Role of mesenchymal stromal cell-derived extracellular vesicles in tumour microenvironment

Abstract Stromal cells, deriving from mesenchymal stromal cells (MSCs), are crucial component of tumour microenvironment and represent key regulators of tumour processes. MSCs can be recruited to the tumour environment and interact with many cellular elements, thus influencing tumour biology. Cell-to-cell communication is in part mediated by the release of extracellular vesicle (EVs). EVs can induce significant molecular changes in recipient cells, delivering bioactive molecules. In this review, we describe the MSC-derived EVs content and discuss their role in different processes related to cancer biology. Furthermore, we summarize chemical or biological EVs modifications aiming to develop more efficient antitumor therapies

ATP-mediated cytotoxicity in microglial cells.

Microglial cells are known to express purinergic receptors for extracellular ATP of both the P2Y and P2X subtypes. Functional studies have shown that both primary mouse microglial cells and the N9 and N13 microglial cell lines express the pore-forming P2Z/P2X7 receptor. Here we identify the presence of this receptor in N9 and N13 cells with a specific polyclonal Ab and show that microglial cells expressing the P2Z/P2X7 receptor are exquisitively sensitive to ATP-mediated cytotoxicity while clones selected for the lack of this receptor are resistant. Transfection of HEK293 cells with P2X7 (but not P2X2) receptor cDNA confers susceptibility to ATP-mediated cytotoxicity. Morphological and biochemical analysis suggests that ATP-dependent cell death in microglial cells occurs by apoptosis. Finally, microglial cells release ATP via a non-lytic mechanism when activated by bacterial endotoxin, thus suggesting the operation of a purinergic autocrine/paracrine loop

Role of GSK-3 signalling in non-promyelocitic acute myeloid leukemia (AML) cell response to chemotherapy

Background and aims: 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/b-catenin, Hedgehog, and AKT. Association of GSK-3 inhibitors with All- trans-retinoic acid (ATRA) significantly improved ATRA-medi- ated differentiation and cell death of acute promyelocytic leukemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemoterapeutic agents. Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocytic AML blast cells (30 samples). AML cellscul- tured 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 stain- ing, respectively. Results: Flow cytometry and Western blot analysis in AML sam- ples revealed highexpression levels of all GSK-3 forms, including total GSK-3\u3b1, (Ser21) GSK-3\u3b1, total GSK-3b, and (Ser 21) GSK- 3b; theseforms were all down-modulated when AML cells were cul- tured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role intransducting microenvironmental 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; inter- estingly,hBM-MSCsor 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 Idarubicinin vitro. Notably, LiCl and AR- A014418 were capable of abrogatinghBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin. Conclusion: Our data clearly demonstrate that GSK-3 inhibition may improve the efficacy of some chemoterapeutic agents used in the treatment of non-promyelocytic AML

Targeting the Endothelin-1 Receptors Curtails Tumor Growth and Angiogenesis in Multiple Myeloma

The endothelin-1 (ET-1) receptors were recently found to mediate pro-survival functions in multiple myeloma (MM) cells in response to autocrine ET-1. This study investigated the effectiveness of macitentan, a dual ET-1 receptor antagonist, in MM treatment, and the mechanisms underlying its activities. Macitentan affected significantly MM cell (RPMI-8226, U266, KMS-12-PE) survival and pro-angiogenic cytokine release by down-modulating ET-1-activated MAPK/ERK and HIF-1α pathways, respectively. HIF-1α silencing abrogated the ET-1 mediated induction of genes encoding for pro-angiogenic cytokines such as VEGF-A, IL-8, Adrenomedullin, and ET-1 itself. Upon exposure to macitentan, MM cells cultured in the presence of the hypoxia-mimetic agent CoCl2, exogenous ET-1, or CoCl2 plus ET-1, down-regulated HIF-1α and the transcription and release of downstream pro-angiogenic cytokines. Consistently, macitentan limited significantly the basal pro-angiogenic activity of RPMI-8226 cells in chorioallantoic membrane assay. In xenograft mouse models, established by injecting NOG mice either via intra-caudal vein with U266 or subcutaneously with RPMI-8226 cells, macitentan reduced effectively the number of MM cells infiltrating bone marrow, and the size and microvascular density of subcutaneous MM tumors. ET-1 receptors targeting by macitentan represents an effective anti-proliferative and anti-angiogenic therapeutic approach in preclinical settings of MM

Rational of targeting WNT/beta-catenin signaling in acute myeloid leukemia (AML)

