precursor, is increasingly associated with the disruption of cell adhesion. In addition to its well-characterized role in plaque formation and synaptic dysfunction, Aβ interacts with various adhesion molecules and extracellular matrix components, thereby impairing neuronal connectivity and integrity. We have shown that pretreatment of SH-SY5Y cells with Aβ42 fibrils affects cell adhesion; however, we did not observe this effect with Aβ42 monomers. Understanding the molecular mechanisms by which Aβ fibrils disrupt cell adhesion pathways may reveal new therapeutic approaches to prevent disease progression.
Keywords: amiloid beta, amyloid-beta fibrils
Published in DiRROS: 20.03.2026; Views: 378; Downloads: 373
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3. Imunosupresija pri glioblastomu : vloga cistatina F in vpliv na celice NKEmanuela Senjor, Ana Mitrović, Marko Jukič, Matic Proj, Stanislav Gobec, Barbara Breznik Vittori, Janko Kos, Milica Perišić, 2024, published scientific conference contribution Keywords: imunosupresija, glioblastom, celice NK, onkologija
Published in DiRROS: 25.02.2026; Views: 493; Downloads: 157
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4. Imunospresija pri glioblastomu : vloga cistatina F in vpliv na celice NKEmanuela Senjor, Ana Mitrović, Marko Jukič, Matic Proj, Stanislav Gobec, Barbara Breznik, Janko Kos, Milica Perišić, 2024, published scientific conference contribution Published in DiRROS: 25.02.2026; Views: 460; Downloads: 136
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6. Targeting cystatin F activation enhances NK cell cytotoxicity in glioblastoma modelsEmanuela Senjor, Anamarija Habič, Urban Švajger, Ana Mitrović, Matic Proj, Andrej Porčnik, Borut Prestor, Miha Jerala, Matic Bošnjak, Stanislav Gobec, Barbara Breznik Vittori, Janko Kos, Milica Perišić, 2025, original scientific article Abstract: Introduction: Glioblastoma (GBM) is a highly invasive brain tumor with limited treatment options and poor prognosis. Natural killer (NK) cells are key effectors of antitumor immunity, capable of eliminating cancer stem-like cells. However, GBM creates an immunosuppressive microenvironment that limits NK cell function. Here, we identify cystatin F as an immunosuppressive factor involved in regulating NK cell granule-mediated cytotoxicity. Methods: We analyzed cystatin F expression in GBM and its correlation with immune exhaustion markers. NK cell activity was compared between GBM patients and healthy donors. In vitro co-cultures of cystatin F-expressing microglial cells and glioblastoma stem-like cells were used to assess NK cell function. To block cystatin F activation from dimeric to active monomeric form, a small-molecule inhibitor of cathepsin V, the activating protease, was applied. Results: Cystatin F expression correlated with immune exhaustion and suppression markers in GBM. NK cells from patients showed reduced cytotoxicity compared to healthy donors. Co-cultures confirmed that cystatin F-expressing microglia impaired NK cell cytotoxicity, while inhibition of cathepsin V restored NK cell function in standard cytotoxicity assays, 3D spheroids, and microfluidic perfused models. Discussion: These results indicate that cystatin F mediates NK cell suppression in GBM. Targeting its activation enhances NK cell cytotoxicity, offering a potential strategy to improve NK-based immunotherapy for glioblastoma.
Keywords: glioblastoma, cystatin F, 3D models
Published in DiRROS: 26.11.2025; Views: 1170; Downloads: 457
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7. Exhausted natural killer cells in adult IgA vasculitisMatija Bajželj, Emanuela Senjor, Nika Boštic, Matjaž Hladnik, Snežna Sodin-Šemrl, Milica Perišić, Janko Kos, Alojz Ihan, Alojzija Hočevar, Andreja Nataša Kopitar, Katja Lakota, 2025, original scientific article Abstract: Introduction. IgA vasculitis nephritis (IgAVN) manifests in up to 84% of adult patients with IgA vasculitis (IgAV) and is associated with an elevated risk of progression to chronic kidney failure. The underlying pathogenic mechanism of adult IgAVN in leukocytes remain largely uncharacterised. Although natural killer (NK) cells were investigated in paediatric IgAV, their specific role in the pathogenesis of adult IgAV has yet to be elucidated. Methods. RNA sequencing of leukocytes from adult IgAV patients and healthy controls (HC) was performed. NK cells’ cytotoxicity was assessed using calcein-AM stained K562 cells, and exocytosis was measured by LAMP-1/CD107a expression. Intracellular perforin and granzyme B were analyzed via flow cytometry, and cytokine secretion was measured by Luminex xMAP. Interferon-induced genes were validated with qPCR. Results. Principal component analysis (PCA) of leukocyte gene expression profiles distinguished IgAV patients from HC. Pathway enrichment analysis showed differences in patients’ subsets - Interferon signalling Reactome pathway was observed only in sample from patients with skin-limited IgAV (sl-IgAV) and was confirmed by increased expression of interferon-induced genes using qPCR. Only in samples from IgAVN patients enrichment of NK cell-mediated cytotoxicity KEGG pathway was found. NK cells from IgAVN patients showed significantly decreased cytotoxicity compared to samples from sl-IgAV patients (p = 2.53 × 10− 2). The % of CD107a+-NK cells significantly increased after stimulation in HC (p = 9.7 × 10− 3) and in sl-IgAV patient samples (p = 2.21 × 10− 2) while only a minor increase was observed in samples of IgAVN patients. IgAVN patients exhibited a decreased % of perforin+ NK cells compared to HC. Following phytohemagglutinin (PHA)/interleukin (IL)-2 stimulation, a significant reduction in intracellular perforin level was observed in HC (p = 2.53 × 10− 2), but not in IgAVN patients NK cells. Interferon (IFN)-ϒ and macrophage inflammatory protein (MIP)-1β were significantly decreased in NK cell culture supernatants from IgAVN patients (p = 2.64 × 10− 2 and p = 2.65 × 10− 2 respectively). Conclusion. Patients with IgAVN exhibited impaired cytotoxic and immunomodulatory functions of NK cells, along with a marked absence of interferon signaling in PBMCs. Further studies are needed to confirm if discrimination of patient subsets based on leukocyte samples might be of clinical use and if deregulated NK function might contribute to the pathogenesis of nephritis in adult IgAV.
Keywords: immunoglobulin A, IgAVN, IgA vasculitis, RNA sequencing, kidney diseases, immunoglobulins, killer cells, adults
Published in DiRROS: 12.11.2025; Views: 802; Downloads: 385
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8. Different glycosylation profiles of cystatin F alter the cytotoxic potential of natural killer cellsEmanuela Senjor, Martina Pirro, Urban Švajger, Mateja Prunk, Jerica Sabotič, Anahid Jewett, Paul J. Hensbergen, Milica Perišić, Janko Kos, 2024, original scientific article Abstract: Cystatin F, a cysteine peptidase inhibitor, is a potent modulator of NK cytotoxicity. By inhibiting granule-mediated cytotoxicity pathway, cystatin F induces formation of non-functional NK cell stage, called split-anergy. We show that N-glycosylation determines the localization and cellular function of cystatin F. Cystatin F mostly exhibited high-mannose glycosylation in U-937 cells, both high-mannose and complex glycosylation in NK-92 and primary NKs, and predominantly complex glycosylation in super-charged NKs. Manipulating N-glycosylation with kifunensine increased high-mannose glycosylation of cystatin F and lysosome localisation, which decreased cathepsin C activity and reduced NK cytotoxicity. Mannose-6-phosphate could significantly reduce the internalization of extracellular cystatin F. By comparing NK cells with different cytotoxic potentials, we found that high-mannose cystatin F was strongly associated with lysosomes and cathepsin C in NK-92 cell line. In contrast, in highly cytotoxic super-charged NKs, cystatin F with complex glycosylation was associated with the secretory pathway and less prone to inhibit cathepsin C. Modulating glycosylation to alter cystatin F localisation could increase the cytotoxicity of NK cells, thereby enhancing their therapeutic potential for treating cancer patients.
Keywords: cystatin F, immunosuppression, NK cells, N-Glycosylation
Published in DiRROS: 04.09.2025; Views: 700; Downloads: 281
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9. Cysteine cathepsins as therapeutic targets in immune regulation and immune disordersEmanuela Senjor, Janko Kos, Milica Perišić, 2023, review article Abstract: Cysteine cathepsins, as the most abundant proteases found in the lysosomes, play a vital role in several processes—such as protein degradation, changes in cell signaling, cell morphology, migration and proliferation, and energy metabolism. In addition to their lysosomal function, they are also secreted and may remain functional in the extracellular space. Upregulation of cathepsin expression is associated with several pathological conditions including cancer, neurodegeneration, and immune-system dysregulation. In this review, we present an overview of cysteine-cathepsin involvement and possible targeting options for mitigation of aberrant function in immune disorders such as inflammation, autoimmune diseases, and immune response in cancer.
Keywords: cysteine cathepsins, inflammation, autoimmune diseases, cancer
Published in DiRROS: 04.09.2025; Views: 1010; Downloads: 575
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10. New inhibitors of cathepsin V impair tumor cell proliferation and elastin degradation and increase immune cell cytotoxicityAna Mitrović, Emanuela Senjor, Marko Jukič, Lara Bolčina, Mateja Prunk, Matic Proj, Milica Perišić, Stanislav Gobec, Janko Kos, 2022, original scientific article Abstract: Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy.
Published in DiRROS: 04.09.2025; Views: 651; Downloads: 485
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