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Abstract Extracellular vesicles (EVs) are a new mechanism of cellular communication, by delivering their cargo into target cells to modulate molecular pathways. EV-mediated crosstalk contributes to tumor survival and resistance to cellular stress. However, the role of EVs in B-cell Acute Lymphoblastic Leukaemia (B-ALL) awaits to be thoroughly investigated. We recently published that ActivinA increases intracellular calcium levels and promotes actin polymerization in B-ALL cells. These biological processes guide cytoskeleton reorganization, which is a crucial event for EV secretion and internalization. Hence, we investigated the role of EVs in the context of B-ALL and the impact of ActivinA on this phenomenon. We demonstrated that leukemic cells release a higher number of EVs in response to ActivinA treatment, and they can actively uptake EVs released by other B-ALL cells. Under culture-induced stress conditions, EVs coculture promoted cell survival in B-ALL cells in a dose-dependent manner. Direct stimulation of B-ALL cells with ActivinA or with EVs isolated from ActivinA-stimulated cells was even more effective in preventing cell death. This effect can be possibly ascribed to the increase of vesiculation and modifications of EV-associated microRNAs induced by ActivinA. These data demonstrate that ActivinA boosts EV-mediated B-ALL crosstalk, improving leukemia survival in stress conditions.

The development of biomimetic nanoparticles (NPs) has revolutionized the concept of nanomedicine by offering a completely new set of biocompatible materials to formulate innovative drug delivery systems capable of imitating the behavior of cells. Specifically, the use of leukocyte-derived membrane proteins to functionalize nanovesicles (leukosomes) can enable their long circulation and target the inflamed endothelium present in many inflammatory pathologies and tumors, making them a promising and versatile drug delivery system. However, these studies did not elucidate the critical experimental parameters involved in leukosomes formulation. In the present study, we approached the preparation of leukosomes using a design of experiment (DoE) method to better understand the influence of experimental parameters on leukosomes features such as size, size distribution, and protein loading. We also validated this formulation technologically and tested its behavior in in vitro colorectal cancer (CRC) models, including CRC patient-derived tumor organoids (PDOs). We demonstrated leukosomes biocompatibility, endothelium adhesion capability, and tumor target in three-dimensional (3D) settings using CRC cell lines. Overall, our study offers a novel conceptual framework for biomimetic NPs using a DoE strategy and consolidates the high therapeutic potential of leukosomes as a viable drug delivery system for anti-inflammatory and antineoplastic applications.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by beta-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has rapidly spread across the globe starting from February 2020. It is well established that during viral infection, extracellular vesicles become delivery/presenting vectors of viral material. However, studies regarding extracellular vesicle function in COVID-19 pathology are still scanty. Here, we performed a comparative study on exosomes recovered from the plasma of either MILD or SEVERE COVID-19 patients. We show that although both types of vesicles efficiently display SARS-CoV-2 spike-derived peptides and carry immunomodulatory molecules, only those of MILD patients are capable of efficiently regulating antigen-specific CD4+ T-cell responses. Accordingly, by mass spectrometry, we show that the proteome of exosomes of MILD patients correlates with a proper functioning of the immune system, while that of SEVERE patients is associated with increased and chronic inflammation. Overall, we show that exosomes recovered from the plasma of COVID-19 patients possess SARS-CoV-2-derived protein material, have an active role in enhancing the immune response, and possess a cargo that reflects the pathological state of patients in the acute phase of the disease.

B-cell precursor-acute lymphoblastic leukemia modulates the bone marrow (BM) niche to become leukemia-supporting and chemo-protective by reprogramming the stromal microenvironment. New therapies targeting the interplay between leukemia and stroma can help improve disease outcome. We identified ActivinA, a TGF-β family member with a well-described role in promoting several solid malignancies, as a factor favoring leukemia that could represent a new potential target for therapy. ActivinA resulted over-expressed in the leukemic BM and its production was strongly induced in mesenchymal stromal cells after culture with leukemic cells. Moreover, MSCs isolated from BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA compared to their normal counterparts. The pro-inflammatory leukemic BM microenvironment synergized with leukemic cells to induce stromal-derived ActivinA. Gene expression analysis of ActivinA-treated leukemic cells showed that this protein was able to significantly influence motility-associated pathways. Interestingly, ActivinA promoted random motility and CXCL12-driven migration of leukemic cells, even at suboptimal chemokine concentrations, characterizing the leukemic niche. Conversely, ActivinA severely impaired CXCL12-induced migration of healthy CD34+ cells. This opposite effect can be explained by the ability of ActivinA to increase intracellular calcium only in leukemic cells, boosting cytoskeleton dynamics through a higher rate of actin polymerization. Moreover, by stimulating the invasiveness of the leukemic cells, ActivinA was found to be a leukemia-promoting factor. Importantly, the ability of ActivinA to enhance BM engraftment and the metastatic potential of leukemic cells was confirmed in a xenograft mouse model of the disease. Overall, ActivinA was seen to be a key factor in conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche.

Abstract Idiopathic Nephrotic Syndrome (INS) is a common childhood glomerular disease requiring intense immunosuppressive drug treatments. Prediction of treatment response and the occurrence of relapses remains challenging. Biofluid-derived extracellular vesicles (EVs) may serve as novel liquid biopsies for INS classification and monitoring. Our cohort was composed of 105 INS children at different clinical time points (onset, relapse, and persistent proteinuria, remission, respectively), and 19 healthy controls. The expression of 37 surface EV surface markers was evaluated by flow cytometry in serum (n = 83) and urine (n = 74) from INS children (mean age = 10.1, 58% males) at different time points. Urine EVs (n = 7) and serum EVs (n = 11) from age-matched healthy children (mean age = 7.8, 94% males) were also analyzed. Tetraspanin expression in urine EVs was enhanced during active disease phase in respect to the remission group and positively correlates with proteinuria levels. Unsupervised clustering analysis identified an INS signature of 8 markers related to immunity and angiogenesis/adhesion processes. The CD41b, CD29, and CD105 showed the best diagnostic scores separating the INS active phase from the healthy condition. Interestingly, combining urinary and serum EV markers from the same patient improved the precision of clinical staging separation. Three urinary biomarkers (CD19, CD44, and CD8) were able to classify INS based on steroid sensitivity. Biofluid EVs offer a non-invasive tool for INS clinical subclassification and “personalized” interventions.