Editorial: Influence of potential diagnostic biomarkers in lung cancer

International audienc

Functional dosing of mesenchymal stromal cell-derived extracellular vesicles for the prevention of acute graft-versus-host-disease

Graft-vs-host-disease (GvHD) is currently the main complication of allogeneic hematopoietic stem cell transplantation. Mortality and morbidity rates are particularly high, especially in steroid-refractory acute GvHD (aGvHD). Immune regulatory human bone marrow mesenchymal stromal cells (hMB-MSCs) represent a therapeutic approach to address this issue. Unfortunately, their effect is hardly predictable in vivo due to several variables, that is, MSC tissue origin, concentration, dose number, administration route and timing, and inflammatory status of the recipient. Interestingly, human bone marrow MSC-derived extracellular vesicles (hBM-MSC-EVs) display many of the hBM-MSC immunoregulatory properties due to their content in paracrine factors that greatly varies according to the collection method. In this study, we focused on the immunological characterization of hBM-MSC-EVs on their capability of inducing regulatory T-cells (T-regs) both in vitro and in a xenograft mouse model of aGvHD. We correlated these data with the aGvHD incidence and degree following hBM-MSC-EV intravenous administration. Thus, we first quantified the EV immunomodulation in vitro in terms of EV immunomodulatory functional unit (EV-IFU), that is, the lowest concentration of EVs leading in vitro to at least threefold increase of the T-regs compared with controls. Second, we established the EV therapeutic dose in vivo (EV-TD) corresponding to 10-fold the in vitro EV-IFU. According to this approach, we observed a significant improvement of both mouse survival and control of aGvHD onset and progression. This study confirms that EVs may represent an alternative to whole MSCs for aGvHD prevention, once the effective dose is reproducibly identified according to EV-IFU and EV-TD definition

Regulative Loop between \u3b2-catenin and Protein Tyrosine Receptor Type \u3b3 in Chronic Myeloid Leukemia

Protein tyrosine phosphatase receptor type \u3b3 (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. \u3b2-catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between \u3b2-catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene \u3b2-Catenin. PTPRG was found to be capable of dephosphorylating \u3b2-catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of \u3b2-catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and \u3b2-catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and \u3b2-catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells

Developmental pathways associated with cancer metastasis: Notch, Wnt, and Hedgehog

Master developmental pathways, such as Notch, Wnt, and Hedgehog, are signaling systems that control proliferation, cell death, motility, migration, and stemness. These systems are not only commonly activated in many solid tumors, where they drive or contribute to cancer initiation, but also in primary and metastatic tumor development. The reactivation of developmental pathways in cancer stroma favors the development of cancer stem cells and allows their maintenance, indicating these signaling pathways as particularly attractive targets for efficient anticancer therapies, especially in advanced primary tumors and metastatic cancers. Metastasis is the worst feature of cancer development. This feature results from a cascade of events emerging from the hijacking of epithelial-mesenchymal transition, angiogenesis, migration, and invasion by transforming cells and is associated with poor survival, drug resistance, and tumor relapse. In the present review, we summarize and discuss experimental data suggesting pivotal roles for developmental pathways in cancer development and metastasis, considering the therapeutic potential. Emerging targeted antimetastatic therapies based on Notch, Wnt, and Hedgehog pathways are also discussed

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

Extracellular Vesicles Mediate Mesenchymal Stromal Cell-Dependent Regulation of B Cell PI3K-AKT Signaling Pathway and Actin Cytoskeleton

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different in silico approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated in vitro as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches

Notch Signaling Molecules as Prognostic Biomarkers for Acute Myeloid Leukemia

The role of Notch signaling in acute myeloid leukemia (AML) is still under investigation. We have previously shown that high levels of Notch receptors and ligands could interfere with drug response. In this study, the protein expression of 79 AML blast samples collected from newly diagnosed patients was examined through flow cytometry. Gamma-secretase inhibitors were used in AML mouse xenograft models to evaluate the contribution of Notch pharmacological inhibition to mouse survival. We used univariate analysis for testing the correlation and/or association between protein expression and well-known prognostics markers. All the four receptors (Notch1–4) and some ligands (Jagged2, DLL-3) were highly expressed in less mature subtypes (M0–M1). Notch3, Notch4, and Jagged2 were overexpressed in an adverse cytogenetic risk group compared to good cytogenetic risk patients. Chi-square analysis revealed a positive association between the complete remission rate after induction therapy and weak expression of Notch2 and Notch3. We also found an association between low levels of Notch4 and Jagged2 and three-year remission following allogeneic stem cell transplantation (HSCT). Accordingly, Kaplan–Meier analysis showed improved OS for patients lacking significant expression of Notch4, Jagged2, and DLL3. In vivo experiments in an AML mouse model highlighted both improved survival and a significant reduction of leukemia cell burden in the bone marrow of mice treated with the combination of Notch pan-inhibitors (GSIs) plus chemotherapy (Ara-C). Our results suggest that Notch can be useful as a prognostic marker and therapeutic target in AML

ROLE OF NOTCH SIGNALING IN STROMA-DEPENDENT ACUTE MYELOID LEUKEMIA CELL CHEMORESISTANCE.

