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// Paul Takam Kamga 1 , Giada Dal Collo 1 , Giulio Bassi 1 , Martina Midolo 1 , Massimo Delledonne 2, 3 , Marco Chilosi 4 , Massimiliano Bonifacio 1 and Mauro Krampera 1 1 Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Verona, Italy 2 Department of Biotechnology, University of Verona, Verona, Italy 3 Personal Genomics S.R.L., Verona, Italy 4 Section of Pathology, Department of Diagnostics and Public Health, University of Verona, Verona, Italy Correspondence to: Mauro Krampera, email: mauro.krampera@univr.it Keywords: Notch signaling; B-acute lymphoblastic leukemia; B-ALL; Alagille syndrome; ALGS Received: January 02, 2018 Accepted: March 11, 2018 Published: April 06, 2018 ABSTRACT 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 γ-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.

// Paul Takam Kamga 1 , Giulio Bassi 1 , Adriana Cassaro 1 , Martina Midolo 1 , Mariano Di Trapani 1 , Alessandro Gatti 1 , Roberta Carusone 1 , Federica Resci 1 , Omar Perbellini 1 , Michele Gottardi 2 , Massimiliano Bonifacio 1 , Armel Herve Nwabo Kamdje 3 , Achille Ambrosetti 1 and Mauro Krampera 1 1 Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Verona, Italy 2 Division of Hematology, Ca’ Foncello Hospital, Treviso, Italy 3 Department of Biomedical Sciences, University of Ngaoundere-Cameroon, Cameroon Correspondence to: Mauro Krampera, email: // Keywords : AML, MSC, Notch, chemoresistance Received : February 10, 2016 Accepted : February 25, 2016 Published : March 07, 2016 Abstract 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.

Background: Epithelial-to-mesenchymal transition (EMT) has a central role in cancer progression and metastatic dissemination and may be induced by local inflammation. We asked whether the inflammation-induced acquisition of mesenchymal phenotype by neoplastic epithelial cells is associated with the onset of mesenchymal stromal cell-like immune-regulatory properties that may enhance tumour immune escape. Methods: Cell lines of lung adenocarcinoma (A549), breast cancer (MCF7) and hepatocellular carcinoma (HepG2) were co-cultured with T, B and NK cells before and after EMT induction by either the supernatant of mixed-lymphocyte reactions or inflammatory cytokines. Results: EMT occurrence following inflammatory priming elicited multiple immune-regulatory effects in cancer cells resulting in NK and T-cell apoptosis, inhibition of lymphocyte proliferation and stimulation of regulatory T and B cells. Indoleamine 2,3-dioxygenase, but not Fas ligand pathway, was involved at least in part in these effects, as shown by the use of specific inhibitors. Conclusions: EMT induced by inflammatory stimuli confers to cancer cells some mesenchymal stromal cell-like immune-modulatory properties, which could be a cue for cancer progression and metastatic dissemination by favouring immune escape.

Chorionic stem cells represent a promising opportunity for regenerative medicine. A deeper understanding of the stimuli that regulate their physiology, could lead to innovative clinical approaches. We revealed the presence of multiple sphingosine-1-phosphate (S1P) receptor isoforms in chorion-derived mesenchymal stem cells (CMSCs). Their activation simultaneously propagated from the plasma membrane through Gi and other heterotrimeric G proteins and further diverged toward extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 and protein kinase D 1. At a functional level, S1P signaling inhibited CMSC migration, while promoting proliferation. Instead, a reduction of cell density was obtained when S1P was combined to treatments that increased cAMP intracellular concentration. Such surprising reduction of cell viability was relatively specific as it was not observed with stromal stem cells from bone marrow. Neither it was observed by activating analogous G proteins with bradykinin nor by inducing cell death via a cAMP-independent pathway. S1P could thus reveal novel keys to improve CMSC differentiation programs acting on cAMP concentration. Furthermore, S1P receptor agonists/antagonists could become instrumental in favoring CMSC engraftment by controlling cell motility.

Master developmental pathways like Notch, Wnt, and Hedgehog are signaling systems that control proliferation, cell death, motility, migration, and stemness. They are commonly activated in many solid tumors, where they drive or contribute to cancer initiation, but also primary and metastatic tumor development. The reactivation of developmental pathways in cancer stroma favors the development of cancer stem cells and allows their maintenance, pointing out these signaling pathways as particularly attractive targets for more efficient anticancer therapies, especially in advanced primary tumors and metastatic cancers. Metastasis, i.e., the colonization of distant organs by tumor cells from a primary site, is the worst feature of cancer development. It results from a cascade of events emerging from hijacking of epithelial–mesenchymal transition, angiogenesis, migration and invasion by transforming cells, and is associated with poor survival, drug resistance, and tumor relapse. In this chapter, 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.

Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells which may reside as pericytes in various tissues and organs, where they may differentiate into specialized cells to replace dying cells and damaged tissues. These cells are commonly found at injury sites, but also in tumors, which are known to behave like “wounds that do not heal.” Herein, we discuss the mechanisms used by MSCs to migrate, home, and repair injured tissues, and we review a number of reports showing that the tumor microenvironment triggers plasticity mechanisms in MSCs to induce malignant neoplastic tissue formation, maintenance, and chemoresistance, as well as tumor growth. The antitumor properties and the therapeutic potential of MSCs are also discussed.

The anti-inflammatory actions of IL-4 are well established through earlier findings. However, the exact mechanism it uses to downregulate the pro-inflammatory cytokine production through monocytes and macrophages is poorly understood. In this study, we examined the effect of IL-4 in the induction of 11β-HSD1 in the two main classes of monocytes, CD14++ CD16- (CD14) and CD14+ CD16+ (CD16). Peripheral Blood Mononuclear Cells (PBMCs) were isolated from 17 healthy donors and were sorted into CD14 and CD16 subpopulations using cell sorting. Effect of IL-4 on 11β-HSD1-enzyme activity was measured in sorted and unsorted monocytes using Homogeneous Time-Resolved Fluorescence (HTRF) and M1/M2 polarization analysis was performed by flow cytometry. Our results indicate that CD14 cells are the major source of 11β-HSD1 enzyme after IL-4 stimulation and that M2 phenotype is not a pre-requisite for its synthesis.

