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Extracellular vesicles (EVs) have emerged as potential vehicles for targeted drug delivery and diagnostic applications. However, achieving consistent and reliable functionalization of EV membranes remains a challenge. Copper-catalyzed click chemistry, commonly used for EV surface modification, poses limitations due to cytotoxicity and interference with biological systems. To overcome these limitations, we developed a standardized method for functionalizing an EV membrane via copper-free click chemistry. EVs derived from plasma hold immense potential as diagnostic and therapeutic agents. However, the isolation and functionalization of EVs from such a complex biofluid represent considerable challenges. We compared three different EV isolation methods to obtain an EV suspension with an optimal purity/yield ratio, and we identified sucrose cushion ultracentrifugation (sUC) as the ideal protocol. We then optimized the reaction conditions to successfully functionalize the plasma-EV surface through a copper-free click chemistry strategy with a fluorescently labeled azide, used as a proof-of-principle molecule. Click-EVs maintained their identity, size, and, more importantly, capacity to be efficiently taken up by responder tumor cells. Moreover, once internalized, click EVs partially followed the endosomal recycling route. The optimized reaction conditions and characterization techniques presented in this study offer a foundation for future investigations and applications of functionalized EVs in drug delivery, diagnostics, and therapeutics.

Introduction: The secretome of mesenchymal stromal cells (MSCs) serves as an innovative tool employed in the regenerative medicine approach. In this particular context, three-dimensional (3D) culture systems are widely utilized to better replicate in vivo conditions and facilitate prolonged cell maintenance during culture. The use of spheroids enables the preservation of the classical phenotypical characteristics of MSCs. However, the distinct microenvironment within the spheroid may impact the secretome, thereby enhancing the angiogenic properties of adult MSCs that typically possess a reduced angiogenic potential compared to MSCs derived from perinatal tissues due to the hypoxia created in the internal region of the spheroid. Methods: In this study, large spheroids (2,600 cells, ∼300 μm diameter) and small spheroids (1,000 cells, ∼200 μm diameter) were used to examine the role of spheroid diameter in the generation of nutrients and oxygen gradients, cellular senescence, and the angiogenic potential of secreted factors and extracellular vesicles (EVs). Results: In this study, we demonstrate that large spheroids showed increased senescence and a secretome enriched in pro-angiogenic factors, as well as pro-inflammatory and anti-angiogenic cytokines, while small spheroids exhibited decreased senescence and a secretome enriched in pro-angiogenic molecules. We also demonstrated that 3D culture led to a higher secretion of EVs with classical phenotypic characteristics. Soluble factors and EVs from small spheroids exhibited higher angiogenic potential in a human umbilical vein endothelial cell (HUVEC) angiogenic assay. Discussion: These findings highlighted the necessity of choosing the appropriate culture system for obtaining soluble factors and EVs for specific therapeutic applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Rovere, Reverberi, Arnaldi, Palamà and Gentili.)

Identifying oncological applications for drugs that are already approved for other medical indications is considered a possible solution for the increasing costs of cancer treatment. Under the hypothesis that nutritional stress through fasting might enhance the antitumour properties of at least some non-oncological agents, by screening drug libraries, we find that cholesterol biosynthesis inhibitors (CBIs), including simvastatin, have increased activity against cancers of different histology under fasting conditions. We show fasting's ability to increase CBIs' antitumour effects to depend on the reduction in circulating insulin, insulin-like growth factor-1 and leptin, which blunts the expression of enzymes from the cholesterol biosynthesis pathway and enhances cholesterol efflux from cancer cells. Ultimately, low cholesterol levels through combined fasting and CBIs reduce AKT and STAT3 activity, oxidative phosphorylation and energy stores in the tumour. Our results support further studies of CBIs in combination with fasting-based dietary regimens in cancer treatment and highlight the value of fasting for drug repurposing in oncology., (© 2023. The Author(s).)