Background and aim: Although recent evidence suggests that aber- rant Wnt signaling can be involved in the neoplastic myeloid cell growth, the role of the Wnt/\u3b2-catenin pathway during the disease development and its contribution to AML chemoresistance are still unclear. Methods: AML primary blast cells(25 samples) and AML cell lines were cultured or co-cultured with human bone marrow mes- enchymal stromal cells (hBM-MSCs),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 signaling components in AML samples. Western Blot and flow cytometry showed the presence of total \u3b2-catenin only in 65% of primary sam- ples analyzed. \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 addi- tion of Wnt 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 syn- ergycally 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 chemoterapeutic agents in combination with Wnt inhibitors reduce proliferation and chemoresistance of AML cellsin culture or co-culture with bone marrow stroma, thus highlighting the role of microenvironmental Wnt/\u3b2-catenin pathway in supporting AML cell survival. Conse- quently, Wnt/\u3b2-catenin signalling may represent a therapeutic targe

CHARACTERIZATION OF MESENCHYMAL STROMAL CELL-DERIVED EXTRACELLULAR VESICLES AND CORRELATION WITH THEIR IMMUNOSUPPRESSIVE PROPERTIES TOWARDS B CELLS

Mesenchymal stromal cells (MSCs) are adult, multipotent stem cells of mesodermal origin. In addition to their stem cell properties, MSCs possess broad immunosuppressive functions influencing both adaptive and innate immune effector cells (IECs). This anti-inflammatory potential is triggered by the release of inflammatory cytokines produced by immune cells in the microenvironment. Indeed, high levels of inflammatory cytokines induce MSCs to become immunosuppressive (primed-MSCs). The immunosuppressive potential of primed-MSCs results from a dynamic interaction between MSCs and IECs mediated by the release of bioactive factors, including extracellular vesicles (EVs). EVs are secreted by many cell types and influence various biological processes, both directly activating cell surface receptors through bioactive ligands and delivering transcription factors, oncogenes, mRNA, and non-coding regulatory RNAs, such as microRNAs (miRNA), into target cells

Molecular characterization of msc-derived extracellular vesicles and correlation with their immunomodulatory potential

Mesenchymal stromal cells (MSCs) are adult, multipotent stem cells of mesodermal origin. In addition to their stem cell properties, MSCs possess broad immunosuppressive functions influencing both adaptive and innate immune effector cells (IECs). This anti-inflammatory potential is triggered by the release of inflammatory cytokines produced by immune cells in the microenvironment. Indeed, high levels of inflammatory cytokines induce MSCs to become immunosuppressive (primed-MSCs). The immunosuppressive potential of primed-MSCs results from a dynamic interaction between MSCs and IECs mediated by the release of bioactive factors, including extracellular vesicles (EVs). EVs are secreted by many cell types and influence various biological processes, both directly activating cell surface receptors through bioactive ligands and delivering transcription factors, oncogenes, mRNA, and non-coding regulatory RNAs, such as microRNAs (miRNA), into target cells. In this study, we have investigated the undisclosed molecular mechanisms regulating the immunosuppressive capabilities of EVs derived from primed-MSCs (primed-EVs), starting from a molecular characterization of their contents by high-throughput approaches. We analyzed miRNAs, mRNAs and proteins differentially expressed in primed-EVs compared to ctrl-EVs. We found miR-155-5p and miR-6723-5p significantly up-regulated in primed-EVs. Enrichment analysis on predicted target genes revealed an involvement of miR-155-5p and miR-6723-5p in different pathways regulating immunomodulatory processes. Moreover, we found IDO mRNA significantly up-regulated in primed-EVs. Furthermore, primed-EVs showed 73 proteins significantly modulated compared to ctrl-EVs. Enrichment analysis on modulated proteins revealed different pathways up and down regulated involved in immunomodulatory properties exerted by primed-EVs. This molecular characterization of MSC-EVs, and the consequent functional validation of candidate molecules, will pave the way for novel therapeutic perspectives for the treatment of inflammatory and autoimmune diseases

The miR-200c/141-ZEB2-TGFβ axis is aberrant in human T-cell prolymphocytic leukemia

T-cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small- to medium-sized prolymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, and no clear T-cell receptor a or β CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of microRNA (miRNA) is aberrant and often heterogeneous in T-PLL. We identified 35 miRNA that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR- 200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFβR3 and aberrant TGFβ1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFβ pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNA in T-PLL and pave the way for new therapeutic targets in this disease