Granulocyte colony-stimulating factor (G-CSF) is a controversial chemical in cardiac cell therapy. Myocardial homing of mobilized bone marrow-derived cells is thought to play a critical role in observed G-CSF-induced cardiac repair; meanwhile, the activation of proliferative potential of cardiac stem cells (CSCs) residing in the heart is a significant challenge. The present study aims to investigate whether G-CSF receptor is expressed in adult resident Sca-1(+) CSCs and determine the effect of G-CSF treatment on the proliferation of CSCs. For cardiac cells isolation, 12-week-old male C57BL/6 mice were anesthetized in a chamber containing 2.5% isoflurane in oxygen, euthanized by CO2 inhalation and then sacrificed by cervical dislocation. Magnetic-activated cell sorting was employed to acquire highly purified Sca-1(+) CSCs. We found that G-CSF receptor was expressed in adult resident Sca-1(+) CSCs by immunofluorescence staining and Western blotting. Exposure of Sca-1(+) cells to G-CSF in the culture medium for 72 h induced time-dependent but self-limiting cell cycle acceleration with a restricted effect on the CSC proliferation. As a result, it has provided a new insight to focus on the association between cardiac G-CSF therapy and adult resident stem cell activation. It may suggest gaining a deeper insight into the mechanisms of the interaction between CSCs and G-CSF to develop a synergistic strategy based on resident stem cell and G-CSF therapy for heart disease.

The anti-inflammatory actions of IL-4 are well established through earlier findings. However, the exact mechanism it uses to downregulate the pro-inflammatory cytokine production through monocytes and macrophages is poorly understood. In this study, we examined the effect of IL-4 in the induction of 11β-HSD1 in the two main classes of monocytes, CD14

Adipose-derived stromal cells (ASCs) are now emerging as a good alternative to bone marrow derived mesenchymal stromal cells (BM-MSC) for cellular therapy. Similarly to BM-MSC, ASCs can be easily isolated as adherent fibroblastoid cell population after processing lipoaspirate samples. Lipoaspiration provides a great number of cells, without extensive manipulation. ASCs express classical mesenchymal markers and only at early passages express CD34. ASCs can differentiate in cells of mesodermal lineages, such as adipocytes, osteocytes and condrocytes. ASCs share with BM-MSC the same ability to inhibit the proliferation of allogeneic, activated immune cells, thus affecting in vivo in animal models the onset and course of rheumatoid arthritis (RA), experimental autoimmune encephalomyelitis (EAE), Crohn's disease (CD), ulcerous colitis (UC) and graft-versus-host disease (GvHD). On the other hand, the main molecular pathway involved in this effect is still unclear. On the basis of this functional property, ASCs are used in different clinical trials to treat RA, CD, UC and GvHD. However, the most promising field of clinical application is represented by bone defect repair. Despite the ability to regenerate injured tissues and to block the progression of inflammatory disorders, some authors reported that ASCs can also induce, in in vivo animal models, the growth and vascularization of solid and hematological tumors. Conversely, ASCs have been shown to hamper tumor cell proliferation, reduce cell viability and induce necrosis. Thus, more accurate studies, collaborative protocols, high standardization of methods, and safety controls are required to exclude transformation of transplanted ASCs.

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.

The aim of this study was to assess the immune modulatory properties of human mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs), and cord blood (CB-MSCs) in the presence of a hydroxyapatite and tricalcium-phosphate (HA/TCP) biomaterial as a scaffold for MSC delivery. In resting conditions, a short-term culture with HA/TCP did not modulate the anti-apoptotic and suppressive features of the various MSC types toward T, B, and NK cells; in addition, when primed with inflammatory cytokines, MSCs similarly increased their suppressive capacities in the presence or absence of HA/TCP. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype with upregulation of OSTERIX and OSTEOCALCIN, similar to what was obtained with dexamethasone and, to a higher extent, with bone morphogenetic protein 4 (BMP-4) treatment. MSC-derived osteoblasts did not trigger immune cell activation, but were less efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells. Interestingly, their suppressive machinery included not only the activation of indoleamine-2,3 dioxygenase (IDO), which plays a central role in T-cell inhibition, but also cyclooxygenase-2 (COX-2) that was not significantly involved in the immune modulatory effect of human undifferentiated MSCs. Since COX-2 is significantly involved in bone healing, its induction by HA/TCP could also contribute to the therapeutic activity of MSCs for bone tissue engineering.

An industrially feasible process for the fast mass-production of molded polymeric micro-patterned substrates is here presented. Microstructured polystyrene (PS) surfaces were obtained through micro injection molding (μIM) technique on directly patterned stamps realized with a new zirconia-based hybrid spin-on system able to withstand 300 cycles at 90 °C. The use of directly patterned stamps entails a great advantage on the overall manufacturing process as it allows a fast, flexible, and simple one-step process with respect to the use of milling, laser machining, electroforming techniques, or conventional lithographic processes for stamp fabrication. Among the different obtainable geometries, we focused our attention on PS replicas reporting 2, 3, and 4 μm diameter pillars with 8, 9, 10 μm center-to-center distance, respectively. This enabled us to study the effect of the substrate topography on human mesenchymal stem cells behavior without any osteogenic growth factors. Our data show that microtopography affected cell behavior. In particular, calcium deposition and osteocalcin expression enhanced as diameter and interpillar distance size increases, and the 4-10 surface was the most effective to induce osteogenic differentiation.

Introduction: Immune system strongly affects cancer cell fate; indeed, immune-checkpoint blockade is emerging as an effective therapeutic strategy in a broad range of tumor types. The effectiveness of these therapies depends on different aspects of tumor biology such as the expression of immune checkpoint proteins, the presence of infiltrating active immune cells and many others. To date, a robust biomarker to predict therapeutic response to these drugs has not been found yet. Among all the strategies used to identify effective biomarkers, a single-cell level molecular profiling of tumor, stromal and infiltrating cells, has not been explored due to technical challenges. Here we present a method for the digital isolation of single PD-L1+ cells from FFPE tissues, along with their genetic characterization for the identification of novel biomarkers. Methods: FFPE tissue sections from two NSCLC patients, were independently tested for PD-L1 expression using IHC VENTANA PD-L1 (SP142) Assay and scored in terms of tumor content (30%, 85%), percentage of PD-L1 positive tumor cells (70%, 95%), PD-L1 intensity, and H-score (130, 175). From the same tissue blocks, additional FFPE sections were dissociated down to cell suspension, and immunofluorescently labelled using Keratin-AlexaFluor®488, Vimentin-AlexaFluor®647 and PD-L1 (clone SP263)-AlexaFluor®546. Four cell populations (Keratin+PD-L1-, Keratin+PD-L1+, Vimentin+PD-L1- and Vimentin+PD-L1+) were identified at the DEPArray™ platform and, for each, we isolated pools of precise number (range 43-121) of cells, along with pure single cells. Recovered cells were whole genome amplified (Ampli1™ WGA), and genome-wide copy-number aberrations (CNA) profiles were obtained by Ampli1™ LowPass kit and IonTorrent sequencing. Results: The expression of PD-L1 on cell membrane was attributed -by means of co-expression of either keratin or vimentin- to tumor or stromal cells, respectively. For both patients, in the tumor populations numerous gains and losses were clearly identified along the genome; Keratin+PD-L1+ and Keratin+PD-L1- pooled cells displayed comparable profiles. Notably, in both patients copy gains were identified on chromosome 9 region including the PD-L1 gene. A variable range of alleles (3 to 6) on the same PD-L1 locus was detected on single tumor cells, along with a high inter-cell heterogeneity in terms of gain extent and genomic regions involved, suggestive of the presence of different tumor clones, hidden in pooled cells data. All the stromal (Vimentin+PD-L1+/-) cells showed flat profiles. Conclusions: This study demonstrates an innovative approach for isolation and genetic profiling of single pure PD-L1+ cells from FFPE tissue. DEPArray™ sorting combined with low-pass whole genome sequencing enables high quality genome-wide profiling of pure single cells and pools isolated from phenotypically distinct populations in FFPE tumor tissue. Citation Format: Giulio Bassi, Chiara Bolognesi, Chiara Mangano, Claudio Forcato, Alberto Ferrarini, Valentina Del Monaco, Paola Tononi, Genny Buson, Gianni Medoro, Manaresi Nicolò, Francesca Fontana. Copy-number profiling of single, digitally-sorted PD-L1 positive cells in NSCLC FFPE tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5607. doi:10.1158/1538-7445.AM2017-5607