Glioblastoma progression in its early stages remains poorly understood. Here, we transfer PDGFB and genetic barcodes in mouse brain to initiate gliomagenesis and enable direct tracing of glioblastoma evolution from its earliest possible stage. Unexpectedly, we observe a high incidence of clonal extinction events and progressive divergence in clonal sizes, even after the acquisition of malignant phenotype. Computational modeling suggests these dynamics result from clonal-based cell-cell competition. Through bulk and single-cell transcriptome analyses, coupled with lineage tracing, we reveal that Myc transcriptional targets have the strongest correlation with clonal size imbalances. Moreover, we show that the downregulation of Myc expression is sufficient to drive competitive dynamics in intracranially transplanted gliomas. Our findings provide insights into glioblastoma evolution that are inaccessible using conventional retrospective approaches, highlighting the potential of combining clonal tracing and transcriptomic analyses in this field., Competing Interests: Declaration of interests The authors declare to have no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The treatment of non-small cell lung cancer (NSCLC) has changed dramatically with the advent of immune checkpoint inhibitors (ICIs). Despite encouraging results, their efficacy remains limited to a subgroup of patients. Circulating immune checkpoints in soluble (s) form and associated with extracellular vesicles (EVs) represent promising markers, especially in ICI-based therapeutic settings. We evaluated the prognostic role of PD-L1 and of two B7 family members (B7-H3, B7-H4), both soluble and EV-associated, in a cohort of advanced NSCLC patients treated with first- ( n = 56) or second-line ( n = 126) ICIs. In treatment-naïve patients, high baseline concentrations of sPD-L1 (>24.2 pg/mL) were linked to worse survival, whereas high levels of sB7-H3 (>0.5 ng/mL) and sB7-H4 (>63.9 pg/mL) were associated with better outcomes. EV characterization confirmed the presence of EVs positive for PD-L1 and B7-H3, while only a small portion of EVs expressed B7-H4. The comparison between biomarker levels at the baseline and in the first radiological assessment under ICI-based treatment showed a significant decrease in EV-PD-L1 and an increase in EV-B7H3 in patients in the disease response to ICIs. Our study shows that sPD-L1, sB7-H3 and sB7-H4 levels are emerging prognostic markers in patients with advanced NSCLC treated with ICIs and suggests potential EV involvement in the disease response to ICIs.

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors. Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O 2 ) or under hypoxia (1% O 2 ) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function. Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1α-induced inflammation and to reduce production of pro-inflammatory cytokines. Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Inflammaging is one of the evolutionarily conserved mechanisms underlying aging and is defined as the long-term consequence of the chronic stimulation of the innate immune system. As macrophages are intimately involved in initiating and regulating the inflammatory process, their dysregulation plays major roles in inflammaging. The paracrine factors, and in particular extracellular vesicles (EVs), released by mesenchymal stromal cells (MSCs) retain immunoregulatory effects on innate and adaptive immune responses. In this paper, we demonstrate that EVs derived from MSCs preconditioned with hypoxia inflammatory cytokines exerted an anti-inflammatory role in the context of inflammaging. In this study, macrophages isolated from aged mice presented elevated pro-inflammatory factor levels already in basal conditions compared to the young counterpart, and this pre-activation status increased when cells were challenged with IFN-γ. EVs were able to attenuate the age-associated inflammation, inducing a decrease in the expression of TNF-α, iNOS, and the NADase CD38. Moreover, we demonstrate that EVs counteracted the mitochondrial dysfunction that affected the macrophages, reducing lipid peroxidation and hindering the age-associated impairment of mitochondrial complex I activity, oxygen consumption, and ATP synthesis. These results indicate that preconditioned MSC-derived EVs might be exploited as new anti-aging therapies in a variety of age-related diseases.

Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rβ1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rβ1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3'-UTR region of the IL-12Rβ1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rβ1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rβ1-positive CLL cells in the spleen. Our findings indicate that IL-12Rβ1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5 + B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5 + B cells, nor in specific B-cell subpopulations or the CD5 + cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bagnara, Colombo, Reverberi, Matis, Massara, Cardente, Ubezio, Agostini, Agnelli, Neri, Cardillo, Vergani, Ghiotto, Mazzarello, Morabito, Cutrona, Ferrarini and Fais.)

Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid-derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O 2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of R26pFUCCI2 mice with cell cycle fluorescent tagging by mutually exclusive nuclear signal. mNVCM were stimulated by fetal versus perinatal hAFSC-CM and hAFSC-EVs to identify the most promising formulation for in vivo assessment in a R26pFUCCI2 neonatal mouse model of myocardial infarction (MI) via intraperitoneal delivery. While the perinatal hAFSC secretome did not provide any significant cardiogenic effect, fetal hAFSC-EVs significantly sustained mNVCM transition from S to M phase by 2-fold, while triggering cytokinesis by 4.5-fold over vehicle-treated cells. Treated mNVCM showed disorganized expression of cardiac alpha-actinin, suggesting cytoskeletal re-arrangements prior to cell renewal, with a 40% significant downregulation of Cofilin-2 and a positive trend of polymerized F-Actin. Fetal hAFSC-EVs increased cardiomyocyte cell cycle progression by 1.8-fold in the 4-day-old neonatal left ventricle myocardium short term after MI; however, such effect was lost at the later stage. Fetal hAFSC-EVs were enriched with a short isoform of Agrin, a mediator of neonatal heart regeneration acting by YAP-related signaling; yet in vitro application of YAP inhibitor verteporfin partially affected EV paracrine stimulation on mNVCM. EVs secreted by developmentally juvenile fetal hAFSC can support cardiomyocyte renewal to some extension, via intercellular conveyance of candidates possibly involving Agrin in combination with other factors. These perinatal derivative promising cardiogenic effects need further investigation to define their specific mechanism of action and enhance their potential translation into therapeutic opportunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Costa, Balbi, Garbati, Palamà, Reverberi, De Palma, Rossi, Paladini, Coviello, De Biasio, Ceresa, Malatesta, Mauri, Quarto, Gentili, Barile and Bollini.)

The study of circulating cancer-derived components (circulome) is considered the new frontier of liquid biopsy. Despite the recognized role of circulome biomarkers, their comparative molecular profiling is not yet routine. In advanced breast cancer (BC), approximately 40% of hormone-receptor-positive, HER2-negative BC cases harbor druggable PIK3CA mutations suitable for combined alpelisib/fulvestrant treatment. This pilot study investigates PIK3CA mutations in circulating tumor DNA (ctDNA), tumor cells (CTCs), and extracellular vesicles (EVs) with the aim of determining which information on molecular targetable profiling could be recollected in each of them. The in-depth molecular analysis of four BC patients demonstrated, as a proof-of-concept study, that it is possible to retrieve mutational information in the three components. Patient-specific PIK3CA mutations were found in both tissue and ctDNA and in 3/4 cases, as well as in CTCs, in the classical population (large-sized CD45-/EpCAM+/- cells), and/or in the "non-conventional" sub-population (smaller-sized CD44+/EpCAM-/CD45- cells). Consistent mutational profiles of EVs with CTCs suggest that they may have been released by CTCs. This preliminary evidence on the molecular content of the different circulating biomaterials suggests their possible function as a mirror of the intrinsic heterogeneity of BC. Moreover, this study demonstrates, through mutational assessment, the tumor origin of the different CTC sub-populations sustaining the translational value of the circulome for a more comprehensive picture of the disease.

Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as a key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant plasma cells. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of anti-apoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for "priming" MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.

Long non-coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naïve B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll-like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients., (© 2021 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

The secretome of mesenchymal stromal cells (MSCs) derived from different tissue sources is considered an innovative therapeutic tool for regenerative medicine. Although adipose tissue-and bone marrow-derived MSCs (ADSCs and BMSCs, respectively) share many biological features, the different tissue origins can be mirrored by variations in their secretory profile, and in particular in the secreted extracellular vesicles (EVs). In this study, we carried out a detailed and comparative characterization of middle- and small-sized EVs (mEVs and sEVs, respectively) released by either ADSCs or BMSCs. Their involvement in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics. Although EVs separated from both cell sources presented similar characteristics in terms of size, concentration, and marker expression, they exhibited different characteristics in terms of protein content and functional effects. ADSC-EVs overexpressed pro-angiogenic factors in comparison to the BMSC-counterpart, and, consequently, they were able to induce a significant increase in endothelial cord outgrowth. On the other hand, BMSC-EVs contained a higher amount of pro-differentiation and chemotactic proteins, and they were able to prompt growth plate organization. The present study highlights the importance of selecting the appropriate cell source of EVs for targeted therapeutic applications., (© 2021 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Circulating autoantibodies of IgG2 isotype predominate in Systemic Lupus Erythematosus (SLE) and concur to the development of the renal lesions characteristic of Lupus Nephritis (LN). Anti-dsDNA and anti-histones IgG2, together with anti-podocyte proteins (i.e., α-enolase) are the major autoantibodies in serum and renal glomeruli of LN patients. The mechanisms underlying autoantibody formation and isotype switching in SLE and LN are unknown. A major issue is how DNA/histones are externalized from cell nucleus, driving the autoimmune response. Neutrophil Extracellular Traps (NETs) have been recently identified as crucial players in this context, representing the main source of DNA and nucleosome proteins. A second key point is what regulates IgG2 isotype switching: in mouse models, T-bet transcription factor has been described as essential for IgG2a class switch. We hypothesized that, in SLE, NET formation is the key mechanism responsible for externalization of autoantigens (i.e., dsDNA, histones 2,3, and α-enolase) and that T-bet is upregulated by NETs, driving, in this way, immunoglobulin class switch recombination (CSR), with production of IgG2 autoantibodies. The data here presented show that NETs, purified from SLE patients, stimulate ex vivo IgG2 isotype class switch possibly through the induction of T-bet. Of note, we observed a prominent effect of NETs on the release of soluble IgG2 in SLE patients', but not in healthy donors' B cells. Our results add important knowledge on the mechanisms of IgG2 class switch in SLE and contribute to further elucidate the role of NETs in LN pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bertelli, Schena, Antonini, Reverberi, Signa, Pedemonte, Consolaro, Gattorno, Negrini, Pupo, Volpi and Ghiggeri.)

