Immunoregulatory properties of bone marrow mesenchymal stromal cell-derived extracellular vesicles

Mesenchymal stromal cells (MSCs) are adult stem cells of mesodermal origin that can be isolated from various tissues, including bone marrow (BM), adipose tissue and amniotic fluid. MSCs express mesenchymal markers, i.e. CD73, CD90, and CD105, and lack expression of hematopoietic markers, such as CD45, CD34, CD11b and CD14. In addition to their tri-lineage differentiation towards adipocytes, chondrocytes and osteoblasts, MSCs modulate the immune response. In fact, MSCs can regulate the proliferation and activation of different immune effector cells (IECs), including T, B and NK cells. The biological effects of MSCs are not exclusively related to their close interaction with target cells by cell-to-cell contact, but can be mediated by molecule release. For instance, MSC immunomodulation may occur through paracrine mechanisms, including indolamine 2,3 dioxygenase, prostaglandin E2, heme-oxygenese-1, and TGF-\u3b2. In the last decade, a key mechanism of cell-to-cell communication of MSCs through extracellular vesicles (EVs) has been clarified. The potential therapeutic role of MSC-derived EVs has been described in different diseases, including cardiovascular disease, acute kidney injury, and lung injury. EVs are molecular shuttles consisting of a phospholipid bilayer containing different molecules, including proteins and different types of RNAs (mRNA and miRNA). EVs are a family of different shedding vesicles, including exosomes (EXs, 50-100 nm), microvesicles (MVs, 100-1000 nm), and apoptotic bodies (ABs, 50-500 nm). EXs originate by multivesicular body and express specific markers, such as CD63, CD9 and Alix. MVs result from the plasmatic membrane and express specific proteins of the cells of origin. To understand whether the MSC immunomodulatory effects are mediated by EV release, we characterized the protein content and immunomodulatory functions towards different immune effector cells of EVs derived from BM-MSCs. In addition, we evaluated the capability of unfractionated PBMCs to internalize MSC-derived EVs. We observed that the rate of EV internalization was higher in B cells and correlated with their capability to reduce B cell proliferation. By using a reproducible and standardized method we showed a new mechanism of MSC-mediated immunosuppression, thus characterizing better the biological function of MSC-derived EVs and paving the way to a possible clinical application of EVs as alternative cell-free therapy

Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T, B and NK cell functions

Mesenchymal stromal cells (MSCs) are multipotent cells, immunomodulatory stem cells that are currently used for regenerative medicine and treatment of a number of inflammatory diseases, thanks to their ability to significantly influence tissue microenvironments through the secretion of large variety of soluble factors. Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease. Here, we used a standardized methodological approach to dissect the immunomodulatory effects exerted by MSC-derived EVs on unfractionated peripheral blood mononuclear cells and purified T, B and NK cells. We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division. Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases

Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia

Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML

Assessment of haematopoietic progenitor cell counting with the Sysmex\uae XN-1000 to guide timing of apheresis of peripheral blood stem cells.

BACKGROUND: Successful peripheral blood stem cell (PBSC) collection depends on optimal timing of apheresis, as usually determined by flow cytometry CD34-positive (+) cell count in peripheral blood (PB). Since this method is costly and labour-intensive, we evaluated the use of the Hematopoietic Progenitor Cell count programme on a Sysmex\uae XN haematologic analyser (XN-HPC) as a rapid and inexpensive alternative for predicting CD34+ cell count in PB samples. MATERIALS AND METHODS: Haematopoietic progenitor cell and CD34+ cell counts were compared using 273 PB samples collected from 78 healthy donors and 72 patients who underwent PBSC transplantation. We assessed the effectiveness of the XN-HPC in safely predicting pre-harvest CD34+ counts. The most efficient cut-off values of XN-HPC were identified. We also evaluated the imprecision (coefficient of variation, CV) and functional sensitivity. RESULTS: Imprecision of the XN-HPC count was <6.3% on daily measurement of three levels of quality control material. Functional sensitivity was 8.9 7106/L. A cut-off value of 6562 7106/L XN-HPC for multiple myeloma (MM) patients and 6530 7106/L for all other subjects had both 100% specificity and 100% positive predictive value for identifying samples with CD34+ cells 6520 7106/L. An XN-HPC threshold of <13 7106/L identified pre-harvest CD34+ cell count <10 7106/L with 100% sensitivity and 100% negative predictive value. DISCUSSION: The XN-HPC is a fast, easy and inexpensive test that can safely improve apheresis workflow thus possibly replacing other more expensive CD34 counts currently performed and promoting optimal timing of PBSC collection