Introduction: Formalin-fixed paraffin-embedded (FFPE) specimens represent an invaluable source of material for precision oncology. However, FFPE samples pose significant challenges for molecular assays, such as targeted Next-Generation Sequencing (NGS), because of the highly variable DNA quality and often limited sample size. Here we present a complete workflow from sample quality control to targeted library preparation to reliably detect sequence and copy number alterations (CNA) through targeted NGS from extremely low input FFPE samples. Materials & methods: Three FFPE specimens from patients with breast (BrCa) or pancreatic cancer, with DNA quality varying over a broad range and with low tumor cellularity (down to 10%) were selected for targeted NGS profiling. The DNA quality was determined using the DEPArray™ FFPE QC Kit, a qPCR-based assay yielding a QC score defined as the ratio between the quantification of a 132 bp amplicon, corresponding approximately to the average length (116bp) of DEPArray OncoSeek amplicons, and a shorter amplicon of 54 bp. The QC scores of the 3 FFPE specimens ranged between 0.23 to 0.74. We then used QC score, multiplied by the ploidy (assessed on DEPArray during cell sorting) and by the number of cells recovered, to estimate the effectively amplifiable template (EAT). Thirty two pure cell populations (21 stromal and 11 tumor), with different EATs (80-300), corresponding to a wide range of number of cells per pool (31-214), were collected with the DEPArray digital sorter. Libraries were prepared from each cell pool, using the single-tube, Illumina-compatible DEPArray OncoSeek panel comprising 63 oncology relevant genes (average 740,000 sequenced amplicons per sample). Results: Variant calling showed sensitivity comprised between 93% and 99% and specificity > 99% for EATs ≥ 80 equivalent to as low as ≈250 pg of DNA. Analysis of CNAs in stromal cells was highly specific (zero false positive at 1.5 fold-change threshold). Moreover, analysis allowed to highly reproducibly identify CNAs in CCND1 (3x), ERBB2 (8x), MYC (3x) and PIK3KA (2x) in both replicates of one BrCa sample. Similarly, ERBB2 amplification (7x) was found in the other BrCa sample (10% tumor cellularity) across two replicates at different EATs (80, 120) corresponding to as low as 59 and 75 cells. As expected by the low tumor content, in the corresponding unsorted sample ERBB2 was below the 2-fold gain threshold with respect to the stromal control, which would not qualify a sample as Her2-amplified. Highlights: Starting from pure intact cells with well characterized DNA quality and ploidy, our workflow allows reliable molecular profiling of sub-nanogram DNA samples by determining with precision the extremely-low minimum amount of cells necessary to obtain highly reproducible sequence variant calling and CNAs detection by targeted NGS. Citation Format: Paola Tononi, Alberto Ferrarini, Genny Buson, Valentina del Monaco, Giulio Bassi, Chiara Mangano, Claudio Forcato, Chiara Bolognesi, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Accurate molecular profiling of sequence and copy number alterations from sub-nanogram FFPE DNA amounts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5349. doi:10.1158/1538-7445.AM2017-5349

Introduction Intra-tumor heterogeneity can hide genomic and genetic features, which may be key driver of disease progression. Routinely, only one biological specimen per patient is generally analyzed, which may only partially represents the genetics of the tumor. Here we report a multi-approach analysis of Circulating Tumor Cells (CTCs) and formalin-fixed paraffin-embedded (FFPE) tumor tissue-derived cells (TCs) obtained from the same patients, to investigate the underlying genetic heterogeneity. Methods Peripheral blood (PB) and FFPE tumor tissue were collected from two advanced lung adenocarcinoma patients, treated with cisplatin-pemetrexed and carboplatin-pemetrexed respectively as first line therapy. The first patient was previously diagnosed an ALK-traslocation and treated with an ALK-inhibitor. PB was enriched with either an EpCAM-based or EpCAM-independent method: the cell output of the latter was stained with Cytokeratin-PE, CD45-APC and DAPI. Matched FFPE sections were obtained from pleural effusion cell blocks for the first patient or from primary tumor tissue for the second one; after dissociation, cells were stained with Vimentin-APC, Keratin-FITC and DAPI. The DEPArray™ platform was used to detect and collect pure single CTCs or TCs, along with WBCs or stromal cells as controls. Whole genome amplified DNA of single CTCs and TCs was used to profile genome-wide copy-number aberrations (CNAs) using the Ampli1™ LowPass kit; single nucleotide variants were analyzed on CTCs WGA products and on pools of TCs using Ampli1™ CHP custom panel and DEPArray™ OncoSeek panel respectively. Results No clinically significant variants were detected in CTCs and FFPE samples; however the copy-number profiles of single TCs and CTCs revealed an overabundance of gains and losses, confirming the aberrant nature of tumor cells. In the first patient, all single cells showed a pattern of shared alterations, with a common amplification of the genome region comprising MYC gene (also confirmed by depth-of-coverage in targeted panel). A hierarchical unsupervised clustering clearly separated WBCs, from the group of TCs and CTCs, characterized by some emerging clones and low inter-cell heterogeneity. The analysis of the copy-number profiles of cells from the second patient showed an opposite situation; unsupervised clustering of low-pass profiles highlighted an independent group formed by single TCs clearly distinct from the highly heterogeneous cluster formed by CTCs. Conclusions The precision granted by analysis of pure cells derived from multiple specimens from the same patient, together with the combination of low-pass whole-genome sequencing and targeted sequencing, reveals unexpected genetic similarities and diversities, and provides fundamental information to understand intra-tumor heterogeneity. Citation Format: Mario Terracciano, Francesco Gelsomino, Francesco Bacchi, Francesca Fontana, Claudio Forcato, Alberto Ferrarini, Michelangelo Fiorentino, Valentina Del Monaco, Giulio Bassi, Chiara Mangano, Chiara Bolognesi, Paola Tononi, Genny Buson, Gianni Medoro, Nicolò Manaresi, Michele Tognetto, Andrea Ardizzoni. Molecular characterization with single-cell resolution of CTCs and FFPE specimens from the same lung adenocarcinoma patients reveals the extent of intra-tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2914. doi:10.1158/1538-7445.AM2017-2914

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygen-level response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis.

