Increased Survival and Migration of CLL B-Cells in the Presence of Marrow Mesenchymal Stromal Cells: Novel Findings for Microenvironment-Targeted Therapies
Authors: Ave Elisa , Frezzato Federica , Gattazzo Cristina , Trimarco Valentina , Martini Veronica , Visentin Andrea , Castelli Monica , Carraro Samuela , Lessi Federica , Cabrelle Anna , Facco Monica , Semenzato Gianpietro , Trentin Livio 
Published: October 2019

Abstract
Abstract 4571

Background.
The accumulation of CD19+/CD5+/CD23+ B cells with a prolonged lifespan in peripheral blood, secondary lymphoid organs and bone marrow (BM) is a peculiar feature of B-cell chronic lymphocytic leukemia (B-CLL). Since CLL cells removed from the in vivo microenvironment and in vitro cultured rapidly undergo spontaneous apoptosis, bidirectional interactions between malignant and by-stander cells may lead to an abnormal microenvironment that confers growth advantages to neoplastic clone. Mesenchymal Stromal Cells (MSCs) are the dominant marrow stromal population in indolent subtype of CLL/small lymphocytic leukemia (SLL) and follicular lymphoma (FL), rather than other aggressive B-cell lymphomas, and are involved in B-CLL cell survival. Despite the phenotypic and cytologic homogeneity, CLL is characterized by extremely variable clinical courses, suggesting that malignant B-cells hold variable degrees of dependency on pro-survival signals coming from the microenvironment. The aim of this study was to assess the role of MSCs in CLL B-cell localization and survival, defining the degree of dependency of leukemic B-cells from external pro-survival signals, with the ultimate goal of identifying patients that mostly benefit microenvironment-targeted therapies.

Methods.
MSCs isolated from the BM of 47 B-CLL patients were expanded ex vivo and characterized through flow cytometry analysis and differentiation cultures. Fresh isolated CLL peripheral blood mononuclear cells were co-cultured with CLL-MSCs or stromal cells and apoptosis were measured by Annexin V test and western blotting analysis (PARP-1 detection). Chemotactic assays were performed.

Results.
The survival of neoplastic cells ranged from 13.3% (±13.2) in leukemic cells cultured in medium alone to 58.5% (±17.2) when leukemic cells were cultured in presence of CLL-MSCs (p<0.01). Transwell experiments showed that the anti-apoptotic effect is mediated by soluble factors produced by MSCs. We investigated whether different CLL clones show a different susceptibility to spontaneous apoptosis when co-cultured in presence of MSCs recovered from B-CLL patients. The detection of the 85KDa cleaved PARP fragment in all CLL B-cells cultured in medium alone confirmed that they underwent spontaneous apoptosis. At the same time, the presence or the lacking of the cleaved fragment of PARP-1 on CLL B-cells after 7 day-co-cultures with MSCs discriminated patients into two groups: non-responder (89 kDa Parp fragment detectable) and responder (89 kDa Parp fragment not detectable) to microenvironment pro-survival signals. Finally, chemotaxis tests showed the ability of MSCs to produce and release molecules promoting the migration and the localization of neoplastic B-cells in bone marrow (Migration Index of leukemic cells: 5.1; Migration Index of normal B cells: 1.9; p<0.01).

Conclusions.
MSCs derived from patients with B-CLL provide survival signals to neoplastic cells, extending their lifespan and producing chemotactic factors that favour their accumulation into BM. On the other hand, CLL cells display heterogeneous responses to environmental pro-survival signals, suggesting that each CLL clone could differently react to the microenvironment protection. The blocking of the cross-talk between malignant clone and accessory cells within the microenvironment might represent an attractive novel strategy for CLL therapy. Our data provide the rationale for tailored therapies which powerfully target the cross-talk with marrow cells, particularly on patients carrying a clone more sensitive to anti-apoptotic signals coming from the microenvironment.

Disclosures:
No relevant conflicts of interest to declare.