We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, anti-fibrotic and proliferative effects. hAFS may also be isolated from III trimester clinical waste samples during scheduled C-sections (perinatal hAFS), thus offering a more easily accessible alternative when compared to fetal hAFS. Nonetheless, little is known about the paracrine profile of perinatal hAFS. Here we provide a detailed characterization of the hAFS total secretome (i.e., the entirety of soluble paracrine factors released by cells in the conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it, from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. hAFS-CM and hAFS-EV formulations were analyzed for protein and chemokine/cytokine content, and the EV cargo was further investigated by RNA sequencing. The phenotype of fetal- and perinatal hAFS, along with their corresponding secretome formulations, overlapped; yet, fetal hAFS showed immature oxidative phosphorylation activity when compared to perinatal ones. The profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. Both cell sources provided formulations enriched with neurotrophic, immunomodulatory, anti-fibrotic and endothelial stimulating factors, and the immature fetal hAFS secretome was defined by a more pronounced pro-vasculogenic, regenerative, pro-resolving and anti-aging profile. Small RNA profiling showed microRNA enrichment in both fetal- and perinatal hAFS-EV cargo, with a stably- expressed pro-resolving core as a reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.

Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Sirtuin 6 (SIRT6) is a NAD + -dependent deacetylase with key roles in cell metabolism. High SIRT6 expression is associated with adverse prognosis in breast cancer (BC) patients. However, the mechanisms through which SIRT6 exerts its pro-oncogenic effects in BC remain unclear. Here, we sought to define the role of SIRT6 in BC cell metabolism and in mouse polyoma middle T antigen (PyMT)-driven mammary tumors., Methods: We evaluated the effect of a heterozygous deletion of Sirt6 on tumor latency and survival of mouse mammary tumor virus (MMTV)-PyMT mice. The effect of SIRT6 silencing on human BC cell growth was assessed in MDA-MB-231 xenografts. We also analyzed the effect of Sirt6 heterozygous deletion, of SIRT6 silencing, and of the overexpression of either wild-type (WT) or catalytically inactive (H133Y) SIRT6 on BC cell pyruvate dehydrogenase (PDH) expression and activity and oxidative phosphorylation (OXPHOS), including respiratory complex activity, ATP/AMP ratio, AMPK activation, and intracellular calcium concentration., Results: The heterozygous Sirt6 deletion extended tumor latency and mouse survival in the MMTV-PyMT mouse BC model, while SIRT6 silencing slowed the growth of MDA-MB-231 BC cell xenografts. WT, but not catalytically inactive, SIRT6 enhanced PDH expression and activity, OXPHOS, and ATP/AMP ratio in MDA-MB-231 and MCF7 BC cells. Opposite effects were obtained by SIRT6 silencing, which also blunted the expression of genes encoding for respiratory chain proteins, such as UQCRFS1, COX5B, NDUFB8, and UQCRC2, and increased AMPK activation in BC cells. In addition, SIRT6 overexpression increased, while SIRT6 silencing reduced, intracellular calcium concentration in MDA-MB-231 cells. Consistent with these findings, the heterozygous Sirt6 deletion reduced the expression of OXPHOS-related genes, the activity of respiratory complexes, and the ATP/AMP ratio in tumors isolated from MMTV-PyMT mice., Conclusions: Via its enzymatic activity, SIRT6 enhances PDH expression and activity, OXPHOS, ATP/AMP ratio, and intracellular calcium concentration, while reducing AMPK activation, in BC cells. Thus, overall, SIRT6 inhibition appears as a viable strategy for preventing or treating BC.