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.

Amniotic Fluid Stem (AFS) cells are broadly multipotent fetal stem cells derived from the positive selection and ex vivo expansion of amniotic fluid CD117/c-kit(pos) cells. Considering the differentiation potential in vitro toward cell lineages belonging to the three germ layers, AFS cells have raised great interest as a new therapeutic tool, but their immune properties still need to be assessed. We analyzed the in vitro immunological properties of AFS cells from different gestational age in coculture with T, B, and natural killer (NK) cells. Nonactivated (resting) first trimester-AFS cells showed lower expression of HLA class-I molecules and NK-activating ligands than second and third trimester-AFS cells, whose features were associated with lower sensitivity to NK cell-mediated lysis. Nevertheless, inflammatory priming with interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) enhanced resistance of all AFS cell types to NK cytotoxicity. AFS cells modulated lymphocyte proliferation in a different manner according to gestational age: first trimester-AFS cells significantly inhibited T and NK cell proliferation, while second and third trimester-AFS cells were less efficient. In addition, only inflammatory-primed second trimester-AFS cells could suppress B cell proliferation, which was not affected by the first and third trimester-AFS cells. Indolamine 2,3 dioxygenase pathway was significantly involved only in T cell suppression mediated by second and third trimester-AFS cells. Overall, this study shows a number of significant quantitative differences among AFS cells of different gestational age that have to be considered in view of their clinical application.

International audience; The aim of this study was to assess the immune modulatory properties of human mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs) and cord blood (CB-MSCs) in the presence of a novel hydroxyapatite and tricalcium-phosphate (HA/TCP) biomaterial as scaffold for MSC delivery. In resting conditions, a short-term culture with HA/TCP did not modulate the anti-apoptotic and suppressive features of the various MSC types towards T, B and NK cells; in addition, when primed with inflammatory cytokines, MSC maintained or not on HA/TCP similarlyincreased their suppressive capacities. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype with up-regulation of OSTERIX and OSTEOCALCIN, similarly to what obtained with dexamethasone and, to a higher extent, BMP-4 treatment. MSC-derived osteoblasts did not trigger immune cell activation, but were less efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells. Interestingly, their suppressive machinery included not only the activation of IDO, which plays a central role in T-cell inhibition, but also COX-2 that was not significantly involved in immune modulatory effect of human undifferentiated MSCs. COX-2 is significantly involved in bone healing, suggesting that its induction by HA/TCP could also contribute to the therapeutic activity of MSC for bone tissue engineering.

International audience; : Allogeneic stem cell (SC)-based therapy is a promising tool for the treatment of a range of human degenerative and inflammatory diseases. Many reports highlighted the immune modulatory properties of some SC types, such as mesenchymal stromal cells (MSCs), but a comparative study with SCs of different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods employed for MSC characterization to compare the immunological properties of bone marrow-MSCs, olfactory ectomesenchymal SCs, leptomeningeal SCs, and three different c-Kit-positive SC types, that is, amniotic fluid SCs, cardiac SCs, and lung SCs. We found that all the analyzed human SCs share a common pattern of immunological features, in terms of expression of activation markers ICAM-1, VCAM-1, HLA-ABC, and HLA-DR, modulatory activity toward purified T, B, and NK cells, lower immunogenicity of inflammatory-primed SCs as compared to resting SCs, and indoleamine-2,3-dioxygenase-activation as molecular inhibitory pathways, with some SC type-related peculiarities. Moreover, the SC types analyzed exert an anti-apoptotic effect toward not-activated immune effector cells (IECs). In addition, we found that the inhibitory behavior is not a constitutive property of SCs, but is acquired as a consequence of IEC activation, as previously described for MSCs. Thus, immune regulation is a general property of SCs and the characterization of this phenomenon may be useful for a proper therapeutic use of SCs.

International audience; Clinical-grade mesenchymal stromal cells (MSCs) are usually expanded from bone marrow (BMMSCs) or adipose tissue (ADSCs) using processes mainly differing in the use of fetal calf serum (FCS) or human platelet lysate (PL). We aimed to compare immune modulatory properties of clinical-grade MSCs using a combination of fully standardized in vitro assays. BMMSCs expanded with FCS (BMMSC-FCS) or PL (BMMSC-PL), and ADSC-PL were analyzed in quantitative phenotypic and functional experiments, including their capacity to inhibit the proliferation of T, B, and NK cells. The molecular mechanisms supporting T-cell inhibition were investigated. These parameters were also evaluated after pre-stimulation of MSCs with inflammatory cytokines. BMMSC-FCS, BMMSC-PL, and ADSC-PL displayed significant differences in expression of immunosuppressive and adhesion molecules. Standardized functional assays revealed that resting MSCs inhibited proliferation of T and NK cells, but not B cells. ADSC-PL were the most potent in inhibiting T-cell growth, a property ascribed to interferon-γ-dependent indoleamine 2,3-dioxygenase activity. MSCs did not stimulate allogeneic T cell proliferation but were efficiently lysed by activated NK cells. The systematic use of quantitative and reproducible validation techniques highlights differences in immunological properties of MSCs produced using various clinical-grade processes. ADSC-PL emerge as a promising candidate for future clinical trials.

