Study of the Quantitative, Functional, Cytogenetic and Immunoregulatory Properties of Bone Marrow Mesenchymal Stem Cells in Patients with B-Cell Chronic Lymphocytic Leukemia

Abstract 3861

The microenvironment in both the bone marrow (BM) and lymph nodes has clearly been implicated in the biology of B-chronic lymphocytic leukemia (B-CLL). The non-hematopoietic components of the BM microenvironment originate from a rare population of multipotent progenitor cells, currently referred to as mesenchymal stem/stromal cells (MSCs). The latter have been shown to support hematopoiesis and to affect B cell proliferation and differentiation, thereby setting the stage for studying MSCs' putative role in CLL pathogenesis. However, this particular field of research has not been extensively investigated and the question as to whether patient MSCs differ from their normal counterparts has not been properly answered. The aim of the present study is to explore whether BM-derived MSCs from CLL patients harbor intrinsic abnormalities, which in turn might contribute to the pathophysiology of the disease.

BM MSCs were thus isolated from 11 patients with B-CLL (Rai stage 0-III) and 16 age- and sex-matched healthy individuals and their quantitative, functional and cytogenetic characteristics were comparatively assessed. BM MSCs were expanded and re-seeded for a total of 6 passages.

Adherent cells from both study groups displayed the same spindle-shape morphology and showed a similar expression of CD29, CD44, CD73, CD90 and CD105 while being negative for CD14, CD34 and CD45. Even though MSC cultures could be established and serially replated from all CLL patients, their growth rate over passages was significantly reduced compared to cultures generated from normal individuals (P < 0.0001). These findings were further substantiated by the MTT assay according to which the number of live cells, at a representative passage (P2), remained significantly lower in CLL patients compared to controls (P <0.0001). The survival characteristics of CLL-derived MSCs at P2 were further evaluated using flow cytometry and 7AAD staining. A statistically significant increase in the proportion of both early and late apoptotic cells was thus documented in B-CLL-derived MSCs as compared to their normal counterparts (P=0.0007 and P=0.0045, respectively). These data suggest that the impaired proliferative potential of CLL-derived MSCs can be attributed, at least in part, to increased cell apoptosis. The frequency of MSCs within the BM mononuclear cell (BMMC) fraction, estimated via the standard CFU-F assay, was significantly reduced in patients, as compared to normal controls (P=0.0039) and this might also be explained by the increased apoptotic rate of CLL-derived MSCs, as well as by BM infiltration by the malignant cells. Regarding MSC differentiation capacity, patient MSCs exhibited normal osteogenic and adipogenic potential as evidenced by cytochemical staining (Von Kossa and Oil Red, respectively) and also by the quantification of osteogenesis (RUNX2, ALP)- and adipogenesis (PPARG, aP2)-related specific gene expression. Furthermore, CLL-derived MSCs did not differ from their normal counterparts in the ability to suppress mitogen-induced T-cell proliferative responses. Since B-CLL cells undergo spontaneous apoptosis in vitro, unless cultured on direct contact with marrow stroma, we evaluated whether ex vivo expanded patient MSCs confer a survival advantage to the malignant clone. Yet, MSCs from both study groups exerted a similar anti-apoptotic effect on CLL cells. CLL-derived MSCs exhibited aberrant production of cytokines that are crucial for B-cell differentiation, survival and apoptosis. More specifically, they secreted significantly lower levels of SDF-1 (P=0.002) and TGF-β1 (P<0.001), as compared to their normal counterparts. Furthermore, CLL-derived MSC supernatants were shown, for the first time, to contain significantly higher levels of APRIL (P=0.0002) and lower levels of BAFF (P=0.0036), as compared to normal MSCs. Finally FISH analysis demonstrated that CLL-derived MSCs did not share any of the cytogenetic abnormalities with CLL cells, thereby suggesting that they are not part of the leukemic clone.

In conclusion, ex-vivo expanded B-CLL-derived MSCs harbor intrinsic qualitative and quantitative abnormalities that may be implicated in disease development and/or progression. We anticipate that our observations will contribute to delineating B-CLL biology and will hopefully provide important clues for the design of appropriate microenvironment-targeted therapies.