Characterization of a new B-ALL cell line with constitutional defect of the Notch signaling pathway

Notch signaling contribution to B-cell acute lymphoblastic leukemia (B-ALL) development is still under investigation. The serendipitous onset of B-ALL in a patient affected by the germinal Notch mutation-dependent Alagille syndrome allowed us to establish a B-ALL cell line (VR-ALL) bearing a genetic loss of function in components of Notch signaling. VR-ALL is a common-type B-ALL cell line, grows in conventional culture medium supplemented with 10% serum, and gives rise, once injected into immunodeficient NOG mice, to a mouse xenograft model of B-ALL. Exome sequencing revealed deleterious mutations in some components of Notch signaling, including Jagged1, Notch1, and Notch2. In addition, VR-ALL is sensitive both in vitro and in vivo to \u3b3-secretase inhibitors (GSIs) as well as conventional anti-leukemic drugs. For all these reasons, VR-ALL may help to gain more insights into the role of Notch signaling in B-ALL

Role of Wnt/\u3b2-Catenin Signalling in Acute Myeloid Leukemia (AML) Cell Response to Chemotherapy

Background: Growing evidences from both preclinical and clinical investigations reveal the critical role of Wnt signalling for the development of many cancers and for their response to chemotherapy. Although recent studies suggest that aberrant Wnt signalling can be involved in the neoplastic myeloid cell growth, the contribution of the Wnt/\u3b2-catenin pathway to AML survival and chemoresistance is still unclear. Aims: In this study, we investigated the contribution of WNT/\u3b2-CATENIN signalling to AML survival and chemoresistance. For this purpose we tested different modulators of Wnt/\u3b2-Catenin pathway for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatment. Methods: AML primary blast cells(30 samples) or AML cell lines cultured alone or in presence of human bone marrow mesenchymal stromal cells (hBM-MSCs), were treated with with Cytarabine (Ara-C) or Idarubicin, in presence or absence of Wnt modulators, including ligands (Wnt3a, Wnt5a/5b), Porcupine inhibitors (IWP-2), LRP6 inhibitors (Niclosamide), or antagonists of TCF/\u3b2-catenin (PKF118-310, PNU-74654). Results: In silico analysis showed the enrichment of Wnt signalling components in AML samples. Western Blot and flow cytometry showed the presence of total \u3b2-catenin only in about 2/3 of primary samples analyzed, while . \u3b2-catenin positive samples had different degree of activation of the pathway, as revealed by the expression of active forms of \u3b2-catenin, including (Ser675)\u3b2-catenin and non-phospho-(Ser33/37/Thr41) \u3b2-catenin. Notably, we found that active forms of \u3b2-catenin increased in AML samples in co-culture with hBM-MSCs, thus suggesting that Wnt signalling could be involved in the crosstalk between bone marrow stroma and AML cells. The addition of Wnt or pharmacological inhibitors, such as IWP-2, PNU-74654 and Niclosamide, to the culture medium of \u3b2-catenin-positive AML samples, either cultured alone or in co-culture with hBM-MSCs, reduced AML cell proliferation with slight effect on cell death. When associated to Idarubicin, all Wnt inhibitors except IWP-2 synergycally induced a dramatic cell death in AML cells in both culture conditions. However, when Idarubicin was replaced by Ara-C the synergism was observed only with Niclosamide and PKF. Cell death was mainly due to apoptosis, as shown by Annexin-V staining. Conclusion: Overall our data show that Wnt inhibitors reduce proliferation and chemoresistance of AML cells in culture or co-culture with bone marrow stroma cells. Wnt/\u3b2-catenin signalling may represent a potential therapeutic strategy to improve AML treatment, overcoming bone marrow stromal-mediated anti-apoptotic and chemoresistance effects