Mesenchymal stromal cells (MSCs) reside in many organs including lung, as shown by their isolation from fetal lung tissues, bronchial stromal compartment, bronchial-alveolar lavage and transplanted lung tissues. It is still controversial whether lung MSCs can undergo mesenchymal-to-epithelial-transition (MET) and possess immune regulatory properties. To this aim, we isolated, expanded and characterized MSCs from normal adult human lung (lung-hMSCs) and compared with human bone marrow-derived MSCs (BM-hMSCs). Our results show that lung-MSCs reside at the perivascular level and do not significantly differ from BM-hMSCs in terms of immunophenotype, stemness gene profile, mesodermal differentiation potential and modulation of T, B and NK cells. However, lung-hMSCs express higher basal level of the stemness-related marker nestin and show, following in vitro treatment with retinoic acid, higher epithelial cell polarization, which is anyway partial when compared to a control epithelial bronchial cell line. Although these results question the real capability of acquiring epithelial functions by MSCs and the feasibility of MSC-based therapeutic approaches to regenerate damaged lung tissues, the characterization of this lung-hMSC population may be useful to study the involvement of stromal cell compartment in lung diseases in which MET plays a role, such as in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

Mesenchymal stem cells (MSCs) are adult non-hematopoietic stem cells originally isolated from bone marrow (BM) (Prockop, 1997), but they are virtually present and can be isolated from almost every tissue of the body (Da Silva et al., 2006), including peripheral blood (Roufosse et al., 2004). This evidence suggests that MSCs could be part of a mesenchymal-stromal cell system diffused throughout the body. The real in vivo counterpart of culture-expanded MSCs is still unknown; however, different Authors suggested that MSCs are a subgroup of vessel-lining pericytes that may contribute to vessel homeostasis by reacting to tissue damage with regenerative processes, locally modulating the inflammatory reaction, and entering systemic circulation to migrate according to cytokine gradients (Crisan et al., 2008). The International Society of Cellular Therapy (ISCT) stated the following three criteria for the definition of MSCs after in vitro expansion (Dominici et al., 2006): (1) the adherence to plastic under standard tissue culture conditions; (2) the expression of a specific combination of cell surface markers; (3) the capability of multilineage differentiation under appropriate in vitro conditions. These criteria are necessary to overcome the problems due to the absence of MSC-specific cell surface markers, the high heterogeneity in terms of differentiation potential, and the similarities to fibroblasts displayed by isolated and expanded MSCs. Consequently, ISTC suggested to define MSCs as “Multipotent Mesenchymal Stromal Cells” instead of “Mesenchymal Stem Cells”. In this Chapter, MSC isolation, expansion and functional characterization will be discussed in details.

Although many literature data are available on the role of Notch signaling in T-cell acute lymphoblastic leukemia (ALL) biology, the importance of this molecular pathway in the development of B-lineage ALL (B-ALL) cells in the BM microenvironment is unknown so far. In this study, we used anti-Notch molecules neutralizing Abs and γ-secretase inhibitor (GSI) XII to investigate the role of the Notch signaling pathway in the promotion of human B-ALL cell survival in presence of stromal cell support. The treatment with combinations of anti-Notch molecule neutralizing Abs resulted in the decrease of B-ALL cell survival, either cultured alone or cocultured in presence of stromal cells from normal donors and B-ALL patients. Interestingly, the inhibition of Notch-3 and -4 or Jagged-1/-2 and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days, similar to what is obtained by blocking all Notch signaling with the GSI XII. Our data suggest that the stromal cell–mediated antiapoptotic effect on B- ALL cells is mediated by Notch-3 and -4 or Jagged-1/-2 and DLL-1 in a synergistic manner.

Mesenchymal stromal cells (MSCs) have been investigated as a treatment for various inflammatory diseases because of their immunomodulatory and reparative properties. However, many basic questions concerning their mechanisms of action after systemic infusion remain unanswered. We performed a detailed analysis of the immunomodulatory properties and proteomic profile of MSCs systemically administered to two patients with severe refractory acute respiratory distress syndrome (ARDS) on a compassionate use basis and attempted to correlate these with in vivo anti-inflammatory actions. Both patients received 2 × 106 cells per kilogram, and each subsequently improved with resolution of respiratory, hemodynamic, and multiorgan failure. In parallel, a decrease was seen in multiple pulmonary and systemic markers of inflammation, including epithelial apoptosis, alveolar-capillary fluid leakage, and proinflammatory cytokines, microRNAs, and chemokines. In vitro studies of the MSCs demonstrated a broad anti-inflammatory capacity, including suppression of T-cell responses and induction of regulatory phenotypes in T cells, monocytes, and neutrophils. Some of these in vitro potency assessments correlated with, and were relevant to, the observed in vivo actions. These experiences highlight both the mechanistic information that can be gained from clinical experience and the value of correlating in vitro potency assessments with clinical effects. The findings also suggest, but do not prove, a beneficial effect of lung protective strategies using adoptively transferred MSCs in ARDS. Appropriate randomized clinical trials are required to further assess any potential clinical efficacy and investigate the effects on in vivo inflammation. Significance This article describes the cases of two patients with severe refractory adult respiratory syndrome (ARDS) who failed to improve after both standard life support measures, including mechanical ventilation, and additional measures, including extracorporeal ventilation (i.e., in a heart-lung machine). Unlike acute forms of ARDS (such in the current NIH-sponsored study of mesenchymal stromal cells in ARDS), recovery does not generally occur in such patients.

Alagille syndrome (ALGS), or arteriohepatic dysplasia, is a congenital multisystem disease due to Notch signalling pathway mutations, most commonly affecting JAGGED-1 (ALGS type 1), and more rarely NOTCH-2 (ALGS type 2), leading to hepatic, lung, renal and ocular dysfunction (chronic cholestasis, peripheral pulmonary artery stenosis, dysplastic kidneys pigmentary retinopathy), and skeletal abnormalies (minor vertebral segmentation, characteristic facies, posterior embryotoxon/anterior segment defects). ALGS is an autosomal dominant disease, but it is characterized also by variable penetrance and clinical expression and somatic/germline mosaicism. A 20-year-old man with ALGS was admitted to the University Hospital of Verona because of pancytopenia. Following analyses led to the diagnosis of Philadelphia chromosome/bcr-abl-negative, CD10-positive, B-lineage acute lymphoblastic leukemia (common B-ALL). In order to identify the genetic components involved in this complex phenotype, we sequenced the exome of a bone marrow sample collected from the patient. By genome interpretation with Knome pipeline applied to the reference genome UCSC hg19, we found missense variants both in NOTCH-2 (E38K) and JAGGED-1 (P871R) genes that are mainly involved in the syndrome, although their effect on protein function was predicted not to be deleterious. However, we detected putative damaging mutations in genes such as PAX5 (R38H) and NOTCH-1 (K1821N), which might be strongly related to the observed disease. In fact, PAX5 is a member of PAX protein family of transcription factors implicated into regulation of early development that binds NOTCH-2 and likely altering its functionality. On the other hand, NOTCH-1 is involved in cell growth and proliferation and thus the predicted alteration of function of the corresponding protein may have an important role in neoplastic transformation. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

In this study, we defined an in vitro inflammatory condition consisting of interleukin-beta and tissue necrosis factor-alpha and showed that naive MSCs in the inflammatory condition, could be turned into pro-inflammatory cells. We investigated pre-treatment conditions to improve MSCs’ anti-inflammatory properties and we found the use of cytokines including INF-gamma could enhance indoleamine 2,3-dioxygenase(IDO) activity and relieve pro-inflammatory changes when encountered the in vitro inflammatory condition. We believe that this result could contribute in the development of cell therapeutics using mesenchymal stem cells with enhanced anti-inflammatory properties.

increased the production of IL-6, COX2, CD54 and CD274. In conclusion, multiparametric flow cytometry enables the simultaneous analysis of intracellular and surface markers to i) determine the licensed status of different batches of MSCs, ii) measure the level of immunomodulatory activity, iii) identify the source of cytokines in co-culture systems and iv) define biomarkers predicting MSC potency.

