Novel frontiers in urogenital cancers: from molecular bases to preclinical models to tailor personalized treatments in ovarian and prostate cancer patients.

Over the last few decades, the incidence of urogenital cancers has exhibited diverse trends influenced by screening programs and geographical variations. Among women, there has been a consistent or even increased occurrence of endometrial and ovarian cancers; conversely, prostate cancer remains one of the most diagnosed malignancies, with a rise in reported cases, partly due to enhanced and improved screening efforts.Simultaneously, the landscape of cancer therapeutics has undergone a remarkable evolution, encompassing the introduction of targeted therapies and significant advancements in traditional chemotherapy. Modern targeted treatments aim to selectively address the molecular aberrations driving cancer, minimizing adverse effects on normal cells. However, traditional chemotherapy retains its crucial role, offering a broad-spectrum approach that, despite its wider range of side effects, remains indispensable in the treatment of various cancers, often working synergistically with targeted therapies to enhance overall efficacy.For urogenital cancers, especially ovarian and prostate cancers, DNA damage response inhibitors, such as PARP inhibitors, have emerged as promising therapeutic avenues. In BRCA-mutated ovarian cancer, PARP inhibitors like olaparib and niraparib have demonstrated efficacy, leading to their approval for specific indications. Similarly, patients with DNA damage response mutations have shown sensitivity to these agents in prostate cancer, heralding a new frontier in disease management. Furthermore, the progression of ovarian and prostate cancer is intricately linked to hormonal regulation. Ovarian cancer development has also been associated with prolonged exposure to estrogen, while testosterone and its metabolite dihydrotestosterone, can fuel the growth of prostate cancer cells. Thus, understanding the interplay between hormones, DNA damage and repair mechanisms can hold promise for exploring novel targeted therapies for ovarian and prostate tumors.In addition, it is of primary importance the use of preclinical models that mirror as close as possible the biological and genetic features of patients' tumors in order to effectively translate novel therapeutic findings "from the bench to the bedside".In summary, the complex landscape of urogenital cancers underscores the need for innovative approaches. Targeted therapy tailored to DNA repair mechanisms and hormone regulation might offer promising avenues for improving the management and outcomes for patients affected by ovarian and prostate cancers.

Influence of the isolation method on characteristics and functional activity of mesenchymal stromal cell-derived extracellular vesicles.

BACKGROUND AIMS: Extracellular vesicle (EV) isolation methods are based on different physicochemical properties and may result in the purification of distinct EV populations. We compared two different isolation methods suitable for producing clinical-grade mesenchymal stromal cell-derived EVs (MSC-EVs)-ion exchange chromatography (IEX) and ultrafiltration (UF)-and evaluated their impact on the composition and functional properties of EVs. METHODS: EVs were purified from conditioned culture medium using an anion exchange resin (IEX) or Amicon filters with a 100-kDa cutoff (UF) (MilliporeSigma, Burlington, MA, USA). We assessed nanoparticle size and distribution by nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) and morphology by transmission electron microscopy. We also measured protein, lipid and total RNA concentration and immunophenotyped both EV populations by flow cytometry (MACSPlex assay; Miltenyi Biotec, Bergisch Gladbach, Germany). Moreover, immunomodulatory activity was tested using a standardized macrophage polarization assay and T-cell stimulation assay. Finally, proteomic analysis and cytokine quantification were carried out to better characterize both EV populations. RESULTS: We found by both TRPS and NTA that IEX and UF yielded a comparable amount of total particles with similar size and distribution. In addition, a similar quantity of lipids was obtained with the two procedures. However, IEX yielded 10-fold higher RNA quantity and a larger amount of proteins than UF. MSC-EVs isolated from IEX and UF were positive for the exosome markers CD9, CD63 and CD81 and showed a comparable surface marker expression pattern. Both populations demonstrated immunomodulatory activity in vitro, as they prevented acquisition of the M1 phenotype in lipopolysaccharide-stimulated macrophages and inhibited acquisition of the activation markers CD69 and CD25 on T cells, but the IEX-EVs exerted a significantly greater immunomodulatory effect on both macrophages and T cells compared with UF-EVs. Proteomic analysis and gene ontology enrichment analysis revealed no major differences between the preparations. Finally, cytokine quantification revealed that IEX-EVs were more enriched in some crucial anti-inflammatory and immunomodulatory cytokines (e.g., IL-2, IL-10, transforming growth factor beta and vascular endothelial growth factor) compared with UF-EVs. CONCLUSIONS: MSC-EVs isolated by IEX and UF displayed similar physicochemical, phenotypic and functional characteristics. In our conditions, both EV populations demonstrated important anti-inflammatory activity in macrophages and T cells. However, IEX-EVs were more potent than UF-EVs, which may indicate the superiority of this method for the production of clinical-grade EVs.

Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia.