Human RNASET2 acts as a powerful oncosuppressor protein in in vivo xenograft-based murine models of human cancer. Secretion of RNASET2 in the tumor microenvironment seems involved in tumor suppression, following recruitment of M1-polarized macrophages. Here, we report a murine Rnaset2 -based syngeneic in vivo assay. BALB/c mice were injected with parental, empty vector-transfected or murine Rnaset2 -overexpressing mouse C51 or TS/A syngeneic cells and tumor growth pattern and immune cells distribution in tumor mass were investigated. Compared to control cells, mouse Rnaset2 -expressing C51 cells showed strong delayed tumor growth. CD86 + M1 macrophages were massively recruited in Rnaset2- expressing C51-derived tumors, with concomitant inhibition of MDSCs and CD206 + M2 macrophages recruitment. At later times, a relevant expansion of intra-tumor CD8 + T cells was also observed. After re-challenge with C51 parental cells, most mice previously injected with Rnaset2 -expressing C51 cells still rejected C51 tumor cells, suggesting a Rnaset2-mediated T cell adaptive immune memory response. These results point at T2 RNases as evolutionary conserved oncosuppressors endowed with the ability to inhibit cancer growth in vivo through rebalance of intra-tumor M1/M2 macrophage ratio and concomitant recruitment of adaptive anti-tumor CD8 + T cells.

Background: B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5 + and CD5 - B cells from the spleen and peripheral blood (PB)., Methods: Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5 + and CD5 - cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution., Results: CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5 + B (0.57%) cells compared to CD5 - B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family., Conclusions: CBS-IG receptors appear to represent a part of the "public" BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.

Osteoarthritis (OA) is a disabling joint disorder causing articular cartilage degeneration. Currently, the treatments are mainly aimed to pain and symptoms relief, rather than disease amelioration. Human bone marrow stromal cells (hBMSCs) have emerged as a promising paracrine mechanism-based tool for OA treatment. Here, we investigate the therapeutic potential of conditioned media (CM) and extracellular vesicles (EVs) isolated from hBMSC and grown in a xeno-free culture system (XFS) compared to the conventional fetal bovine serum-culture system (FBS) in an in vitro model of OA. First, we observed that XFS promoted growth and viability of hBMSCs compared to FBS-containing medium while preserving their typical phenotype. The biological effects of the CM derived from hBMSC cultivated in XFS- and FBS-based medium were tested on IL-1α treated human chondrocytes, to mimic the OA enviroment. Treatment with CM derived from XFS-cultured hBMSC inhibited IL-1α-induced expression of IL-6, IL-8, and COX-2 by hACs compared to FBS-based condition. Furthermore, we observed that hBMSCs grown in XFS produced a higher amount of EVs compared to FBS-culture. The hBMSC-EVs not only inhibit the adverse effects of IL-1α-induced inflammation, but play a significant in vitro chondroprotective effect. In conclusion, the XFS medium was found to be suitable for isolation and expansion of hBMSCs with increased safety profile and intended for ready-to-use clinical therapies., (Copyright © 2020 Palamà, Shaw, Carluccio, Reverberi, Sercia, Persano, Pisignano, Cortese, Barry, Murphy and Gentili.)