Stromal cells are essential components of the bone marrow (BM) microenvironment regulating and supporting the survival of different tumors, including B-cell acute and chronic lymphocytic leukemia (B-ALL and CLL), and acute myeloid leukemia (AML). In this study, we investigated the role of Notch signalling in human BM-mesenchymal stromal cell (hBM-MSC)-promoted ALL, CLL and AML survival and chemoresistance. The block of Notch signalling through γ-secretase inhibitor (GSI) XII reverted the protective effect mediated by co-culture with BM-MSC. The treatment with combinations of anti-Notch neutralizing Abs resulted in the decrease of B-ALL cell survival, either cultured alone or cocultured in presence of BM-MSC from normal donors and B-ALL patients. The inhibition of Notch-3 and -4 or Jagged-1/-2 and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days, similar to what is obtained by blocking all Notch signaling with the GSI XII. The same Notch receptors are involved in CLL survival except for Notch-1 that, in CLL, mediates a synergistic effect with other Notch receptors in inducing the anti-apoptotic phenotype. Some preliminary data showed that Notch system is involved in survival and chemoresistance of acute myeloid leukemia blasts. Overall, our findings show that stromal cell-mediated Notch signaling has a role in promoting ALL, CLL and AML survival and resistance to chemotherapy. Therefore, the target of Notch pathway activation may represent a useful strategy to overcome drug resistance and improve the efficacy of conventional treatments. Disclosures: No relevant conflicts of interest to declare.

Allogeneic stem cell-based therapy is a promising tool for the treatment of a range of human degenerative and inflammatory diseases. Many reports highlighted the immune modulatory properties of some stem cell (SC) types, such as mesenchymal stromal cells (MSCs), but a comparative study with SCs of different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods employed for MSC characterization to compare the immunological properties of bone marrow-MSCs, olfactory ecto-mesenchymal stem cells, leptomeningeal stem cells, and three different c-Kit-positive SC types, i.e. amniotic fluid SCs, cardiac SCs, and lung SCs. We found that all the analyzed human SCs share a common pattern of immunological features, in terms of expression of activation markers, modulatory activity towards immune effector cells, immunogenicity and molecular inhibitory pathways, with some SC type-related peculiarities. In addition, we found that the inhibitory behaviour is not a constitutive property of SCs, but is acquired as a consequence of immune effector cell activation, as previously described for MSCs. Thus, immune regulation is a general property of stem cells and the characterization of this phenomenon may be useful for a proper therapeutical use of SCs. Disclosures: No relevant conflicts of interest to declare.

Human bone marrow Mesenchymal Stromal Cells (MSC) are potent modulators of T cell activation and proliferation, mainly through the production of partially defined soluble factors, including the IFN-g-induced tryptophan-degrading enzyme IDO, a key immunosuppressive effector pathway. Actually, MSC may affect the functions of virtually all immune effector cells, including B cells. However, current literature concerning MSC immunomodulatory activity on B cells is still controversial, due to both biological peculiarities of B cells, which do not produce IFN-γ, a key MSC-triggering cytokine, and to different and poorly comparable experimental approaches. Human purified B cells, either resting or activated for 4 days with a stimulation cocktail (CD40 ligand + enhancer polyhistidine mAb MAB050 + rhIL-2 + mouse F(ab’)2 anti-human IgM/IgA/IgG + CpG oligodeoxynucleotide 2006) were co-cultured with MSC, either at resting conditions or following inflammatory priming (MSC pre-incubation with IFN-γ + TNF-α for 48 hours), or with MSC supernatants. CD27-positive (memory) and CD27-negative (naïve) B cell survival, proliferation, and intracellular activation status (through signaling network analysis by Phosphoflow) were assessed. Our results showed that MSC are normally supportive cells, not intrinsically capable of suppressing B cell proliferation, and require inflammatory priming to acquire B cell inhibitory potential. Inflammatory-primed MSC impair significantly activated B cell growth in a cell contact-independent manner, as supernatant is sufficient to provide maximal inhibition of B cell proliferation. B cell inhibition by MSC is not related to either induction of B cell apoptosis or early signaling events necessary for B cell activation. In addition, IDO pathway triggered in IFN-γ-primed MSC seems to have a role also in B cell inhibition by interfering with the tryptophan metabolism. Overall, B cell behavior following the interaction with MSC depends on the functional state of both B cells and MSC. The role of IDO in B cell regulation needs further investigation, as it may be relevant to develop new therapeutic approaches in pathological conditions related to B cell hyper-activation. Disclosures: No relevant conflicts of interest to declare.