Studi preclinici e clinici hanno dimostrato il ruolo di Notch nello sviluppo di vari tipi di cancro e nella loro risposta alla chemioterapia. Di recente, il nostro gruppo di ricerca ha mostrato che l\u2019inibizione di Notch sopprime la chemioresistenza mediata dallo stroma nei confronti delle neoplasie linfoidi.Tuttavia, il ruolo di Notch nelle leucemie acute mieloidi (AML), e il suo contributo nell\u2019interazione tra cellule leucemiche e cellule stromali midollari rimane ancora controverso.In questo studio, abbiamo valutato il ruolo mediato dall\u2019attivazione di Notch nella proliferazione, sopravvivenza e chemioresistenza di cellule di AML in co-coltura con cellule stromali mesenchinali isolate da midollo osseo di donatori sani (hBM-MSC) e pazienti con AML (hBM-MSC*). L\u2019espressione del recettore Notch e dei suoi ligandi era simile tra hBM-MSC e hBM-MSC*, ad eccezione di Notch1 e Jagged1 che invece erano maggiormente espressi nelle hBM-MSC*. Inoltre, almeno il 50% dei pazienti di AML mostravano una basale attivazione della via del Notch, e la sua attivazione ed espressione era modulata dopo co-coltura con hBM-MSC*. In particolare, hBM-MSC* supportavano maggiormente la proliferazione delle cellule di AML e inducevano chemioresistenza rispetto alle hBM-MSC. In presenza di chemioterapici, sia il trattamento con inibitori che quello con anticorpi bloccanti del Notch, riduceva significativamente l\u2019effetto supportivo mediato dalle hBM-MSC e hBM-MSC* nei confronti dei blasti leucemici di AML. L\u2019inibizione specifica di Notch1, 2, 3 o di Jagged1, 2 riduceva parzialmente la chemioresistenza delle cellule di AML in co-coltura con le hBM-MSC. Al contrario, si aveva un recupero totale della chemiosesitivit\ue0 quando alle co-colture era aggiunto l\u2019inibitore di Notch4.In un secondo momento, per studiare pi\uf9 a fondo i meccanismi alla base dell\u2019attivazione del Notch, mediante l\u2019uso di plasmidi shRNA, abbiamo effettuato un silenziamento di RBP-jK, il principale fattore di trascrizione della via del Notch. Il silenziamento eseguito su due linee di AML, ovvero HL-60 e THP1, induceva una maggiore sensibilit\ue0 al trattamento con i farmaci rispetto alle cellule controllo infettate con un shRNA non specifico. Inoltre, il knock-down di RBP-jk correlava con i bassi livelli di proteine coinvolte nella sopravvivenza cellulare, come AKT, STAT3 and NF-\u3baB. Analogamente, il blocco farmacologico del Notch attraverso gli inibitori della gamma-secretasi, sommato all\u2019effetto sinergico mediato dalla chemioterapia, attivava l\u2019asse Bax/caspasi-3 riducendo l\u2019espressione di AKT, STAT3 and NF-\u3baB in cellule di AML co-coltivate con hBM-MSC.Nel complesso, i nostri risultati suggeriscono che l\u2019inibizione del Notch potrebbe rappresentare un potenziale target terapeutico non soltanto per le neoplasie linfoidi ma anche per le AML, bypassando la chemioresistenza indotta dallo stroma.Both preclinical and clinical investigations suggest that Notch signaling 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.In this study, 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*). hBMMSCs and hBM-MSCs* showed similar expression of Notch receptors and ligands, except for Notch1 and Jagged1 that were expressed at higher level by hBM-MSCs*. In addition, at least 50% of AML samples showed basal Notch activation, and the expression and activation pattern was modulated after co-culture with hBM-MSCs*. Notably, hBM-MSCs* were more efficient in supporting AML proliferation and inducing AML chemoresistance, as comparedto hBM-MSCs. Treatment with either Notch inhibitors or combinations of Notch receptorblocking antibodies significantly lowered the supportive role of hBM-MSCs and hBMMSCs* towards AML blast cells in presence of chemotherapeutic agents. The specific blockade of Notch1, 2, 3 or Jagged1, 2 reduced partially the chemoresistance, while Notch4 blockade restored completely primary AML cell chemosensitivity when in co-culture withhBM-MSCs. With the aim to unravel the mechanisms underlying Notch activation, we silenced the main Notch transcription factor RBP-jK through shRNA plasmids in two AML cells lines, namely HL-60 and THP1. Consistently, silenced cells were more sensible to drug treatment than cells infected with non-specific shRNA. Interestingly, RBP-jk knock-down was associated with low levels of pro-survival proteins, such as AKT, STAT3 and NF-\u3baB.Accordingly, we found that Notch pharmacological blockade through gamma-secretase inhibitors in synergy with chemotherapy activates Bax/caspase-3 axis by lowering the protein level of AKT, STAT3 and NF-\u3baB in AML cells in co-culture with hBM-MSCs.Our results suggest that Notch signaling inhibition, by overcoming the stromal-mediated promotion of chemoresistance, may represent a potential therapeutic target not only for lymphoid neoplasms but also for AML

Unlocking the Potential of Biomarkers for Immune Checkpoint Inhibitors in Cancer Therapy

Immune checkpoint inhibitors (ICIs) are pharmaceutical agents capable of disrupting immune checkpoint signaling, leading to T-cell activation and a robust anti-tumor response [...