The high invasive phenotype of glioblastoma is one of the main causes of therapy inefficacy and tumor relapse. Cell adhesion molecules of the cadherin family are involved in cell migration and are known as master regulators of epithelial tumor invasiveness, but their role in glioblastoma is less understood. In particular, we recently demonstrated, in the syngeneic murine model, the occurrence of a previously undescribed cadherin switch between Cdh2 and Cdh4 during gliomagenesis, which is necessary for the acquisition of the highly infiltrative and tumorigenic phenotype of these cells. In the present study, we tested the role of Cdh4 in human gliomas. Our results on patient-derived glioma cells demonstrate a positive correlation between Cdh4 expression levels and the loss of cell-cell contact inhibition of proliferation controls that allows cells to proliferate over confluence. Moreover, the silencing of Cdh4 by artificial microRNAs induced a decrease in the infiltrative ability of human glioma cells both in vitro and in vivo. More strikingly, Cdh4 silencing induced an impairment of the tumorigenic potential of these cells after orthotopic transplantation in immunodeficient mice. Overall, we conclude that in human glioblastoma, Cdh4 can also actively contribute in regulating cell invasiveness and malignancy.

The mutual reshape of tumor and immune system cells during tumor progression is a widely accepted notion in different cancers including gliomas. The importance of this phenomenon in shaping glioma progression and the mechanisms governing it, however, are not fully elucidated. Taking advantage of a well-characterized in vivo glioma model we performed an analysis of glioma cells transcriptomes at different stages of progression and unveiled the reorganization of glioma-immune system interactions. Specifically, we show that the inability of low-grade glioma cells to orthotopically graft in syngeneic immunocompetent mice, positively correlates with the abundance of infiltrating lymphocytes in donor tumors and with a highly immunostimulatory transcriptional profile. Notably, during tumor progression glioma cells downregulate these genes and the immune infiltrate shifts towards a pro-tumorigenic phenotype. Challenging low-grade gliomas by grafting into immunodeficient hosts revealed the crucial role of the adaptive immune system in constraining glioma progression. Finally, we observed that although progression still takes place in immunodeficient mice, it is slower, likely due to a milder selection thus reinforcing the view of a pivotal role for the immune system in regulating glioma progression., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This unit describes protocols for isolating subpopulations of extracellular vesicles (EVs) purified from human adipose tissue-derived mesenchymal stromal cells by density gradient centrifugation and for characterizing them by flow cytometry (FCM). Determining the optimal strategy for isolating EVs is a critical step toward retrieving the maximal amount while ensuring the recovery of different vesicular subtypes. The first protocol details density gradient centrifugation to isolate both exosomes and microvesicles. In the second protocol, characterization of EV subpopulations by FCM is depicted, taking advantage of non-conventional modalities, in accordance with the latest technical indications. The procedures described here can be easily reproduced and can be employed regardless of the cell type used to obtain EVs. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Background: Restoration of damaged tissues through the activation of endogenous progenitors is an attractive therapeutic option. A deep evaluation of the intrinsic stem/progenitor cell properties as well as the reciprocal interactions with injured environments is of critical importance., Methods: Here, we show that bone marrow stromal cell antigen 2 (BST2) allows the isolation of a population of circulating progenitors, the circulating healing (CH) cells, characterized by a distinctive core signature. The bone marrow (BM) origin of BST2 pos CH cells has been strengthened by the co-expression of leptin receptor, the hallmark of a subpopulation of BM-skeletal stem cells., Results: BST2 pos CH cells retained the capacity to (i) respond to injury signals generated by a bone fracture, (ii) modify the expression of cell motility genes following damage, and (iii) react to hepatocyte growth factor-activator (HGFA), an injury-related stimulus sufficient to induce their transition into G ALERT , a state in which cells are functionally activated and participate in tissue repair., Conclusions: Taken together, these results could pave the way for the identification of new strategies to enhance and potentiate endogenous regenerative mechanisms for future therapies.

The unit describes protocols for isolating and characterizing extracellular vesicles (EVs) derived from human adipose tissue-derived mesenchymal stromal cells (MSCs). EVs are a mixed population of membrane-surrounded structures with overlapping composition and size. Advances made in recent years have led to a better understanding of the biological role of EVs. In particular, they can be considered key factors responsible for MSC-paracrine activity, mediating their anti-inflammatory effects towards innate immune cells, such as macrophages. The topics comprise description of the MSC-conditioned medium containing vesicles preparation, EV isolation, and characterization mainly by specifically set up flow cytometry and electron microscopy approaches, and in vitro methodologies involved in testing the EV anti-inflammatory capacity. The procedures described here can be easily reproduced and can be employed regardless of the type of progenitor cells used to secrete EVs. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rβ1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rβ1 chain when cocultured with activated T cells or CD40L + cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)