Bone is one of the most frequently transplanted tissues, with allografts and autografts accounting for more than 80% of total grafts. Synthetic biomaterials in association with autologous or allogeneic mesenchymal stromal cells (MSCs) represent a valid alternative in orthopaedic and maxillofacial surgery. Aim of REBORNE consortium is to perform clinical trials using standardized protocols based on advanced biomaterials and MSCs. Aim of our Unit was to assess MSC immune modulatory properties in presence of a novel biomaterial consisting of hydroxyapatite and tricalcium-phosphate (HA/TCP, Biomatlante, France) as scaffold for MSC delivery. We assessed immune modulatory properties, in terms of immunophenotype, inhibitory and anti-apoptotic effects, of MSCs of different origin when associated with the HA/TCP biomaterial, including bone marrow-derived mesenchymal stromal cells (BM-MSCs), adipose-derived MSCs (ASCs) and cord blood-derived MSCs (CB-MSCs). The culture of all MSCs with HA/TCP, did not modulate the anti-apoptotic and suppressive features of all MSCs toward sorted-T, B and NK lymphocytes as compared to standard culture setting. When exposed to inflammatory cytokines, such as IFN-γ and TNF-α, all MSCs were induced to acquire the suppressive phenotype, characterized by MHC-1 and CAM molecules up-regulation, and de novo expression of HLA-DR molecules. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype, as indicated by the up-regulation of osterix and osteocalcin transcription. The differentiation process is promoted in all MSC types, but especially in BM-MSCs, by dexamethasone and, to a higher extent, BMP-4 treatment. In view of using MSCs for advanced therapies in allogeneic setting, we evaluated the immunogenicity and immune modulatory features of pre-differentiated MSCs in association with HA/TCP towards both innate and adaptive immune cells, as well as the molecular pathways involved in MSC-mediated immune regulation. MSC-derived osteoblasts did not induce immune cell activation, as demonstrated by co-culture of unstimulated immune effector cells with pre-differentiated MSCs. We found a lower suppressive capability of pre-differentiated MSCs towards stimulated T and NK cells. However, some inhibitory effects could be exerted by BMP-4 treated-MSCs, and could be related to the presence of undifferentiated MSCs in culture, which could inhibit lymphocyte proliferation. The balance between the number of bone depositing osteocyte-like MSCs and immunosuppressive undifferentiated MSCs could be useful, in vivo, to promote bone healing and to reduce local inflammation, respectively. We then investigated in pre-differentiated MSCs the molecular mechanisms involved in the inhibition of T cell proliferation, by interfering with IDO and COX-2 activation. We found that pre-differentiated MSCs switched on their suppressive machinery by activating both IDO, which plays a central role in immune regulation, and COX-2, whose role is not significant in undifferentiated MSCs. COX-2 is produced rapidly as inflammation mediator after fracture, and recent data highlighted the role of COX-2 in improving fracture healing. In conclusion, this study increases the knowledge on MSC biology and gives pre-clinical data concerning the use of allogeneic MSC in combination with ceramic scaffolds to treat bone defects. Disclosures: No relevant conflicts of interest to declare.

Alagille syndrome (ALGS), or arteriohepatic dysplasia, is a congenital multisystem disease due to Notch signalling pathway mutations, most commonly affecting JAG1 (ALGS type 1), and more rarely NOTCH2 (ALGS type 2), leading to hepatic, lung, renal and ocular dysfunction (chronic cholestasis, peripheral pulmonary artery stenosis, dysplastic kidneys pigmentary retinopathy), and skeletal abnormalies (minor vertebral segmentation, characteristic facies, posterior embryotoxon/anterior segment defects). ALGS is an autosomal dominant disease, but it is characterized also by variable penetrance and clinical expression and somatic/germline mosaicism. A 20-year-old man with ALGS was admitted to the University Hospital of Verona because of pancytopenia. Following analyses led to the diagnosis of Philadelphia chromosome/bcr-abl-negative, CD10-positive, B-lineage acute lymphoblastic leukemia (common B-ALL). In order to identify the genetic components involved in this complex phenotype, we sequenced the exome of a bone marrow sample collected from the patient. By genome interpretation with Knome pipeline applied to the reference genome UCSC hg19, we found missense variants both in NOTCH2 (E38K) and JAG1 (P871R) genes that are mainly involved in the syndrome, although their effect on protein function was predicted not to be deleterious. However, we detected putative damaging mutations in genes such as PAX5 (R38H) and NOTCH1 (K1821N) which might be strongly related to the observed disease. In fact, PAX5 is a member of PAX protein family of transcription factors implicated into regulation of early development, that binds NOTCH2 and likely altering its functionality. On the other hand, NOTCH1 is involved in cell growth and proliferation and thus the predicted alteration of function of the corresponding protein may have an important role in neoplastic transformation. Disclosures: No relevant conflicts of interest to declare.

Developing a therapeutic strategy for lung regeneration still remains complex. Stem cell-based therapeutical approaches have been suggested as a potential tool; among them, human mesenchymal stromal cells (MSC) possess some promising features to this aim. MSC are stem cells residing in many tissue, i.e. bone marrow (BM), adipose tissue, cord blood, lung, etc., and they are capable of differentiating into different cell types of mesodermal origin, such as fat, bone and cartilage. To assess MSC epithelial differentiation potential, through a partially known process called Mesenchymal to Epithelial Transition (MET), MSC were collected from bone (BM) aspirates and lung biopsies after informed consent. MSC were characterized by immunophenotyping and clonogenicity assay. MSC mesodermal differentiation potential was assessed by testing their ability to differentiate into adipocytes, osteoblasts and chondrocytes. MSCs at different culture passages were induced to acquire the epithelial phenotype by culturing in presence of retinoic acid. The epithelial differentiation was checked by quantitative RT-PCR, immunofluorescence and a functional assay based on the Trans Epithelial Electrical Resistance Measurement (TEER). In presence of retinoic acid, MSC from BM and, mostly, lung upregulated a panel of general epithelial genes (cytokeratin 18, occludin, tight junction protein and claudin) and downregulated some specific mesenchymal markers (smooth muscle actin, snail2, vimentin, THY1), as detected by quantitative RT-PCR. Immunofluorescence confirmed the presence of E-cadherin4, occludin and cytokeratin 18 in a small number of cells (about 0,2 %). Trans Epithelial Electrical Resistance (TEER) measurement confirmed that MSC can acquire in vitro partial epithelial polarization after retinoic acid treatment. These data show that MSC can be induced to differentate into cells resembling some morphological, phenotypical and functional properties of epithelial cells. BM-MSC are less prone to acquire an epithelial phenotype as compared to hLung-MSC. Additional in vivo studies on mouse model with lung damage are in progress. Disclosures: No relevant conflicts of interest to declare.

Stromal cells are essential components of the bone marrow (BM) microenvironment that regulate and support the survival of different tumors, including chronic lymphocytic leukemia (CLL). In this study, we investigated the role of Notch signaling in the promotion of survival and chemoresistance of human CLL cells in coculture with human BM-mesenchymal stromal cells (hBM-MSCs) of both autologous and allogeneic origin. The presence of BM-MSCs rescued CLL cells from apoptosis both spontaneously and following induction with various drugs, including Fludarabine, Cyclophosphamide, Bendamustine, Prednisone and Hydrocortisone. The treatment with a combination of anti-Notch-1, Notch-2 and Notch-4 antibodies or γ-secretase inhibitor XII (GSI XII) reverted this protective effect by day 3, even in presence of the above-mentioned drugs. Overall, our findings show that stromal cell-mediated Notch-1, Notch-2 and Notch-4 signaling has a role in CLL survival and resistance to chemotherapy. Therefore, its blocking could be an additional tool to overcome drug resistance and improve the therapeutic strategies for CLL.

