Hematol Oncol Clin North Am

Hematol Oncol Clin North Am. of apoptotic mechanisms but also to signals delivered by accessory cells at the sites of the disease activity. In tissue microenvironment, CLL B cells reside in close contact with T lymphocytes, stromal MGC33310 cells, mesenchymal stromal cells (MSCs), endothelial cells, follicular dendritic cells and macrophages. Interactions among these components of the microenvironment regulate the trafficking, survival, and proliferation of leukemic B cells in a way that depends both on direct cell-cell contact and/or on the exchange of soluble factors [12]. Moreover, once resident in stromal environment, CLL cells are protected from different therapeutic interventions [13-15]. Among bone marrow stromal cells, MSCs show a bidirectional cross-talking with neoplastic B cells. Leukemic cells are supported by stromal cells and, in turn, are also able to activate and induce stromal cell to proliferate and release several mediators (i.e., CXCL12, CXCL13, CCL19 and CCL21) which sustain the ongoing process [16-18]. These interactions drive CLL B cells into tissue microenvironment, where malignant cells experience the survival and proliferation signals mediated by the B cell receptor (BCR) and other pathways [15]. Nevertheless, these complex cellular and molecular mechanisms are not yet completely defined. Although in healthy subjects MSCs represent a small fraction of the stromal cell population, immunohistochemistry studies performed in patients with several lymphoproliferative diseases showed that SMA+ mesenchymal stromal cells, which represent the counterpart of MSCs, are the dominant stromal cell population in CLL microenvironment [19]. These observations support a crucial role of MSCs on the mechanisms favoring malignant cells and disease progression in CLL. In the last years, the modulation of tumor microenvironment is becoming a promising therapeutic strategy in CLL treatment, demonstrated by the use of an increased number of SB-742457 compounds (i.e. thalidomide, lenalidomide, plerixafor and natalizumab) [20, 21], affecting molecules involved in the compartimentalization of tumor cells. More recently, several small molecules have been developed to inhibit a variety of kinases in the BCR pathway, including Lyn, Syk, Btk and PI3K, which are crucial not only for the activation of multiple survival pathways (such as Akt, Erk, NF-kB) but also for chemokine-mediated migration and adhesion of B cells in the microenvironment [22]. Thus, the understanding of the interactions between CLL B cells and the microenvironment is mandatory to define more effective therapies for CLL. In this context, the main aim of this study was to investigate the impact of MSCs on CLL B cell survival in order to verify whether MSCs protect leukemic B cells from spontaneous apoptosis both at basal conditions and after Fludarabine and Cyclophosphamide containing regimen therapy. We also tested the effect of two kinase inhibitors, Bafetinib (dual BCR-Abl/Lyn inhibitor) and Ibrutinib (Btk inhibitor), known to reduce neoplastic B cell viability [23], on CLL B cells in presence of MSCs. Moreover, the investigation of soluble factors, mainly cytokines and chemokines, which could be involved in leukemic cell survival, was performed. Our data clearly demonstrated that MSCs display a pro-survival effect on leukemic B cells from CLL patients and that CLL clones displayed a variable degree of responsiveness to microenviromental stimuli, suggesting that same clones are dependent SB-742457 and other are independent from MSC pro-survival capability. This observation might be relevant in order to identify patients who may benefit of compounds targeting CLL microenvironment. RESULTS Mesenchymal stromal cells from CLL patients display phenotypic profile and differentiation capability of MSCs from normal subjects MSCs were obtained from the bone marrow of 46 CLL patients by plastic adhesion as previously described [24, 25]. The adherent fraction leads to the formation of high proliferating spindle-shaped colonies, reaching the confluence in 30 days (Figure S1A). Flow cytometry analysis showed that MSCs were positive for CD90, CD73, CD105, and negative for CD14, CD34, CD45 and CD31 (Figure S1B). MSC ability to differentiate in adipocytes and osteocytes was tested using specific conditioned media. Adipogenic differentiation was demonstrated by the detection of lipid vesicles in the cytoplasm of (pre)adipocytes, stained with Oil Red. Osteogenic differentiation was documented by the increased expression of mRNA coding for Core Binding Factor 1 (CBFA1) and the deposition of mineralized matrix, shown by the Von Kossa staining (Figure SB-742457 S1C). The cell adhesion, the immunophenotype and the SB-742457 differentiation ability of stromal cells generated in our cultures are in accordance to the criteria required for MSC characterization [26, 27]. MSCs from CLL patients support neoplastic B cell survival We tested the effect of MSCs on the survival of leukemic B cells obtained from 30 CLL patients and normal.