Sabina Di Matteo

Science is dynamic, and novel models to better understand experimental observations are very important for its advancement. In the context of cell biology, knowledge about how cells behave in response to extracellular stimuli, and how these processes are dysregulated in pathologic conditions, is one of the challenges of biological research nowadays. From a theoretical point of view, the discovery of the double-helix model of DNA structure led to the idea that most aspects of a cell’s behavior could be understood by knowing the sequence and the rules regulating the expression of genes. A paradigm of this way of thinking is the theory of the “selfish gene” by Richard Dawkins. Indeed, this approach is being found to have several limitations, especially in the interpretations of complex pathologies, such as cancer, metabolic or neurodegenerative diseases. For this reason, a paradigm shift should be considered to provide a better modeling of the biological processes that control cellular behavior and physiology. In particular, a central role should be given to metabolism as a relay between the environment and microenvironment and cell function, by driving epigenetic modifications. For these reasons, a new “selfish cell” model should be introduced, which could prove useful to better explain complex diseases and find more effective translational approaches.

Cancer remains one of the leading causes of morbidity and mortality worldwide, with lung, breast, and colorectal cancers among the most prevalent and lethal malignancies. In recent years, extracellular vesicles (EVs) have emerged as important mediators of intercellular communication and promising tools in oncology. EVs are membrane-bound vesicles released by most cell types and carry diverse biomolecules, including nucleic acids, proteins, lipids, and metabolites derived from their parent cells. Their presence in biological fluids makes them attractive candidates for liquid biopsy applications and minimally invasive cancer diagnosis. In addition, EVs have gained considerable attention as therapeutic platforms due to their biocompatibility, stability, and ability to deliver functional cargo to recipient cells. Beyond mammalian EVs, plant-derived extracellular vesicles (PDEVs) are increasingly being investigated as scalable and potentially safe nanocarriers for biomedical applications. This review summarizes current advances in the use of EVs for cancer diagnosis and therapy, with particular emphasis on their role as biomarkers, drug-delivery systems, and emerging therapeutic agents. Furthermore, the review discusses current challenges and future perspectives related to EV isolation, characterization, and clinical translation in oncology.

Nitric oxide (NO) plays an important role in neuronal communication, synaptic plasticity and vascular regulation. Due to its important function in neuronal homeostasis, NO imbalance is associated with neurodegeneration. Specifically, in Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and frontotemporal lobar degeneration (FTLD), an excessive amount of NO, mostly produced by inducible NO synthase (iNOS), reacts with superoxide to form peroxynitrite, driving oxidative/nitrosative stress, mitochondrial dysfunction, and aberrant protein modifications. In AD, NO dysregulation promotes amyloid-β (Aβ) accumulation, tau hyperphosphorylation and synaptic loss, creating a self-perpetuating cycle of neuronal damage. NO’s dual role, protective at physiological levels but harmful if overproduced, underscores the therapeutic potential of antioxidant compounds that restore the balance of NO/NOS (especially iNOS) while preserving physiological functions. However, despite the emerging role of antioxidant-based therapeutic approaches, clinical translation is limited by the complexity of NO signaling and the absence of safe, specific NOS inhibitors. By targeting the molecular switch from protective to toxic, NO activity may offer new personalized treatment avenues for neurodegenerative diseases.

A major function of the intrahepatic biliary epithelium is bicarbonate excretion in bile. Recent reports indicate that budesonide, a corticosteroid with high receptor affinity and hepatic first pass clearance, increases the efficacy of ursodeoxycholic acid, a choleretic agent, in primary biliary cholangitis patients. We have previously reported that bile ducts isolated from rats treated with dexamethasone or budesonide showed an enhanced activity of the Na⁺/H⁺ exchanger isoform 1 (NHE1) and Cl⁻/HCO₃⁻ exchanger protein 2 (AE2) . Increasing the delivery of steroids to the liver may result in three beneficial effects: increase in the choleresis, treatment of the autoimmune or inflammatory liver injury and reduction of steroids’ systemic harmful effects. In this study, the steroid dexamethasone was loaded into nanohydrogels (or nanogels, NHs), in order to investigate corticosteroid-induced increased activities of transport processes driving bicarbonate excretion in the biliary epithelium (NHE-1 isoform) and to evaluate the effects of dexamethasone-loaded NHs (NHs/dex) on liver injury induced by experimental cholestatis. Our results showed that NHs and NHs/dex do not reduce cell viability in vitro in human cholangiocyte cell lines. Primary and immortalized human cholangiocytes treated with NHs/dex show an increase in the functional marker expression of NHE1 cholangiocytes compared to control groups. A mouse model of cholangiopathy treated with NHs/dex shows a reduction in markers of hepatocellular injury compared to control groups (NHs, dex, or sham group). In conclusion, we believe that the NHs/dex formulation is a suitable candidate to be investigated in preclinical models of cholangiopathies. Graphical abstract