Abstract 1924 Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. Allografts and autografts account for more than 80% of total graft volume, despite their considerable disadvantages, including the risk of disease transfer and immunologic rejection, limited supply of bone, costs and complications. Significant growth opportunities exist for synthetic bone grafts in association with mesenchymal stromal cells (MSC) from autologous or allogeneic sources as alternatives to biological bone grafts in orthopaedic and maxillofacial surgery. The objective of REBORNE is to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. To reach this goal, five phase I clinical studies with 20 patients have been planned in 12 clinical Centers spread in 8 European countries. Aim of the Immunological Unit of Reborne is to assess the MSC immunomodulatory properties in presence of the biomaterial used as scaffold for MSC delivery. All the functional experiments were performed in parallel, by comparing the effects of standard culture conditions and three-dimensional culture setting using MBCP (Biomatlante). Material and methods: Bone marrow MSC were provided from REBORNE Consortium Centers. To perform proliferation assays, different immune effector cells (T, B and NK cells) were stained with CFSE according to manufacturer's protocol. Active caspase-3 cell staining was used for survival quantification of immune effector cells after co-culture experiments. Differentiation potential was evaluated by culturing MSC with two different media containing either bone morphogenetic protein 4 (BMP4) or dexamethasone. After three weeks, osteogenic differentiation was quantified by qRT-PCR, alkaline phosphatase activity and alizarin red staining. Results: We found that primed MSC, pre-treated with the inflammatory cytokines IFNg and TNFa, displayed upregulation of HLA-ABC, CD54, CD106 and de novo expression of HLA-DR, both in standard culture conditions and in association with MBCP. Immune effector cells could be cultured and collected even in presence of MBCP and no significant differences were found between standard- (MSC + effector cells) and 3D-coculture conditions (MSC + MBCP + effector cells), in terms of immune effector cell proliferation. In both experimental conditions MSC suppressed T and NK cell proliferation (% suppression: MSC + T = 68.4; MSC + MBCP + T = 62.4; MSC + NK = 17.5; MSC + MBCP + NK = 20.2) and increased B cell proliferation (MSC + B = +13%; MSC + MBCP + B = +12.3%). In addition, immune effector cells viability was not affected by MBCP and MSC co-culture increased their survival even in presence of MBCP; in fact, in each culture condition the percentage of inhibition of T, B and NK cell apoptosis was higher than 20% in comparison to immune effector cells cultured without MSC. Dexamethasone and BMP4 were capable of inducing MSC differentiation into osteoblast-like cells, as confirmed by qRT-PCR analysis. We demonstrated that BMP4-based medium led to fully differentiated osteoblasts (Osterix+, RUNX2+, DLX5+ and alkaline-phosphatase+). Moreover, MBCP was more efficient in increasing osteoblastic differention as compared to standard culture conditions, as shown by the higher expression of Osteocalcin and Osterix. These data show that the association of MBCP and MSC does not affect MSC properties and suggest that it could be a treatment of choice of bone defects instead of allograft and autograft transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4049 Aim of the European Consortium CASCADE is to standardize GMP-grade production and clinical use of Mesenchymal Stromal Cells (MSC) to treat skin and corneal wounds. MSC possess immunogenicity and immunomodulatory properties that must be carefully addressed before clinical use. CASCADE Immunological Unit is aimed to set up and validate a wide panel of functional assays to fully characterize in a standardized and reproducible manner the immunomodulatory properties of MSC obtained inside CASCADE Units from bone marrow, adipose tissue, cord blood, and amniotic membrane (BM, AT, CB, AM) through different GMP-grade expansion protocols including platelet lysate- and fetal calf serum-based culture conditions. Immune cells were isolated using indirect immunomagnetic depletion; samples with less than 96% of purity were discarded. For the experiments, MSC were expanded in the same medium used for production and harvested at 70% confluence. Primed MSC were obtained by 48h-treatment with 10 ng/ml of rh-INFγ and 15 ng/ml of rh-TNFα. Cocultures were set up by plating primed or unprimed MSC in 96 or 48 flat bottomed – well plates; CFSE-stained T, B, NK cells were seeded at different effector cell:MSC ratios. Cells were harvested after 4 or 6 days of coculture for proliferation evaluation by FACS analysis. T cells were stimulated with mitogenic αCD3 plus αCD28 antibodies at 0.5 μg/ml each; B cells were activated with CD40L at 50 ng/ml, its enhancer at 5 g/ml, IL-2 20 UI/ml, CpG 2006 2.5 μg/mL, and F(ab')2 anti-IgM/IgA/IgG 2 μg/mL; NK cells were activated with 100 U/ml rh-IL2. To identify the molecular mechanisms involved in immunomodulatory properties of MSC, coculture of immune effector cells and MSC were performed in the presence of specific inhibitors, after identifying their non-toxic and effective concentrations: 1 mM for L-1MT (IDO inhibitor), 2 μM for snPP (HO-1 inhibitor), 5 μM for NS-398 (COX2 inhibitor), 1 mM for L-NMMA (iNOS inhibitor) and 10 μg/ml for anti-IFNγ neutralizing antibody. We also studied the capacity of resting and primed MSC to sustain the survival of unstimulated T, B, and NK cells through the evaluation of the percentage of caspase-3negCD45pos viable immune cells after 4 to 6 days in culture with or without MSC. For MSC immunogenicity assay, the proliferation of allogeneic T was evaluated at day 5 of culture by incorporation of 3H-Thymidine; in addition, NK cells were activated for 2 days with 100 U/ml of rh-IL2 whereas resting or primed MSC were loaded with non radioactive fluorophore (BaTDA) or with Cr51 and used as target cells. Inflammatory milieu significantly upregulated MHC class I and II, CD54, CD106, CD40, CD274, CD112, CD155 expression, and downregulated NKG2D ligands (ULBP 1–3, MICA/B) and mesenchymal markers (CD73, CD90, CD105). AT-derived MSC expressed less MHC class II, CD200 and CD106 molecules than BM-MSC. MSC coculture inhibited T and NK cell proliferation without inducing apoptosis, and this effect was greater in presence of primed MSC. On the contrary, only primed MSC were capable of suppressing B cell proliferation. In addition, MSC inhibited apoptosis of resting T, B, and NK cells, while inflammatory priming increased their pro-survival activity. T cell/MSC coculture showed that activation of IDO and HO-1 was the main mechanism involved in MSC immune modulation, as the addition of specific inhibitors (L-1-MT and snPP) significantly reverted the phenomenon. MSC never promoted allogeneic T cell proliferation; by contrast, IL-2-activated NK cells could efficiently recognize and kill allogenic unprimed MSC. However, MSC became insensitive to NK cells once primed with inflammatory cytokines. Some differences were observed depending on the origin and culture conditions of clinical-grade MSC. All the experimental protocols to assess MSC inhibitory effects on immune effector cells have been standardized and will be applied for the release of GMP-grade MSC produced inside the CASCADE Consortium. Disclosures: No relevant conflicts of interest to declare.