Inhibition of GSK-3 Signalling Enhances Sensitivity of Non-Promyelocitic Acute Myeloid Leukemia (AML) Cell to Chemotherapy

Background: GSK-3 is a serine-threonine kinase involved in metabolic regulation as well as in the control of many pathways associated to cancer development, including Notch Wnt/\u3b2-catenin, Hedgehog, and AKT. It has been demonstrated that association of GSK-3 inhibitors with All-trans-retinoic acid (ATRA) significantly improves ATRA-mediated differentiation and cell death of acute promyelocytic (APL) leukaemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemotherapeutic agents. Aims: In this study, we aim to validate GSK-3 signalling as potent successful therapeutic target in non-promyelocytic AML. For this purpose we tested different GSK-3 for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatments. Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocyticAML blast cells (30 samples). AML cellscultured alone or in presence ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs) were treated with GSK-3 inhibitors, including LiCL, AR-A014418, SB 216763, in association or not with Cytarabine (Ara-C) or Idarubicin. Cell proliferation and cell death were measured by CFSE dilution and TOPRO-3/Annexin-V staining, respectively. Results: Flow cytometry and Western blot analysis in AML samples revealed high expression levels of all GSK-3forms, including total GSK-3\u3b1, (Ser21) GSK-3\u3b1, total GSK-3\u3b2, and (Ser 21) GSK-3\u3b2; theseforms were all down-modulated when AML cells were cultured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role in transducting micro-environmental signals in AML cells interacting with bone marrow stroma. The treatment of AML cells with increasing concentrations of each GSK-3 inhibitors decreased AML cell viability in a dose-dependent manner; interestingly, hBM-MSCs or peripheral blood mononuclear cells were less sensitive to GSK-3inhibitors. The addition of each inhibitor increased dramatically the AML cell apoptotic rate induced by the addition of Ara-C or Idarubicin in vitro. Notably, LiCl and AR-A014418 were capable of abrogating hBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin. Conclusion: Overall our data clearly demonstrated that inhibition of GSK-3 reduced proliferation and chemoresistance of non promyelocytic AML cells. Thus GSK-3 inhibition represents a therapeutic strategy not only for APL but also for other AML subtypes

Notch singalling inhibition as a multi-target therapy to overcome bone marrow microenvironment-mediated drug resistance in AML