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12 days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2 months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications.

Background: Neuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal elements recruited or generated by the cancer cells favor the development of an immune-suppressive microenvironment. Herein, we investigated in NB cell lines and in NB biopsies, the presence of cancer cells with mesenchymal phenotype and determined the immune-suppressive properties of these tumor cells on natural killer (NK) cells. Methods: We assessed the mesenchymal stromal cell (MSC)-like phenotype and function of five human NB cell lines and the presence of this particular subset of neuroblasts in NB biopsies using flow-cytometry, immunohistochemistry, RT-qPCR, cytotoxicity assays, western blot and silencing strategy. We corroborated our data consulting a public gene-expression dataset. Results: Two NB cell lines, SK-N-AS and SK-N-BE(2)C, exhibited an unprecedented MSC phenotype (CD105⁺/CD90⁺/CD73⁺/CD29⁺/CD146⁺/GD2⁺/TAZ⁺). In these NB-MSCs, the ectoenzyme CD73 and the oncogenic/immune-regulatory transcriptional coactivator TAZ were peculiar markers. Their MSC-like nature was confirmed by their adipogenic and osteogenic differentiation potential. Immunohistochemical analysis confirmed the presence of neuroblasts with MSC phenotype (CD105⁺/CD73⁺/TAZ⁺). Moreover, a public gene-expression dataset revealed that, in stage IV NB, a higher expression of TAZ and CD105 strongly correlated with a poorer outcome. Among the NB-cell lines analyzed, only NB-MSCs exhibited multifactorial resistance to NK-mediated lysis, inhibition of activating NK receptors, signal adaptors and of NK-cell cytotoxicity through cell-cell contact mediated mechanisms. The latter property was controlled partially by TAZ, since its silencing in NB cells efficiently rescued NK-cell cytotoxic activity, while its overexpression induced opposite effects in non-NB-MSC cells. Conclusions: We identified a novel NB immunoregulatory subset that: (i) displayed phenotypic and functional properties of MSC, (ii) mediated multifactorial resistance to NK-cell-induced killing and (iii) efficiently inhibited, in coculture, the cytotoxic activity of NK cells against target cells through a TAZ-dependent mechanism. These findings indicate that targeting novel cellular and molecular components may disrupt the immunomodulatory milieu of the NB microenvironment ameliorating the response to conventional treatments as well as to advanced immunotherapeutic approaches, including adoptive transfer of NK cells and chimeric antigen receptor T or NK cells.