Oxidative stress and fibrosis are important stress responses that characterize bronchopulmonary dysplasia (BPD), a disease for which only a therapy but not a cure has been developed. In this work, we investigated the effects of mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) on lung and brain compartment in an animal model of hyperoxia-induced BPD. Rat pups were intratracheally injected with MSC-EVs produced by human umbilical cord-derived MSC, following the Good Manufacturing Practice-grade (GMP-grade). After evaluating biodistribution of labelled MSC-EVs in rat pups left in normoxia and hyperoxia, oxidative stress and fibrosis investigation were performed. Oxidative stress protection by MSC-EVs treatment was proved both in lung and in brain. The lung epithelial compartment ameliorated glycosaminoglycan and surfactant protein expression in MSC-EVs-injected rat pups compared to untreated animals. Pups under hyperoxia exhibited a fibrotic phenotype in lungs shown by increased collagen deposition and also expression of profibrotic genes. Both parameters were reduced by treatment with MSC-EVs. We established an in vitro model of fibrosis and another of oxidative stress, and we proved that MSC-EVs suppressed the induction of αSMA, influencing collagen deposition and protecting from the oxidative stress. In conclusion, intratracheal administration of clinical-grade MSC-EVs protect from oxidative stress, improves pulmonary epithelial function, and counteracts the development of fibrosis. In the future, MSC-EVs could represent a new cure to prevent the development of BPD.

Biodistribution of Intratracheal, Intranasal, and Intravenous Injections of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles in a Mouse Model for Drug Delivery Studies.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are extensively studied as therapeutic tools. Evaluation of their biodistribution is fundamental to understanding MSC-EVs' impact on target organs. In our work, MSC-EVs were initially labeled with DiR, a fluorescent lipophilic dye, and administered to BALB/c mice (2.00 × 1010 EV/mice) through the following routes: intravenous (IV), intratracheal (IT) and intranasal (IN). DiR-labeled MSC-EVs were monitored immediately after injection, and after 3 and 24 hours (h). Whole-body analysis, 3 h after IV injection, showed an accumulation of MSC-EVs in the mice abdominal region, compared to IT and IN, where EVs mainly localized at the levels of the chest and brain region, respectively. After 24 h, EV-injected mice retained a stronger positivity in the same regions identified after 3 h from injection. The analyses of isolated organs confirmed the accumulation of EVs in the spleen and liver after IV administration. Twenty-four hours after the IT injection of MSC-EVs, a stronger positivity was detected selectively in the isolated lungs, while for IN, the signal was confined to the brain. In conclusion, these results show that local administration of EVs can increase their concentration in selective organs, limiting their systemic biodistribution and possibly the extra-organ effects. Biodistribution studies can help in the selection of the most appropriate way of administration of MSC-EVs for the treatment of different diseases.

Macrophage bioassay standardization to assess the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles.

BACKGROUND AIMS: Owing to the lack of biological assays, determining the biological activity of extracellular vesicles has proven difficult. Here the authors standardized an in vitro assay to assess the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) based on their ability to prevent acquisition of the M1 phenotype in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Induction of tumor necrosis factor alpha, IL-1ß, IL-6 and inducible nitric oxide synthase (iNOS) characterizes the M1 phenotype. Nitric oxide released by iNOS turns into nitrite, which can be easily quantitated in culture media by Griess reaction. METHODS: The authors first tested different assay conditions in 96-well plates, including two seeding densities (2 × 104 cells/well and 4 × 104 cells/well), four LPS doses (1 ng/mL, 10 ng/mL, 100 ng/mL and 1000 ng/mL) and two time points (16 h and 24 h), in order to determine the best set-up to accurately measure nitrite concentration as an index of M1 macrophage polarization. RESULTS: The authors found that seeding 2 × 104 cells/well and stimulating with 10 ng/mL LPS for 16 h allowed the inhibition of nitrite production by 60% with the use of dexamethasone. Using these established conditions, the authors were able to test different MSC-sEV preparations and generate dose-response curves. Moreover, the authors fully analytically validated assay performance and fulfilled cross-validation against other M1 markers. CONCLUSIONS: The authors standardized a quick, cheap and reproducible in vitro macrophage assay that allows for the evaluation and estimation of the anti-inflammatory activity of MSC-sEVs.

Temperature-Related Effects of Myocardial Protection Strategies in Swine Hearts after Prolonged Warm Ischemia.

Insufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after circulatory death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitably longer period of unprotected warm ischemia between circulatory arrest and graft procurement. In this scenario, we aim to improve heart preservation after a warm ischemic period of 20 min by testing different settings of myocardial protective strategies. Pig hearts were collected from a slaughterhouse and assigned to one of the five experimental groups: baseline (BL), cold cardioplegia (CC), cold cardioplegia + adenosine (CC-ADN), normothermic cardioplegia (NtC + CC) or normothermic cardioplegia + cold cardioplegia + adenosine (NtC-ADN + CC). After treatment, tissue biopsies were taken to assess mitochondrial morphology, antioxidant enzyme activity, lipid peroxidation and cytokine and chemokine expressions. NtC + CC treatment significantly prevented mitochondria swelling and mitochondrial cristae loss. Moreover, the antioxidant enzyme activity was lower in this group, as was lipid peroxidation, and the pro-inflammatory chemokine GM-CSF was diminished. Finally, we demonstrated that normothermic cardioplegia preserved mitochondria morphology, thus preventing oxidative stress and the subsequent inflammatory response. Therefore, normothermic cardioplegia is a better approach to preserve the heart after a warm ischemia period, with respect to cold cardioplegia, before transplantation.

Engineered EVs for Oxidative Stress Protection.

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein.

Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis.

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1ß, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.