Background: Several pro-survival proteins promoting resistance to chemother- apy, such as BCl-2, STAT3,NF\u3baB and AKT,are over-expressed in AML cells, thus representing the basis for targeted therapies. However, only multi-target drug strategy may lead to the modulation of the pro-survival protein network, due to the simultaneous activation of alternative pathways. Notch signalling is a master developmental pathway that controls tumour cell survival by interacting with pro-survival proteins, such as \u3b2-catenin, BCL-2, STAT3, NF\u3baB, and AKT, thus representing an ideal target to interfere with all these pathways in different cancer systems. We recently showed that Notch inhibition was capable of abrogating microen- vironment-mediated AML cell chemo-resistance (P550, EHA20); however, little is known about the mechanism involved. Aims: We studied the mechanisms underlying microenvironmental, Notch- mediated AML chemo-resistance by investigating the contribution of BCl-2, STAT3, NF\u3baB and AKT. Using in silico and in vitro approaches we analyzed the expression changes of these proteins in ex-vivo AML cell samples in condition of pharmacological or genetical Notch down-regulation, as well as in AML cells either cultured alone or co-cultured with human bone marrow mesenchymal stromal cells (hBM-MSCs) in presence of chemoterapeutic agents, such as cytarabine (Ara-C) and Idarabucin. Methods: Cells were obtained from bone marrow (33) and peripheral blood (22) samples of AML patients. hBM-MSCs were expanded from bone marrow of 20 healthy donors (BM-MSCs) and 20 AML patients (BM-MSCs*). Gene set enrich- ment analysis (GSEA) were perform using GEOR tools on AML expression array of 304 patients previously deposited in Gene Expression Omnibus (GSE10358). Genetic inhibition of Notch signalling was achieved in AML cell lines (HL-60 and THP1) by infecting cells with lentiviral particles carrying shRNA for either RBP- jK or MALM1, two mediators of Notch signaling. Pharmacological Inhibition of Notch in AML was achieved by using Gamma secretase Inhibitors (GSIs), Notch transcription factors Inhibitor SAHM1, and combination of Notch blocking anti- bodies.Ara-C, and idarubicin were added to culture supernatants at different concentrations. Cell viability was evaluated by Annexin-V/Propidium Iodide (PI). Protein levels were analyzed by intracellular staining with corresponding fluo- rophore conjugated antibodies, followed by flow cytometry analysis. Results: In silico Gene set enrichment analysis and flow cytometry analysis showed that AML samples highly expressed Notch1, Jagged1, STAT3, NF\u3ba-B and AKT genes and proteins. Notably, higher levels of Notch1 were found in patients with poor cytogenetic prognosis, while STAT3, NF-\u3baB and AKT were uniformly expressed by AML patients. Protein analysis revealed low levels of pro-survival proteins AKT, STAT3 and NF-\u3baB in RBP-jk and MALM1 knock- down cells, as compared to control cells infected with non specific shRNA. We then verified that genetic (shRNA) and pharmacological inhibition of Notch, by using either GSIs or Notch receptor blocking antibodies, was capable of sen- sitizing AML cells, either cultured alone or in presence of hBM-MSCs, to ARA- C or idarubucine. Additionally, we found that hBM-MSC-dependent induction of AML chemoresistance was associated to increase of AKT, NF-\u3baB and STAT3 protein levels in AML cells. Similarly, Notch inhibition with GSIs prevented hBM- MSC-mediated increase of AKT, NF-\u3baB and STAT3, thus restoring sensibility of AML cells to Idarubucin treatment. Summary/Conclusions: These results suggest that inhibition of Notch sig- nalling is sufficient to reduce protein levels of AKT, STAT3 and NF-\u3baB proteins involved in AML chemoresistance, thus making the pro-survival core network controlled by Notch a potential target for specific Notch targeted therapies

Inhibition of Notch signaling enhances chemosensitivity in B cell precursor acute lymphoblastic leukemia

Notch3 and Notch4 support survival of primary B cell acute lymphoblastic leukemia (B-ALL) cells, suggesting a role for Notch signaling in drug response. Here we used in vitro, in silico, and in vivo mouse xenograft model-based approaches to define the role of the Notch pathway in B-ALL chemosensitivity. We observed significant Notch receptor and ligand expression in B-ALL primary cells and cell lines. Primary leukemia cells from high-risk patients overexpressed Notch3, Notch4, and Jagged2 while displaying a reduction in expression levels of Notch1-4 following chemotherapy. We then analyzed in vitro, cell survival of B-ALL cells treated with conventional chemotherapeutic agents alone or in combination with Notch signaling inhibitors. Gamma-secretase inhibitors (GSI) and anti-Notch4 were all capable of potentiating drug-induced cell death in B-ALL cells by upregulating intracellular levels of reactive oxygen species, which in turn modulated mTOR, NF-\u3baB, and ERK expression. In NOG-mouse-based xenograft models of B-ALL, co-administration of the Notch inhibitor GSI-XII with the chemotherapeutic agent Ara-C lowered bone marrow leukemic burden compared to DMSO or Ara-C alone, thus prolonging mouse survival. Overall, our results support the potential effectiveness of Notch inhibitors in B-ALL patients