Soluble interleukin (IL)-15 exists under two forms: as monomer (sIL-15) or as heterodimeric complex in association with sIL-15Rα (sIL-15/IL-15Rα). Both forms have been successfully tested in experimental tumor murine models and are currently undergoing investigation in phase I/II clinical trials. Despite more than 20 years research on IL-15, some controversial issues remain to be addressed. A first point concerns the detection of the sIL-15/IL-15Rα in plasma of healthy donors or patients with cancer and its biological significance. The second and third unsolved question regards the protumorigenic role of the IL-15/IL-15Rα complex in human cancer and the detrimental immunological consequences associated to prolonged exposure of natural killer (NK) cells to both forms of soluble IL-15, respectively. Data suggest that in vivo prolonged or repeated exposure to monomeric sIL-15 or the soluble complex may lead to NK hypo-responsiveness through the expansion of the CD8⁺/CD44⁺ T cell subset that would suppress NK cell functions. In vitro experiments indicate that soluble complex and monomeric IL-15 may cause NK hyporesponsiveness through a direct effect caused by their prolonged stimulation, suggesting that this mechanism could also be effective in vivo. Therefore, a better knowledge of IL-15 and a more appropriate use of both its soluble forms, in terms of concentrations and time of exposure, are essential in order to improve their therapeutic use. In cancer, the overproduction of sIL-15/IL-15Rα could represent a novel mechanism of immune escape. The soluble complex may act as a decoy cytokine unable to efficiently foster NK cells, or could induce NK hyporesponsiveness through an excessive and prolonged stimulation depending on the type of IL-15Rα isoforms associated. All these unsolved questions are not merely limited to the knowledge of IL-15 pathophysiology, but are crucial also for the therapeutic use of this cytokine. Therefore, in this review, we will discuss key unanswered issues on the heterogeneity and biological significance of IL-15 isoforms, analyzing both their cancer-related biological functions and their therapeutic implications.

Background and Aims: Cholangiocarcinoma (CCA) is a very aggressive cancer showing the presence of high cancer stem cells (CSCs). Doublecortin‐like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumors. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA). Approach and Results: Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5 [leucine‐rich repeat‐containing G protein‐coupled receptor], CD [clusters of differentiation] 90, EpCAM [epithelial cell adhesion molecule], CD133, and CD13), and primary cell cultures were prepared. DCLK1 expression was analyzed in CCA cell cultures by real‐time quantitative PCR, western blot, and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2‐IN‐1) on cell proliferation (MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium] assay, cell population doubling time), apoptosis, and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and real‐time quantitative PCR. DCLK1 serum concentration was analyzed by enzyme‐linked immunosorbent assay. We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCA^LGR5+ and in iCCA^CD133+ cells compared with unsorted cells. LRRK2‐IN‐1 showed an anti‐proliferative effect in a dose‐dependent manner. LRRK2‐IN‐1 markedly impaired cell proliferation, induced apoptosis, and decreased colony formation capacity and colony size in both iCCA and pCCA compared with the untreated cells. In situ analysis confirmed that DCLK1 is present only in tumors, and not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of patients with iCCA (high), pCCA (high), HCC (low), and cirrhosis (low), but it was almost undetectable in healthy controls. Conclusions: DCLK1 characterizes a specific CSC subpopulation of iCCA^CD133+ and pCCA^LGR5+, and its inhibition exerts anti‐neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis.

Human biliary tree stem/progenitor cells (hBTSCs), reside in peribiliary glands, are mainly stimulated by primary sclerosing cholangitis (PSC) and cholangiocarcinoma. In these pathologies, hBTSCs displayed epithelial-to-mesenchymal transition (EMT), senescence characteristics, and impaired differentiation. Here, we investigated the effects of cholest-4,6-dien-3-one, an oxysterol involved in cholangiopathies, on hBTSCs biology. hBTSCs were isolated from donor organs, cultured in self-renewal control conditions, differentiated in mature cholangiocytes by specifically tailored medium, or exposed for 10 days to concentration of cholest-4,6-dien-3-one (0.14 mM). Viability, proliferation, senescence, EMT genes expression, telomerase activity, interleukin 6 (IL6) secretion, differentiation capacity, and HDAC6 gene expression were analyzed. Although the effect of cholest-4,6-dien-3-one was not detected on hBTSCs viability, we found a significant increase in cell proliferation, senescence, and IL6 secretion. Interestingly, cholest-4.6-dien-3-one impaired differentiation in mature cholangiocytes and, simultaneously, induced the EMT markers, significantly reduced the telomerase activity, and induced HDAC6 gene expression. Moreover, cholest-4,6-dien-3-one enhanced bone morphogenic protein 4 (Bmp-4) and sonic hedgehog (Shh) pathways in hBTSCs. The same pathways activated by human recombinant proteins induced the expression of EMT markers in hBTSCs. In conclusion, we demonstrated that chronic exposition of cholest-4,6-dien-3-one induced cell proliferation, EMT markers, and senescence in hBTSC, and also impaired the differentiation in mature cholangiocytes.