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P. Giannoni et al.
peripheral blood lymphocytes was profiled on a GeneChip Gene 1.0 ST array.21
Statistics
Two-sided Student t-tests, Mann-Whitney U tests, or χ2 tests with Fisher correction were used. All the data sets had a normal distribution. The levels of statistical significance are denoted by *P≤0.05, **P≤0.01 or ***P≤0.001.
Results
The terminal differentiation of osteo-induced bone mar- row stromal cells is inhibited by the addition of CLL cells, CLL-conditioned media or CLL-sera
Normal donor BMSC were induced to differentiate toward osteoblasts and then co-cultured with CLL cells (contact/transwell), CLL-cm or CLL-sr (Online Supplementary Figure S1A). After 5 days of co-culture, osteo- induced BMSC were evaluated for RUNX2, osteocalcin, DKK1 and osteopontin mRNA levels by quantitative RT- PCR. RUNX2, the master gene regulating the osteoblasto- genic cascade, appeared significantly downregulated by the presence of CLL cells, CLL-cm or CLL-sr, but not by normal B cells (Figure 1). Osteocalcin levels, which are normally high in fully differentiated osteoblasts, were also signifi- cantly reduced. In contrast, DKK-1, a canonical WNT inhibitor, appeared highly upregulated with a clear trend in all the experimental conditions. Osteopontin mRNA was also enhanced, although even by the presence of normal B cells. Collectively, these findings indicate that CLL cells affect the expression of the major osteoblast-related genes and inhibit differentiation of BMSC towards osteoblasts.
Reduced extracellular matrix deposition by osteo-induced bone marrow stromal cells cultured with CLL cells, CLL-conditioned media or CLL sera
The inhibition of osteoblast differentiation by CLL cells was also confirmed through the evaluation of matrix min- eralization levels. According to Alizarine Red staining, min- eralized areas appeared consistently reduced, with the strongest inhibition of matrix deposition occurring in the presence of CLL-cm or CLL-sr (Figure 2A, B). Based on lit- erature data22-24 we investigated whether IL-6, IL-11 and TNFα could represent candidate cytokines mediating the inhibition of osteoblast differentiation. The effect of an anti-GP130 monoclonal antibody was also tested because this molecule, shared by different cytokines of the IL-6 fam- ily, is pivotal in signal transduction. Monoclonal antibodies neutralizing the above mentioned cytokines (IL-11, TNFα) or blocking binding to their receptors (IL-6R, GP130) were thus added, together with CLL-cm, to cultures of osteo- induced BMSC. All these monoclonal antibodies, but not an isotype-specific control (Online Supplementary Figure S2A), counteracted suppression of in vitro mineralized matrix dep- osition, which was significantly higher after the addition of infliximab (Figure 2C, D). Cultures of osteo-induced BMSC with conditioned media from the TNFα-, IL-6-, or IL-11- RNA-silenced MEC-1 cell line or primary leukemic cells (BA101), further confirmed involvement of these cytokines in the impairment of osteoblast differentiation (Figure 2E-H and Online Supplementary Figure S2A, B). Moreover the increase in mineralized matrix deposition, detected in osteo-induced BMSC cultured with CLL-cm + neutralizing monoclonal antibodies, was paralleled by the restoration of
higher levels of both RUNX2 and osteocalcin mRNA (Online Supplementary Figure S2C, D). Evidence of decreased pSTAT3(ser727) and AKT expression in CLL-cm-treated BMSC versus CLL-cm + Infliximab or in basal conditions, suggests the participation of these signaling pathways in the regula- tion of osteogenesis by CLL-released TNFα (Online Supplementary Figure S2E, F).
The observation of higher mRNA expression for IL-11 and IL-11 receptor A (IL-11RA) in CLL cells than in control B cells, by gene expression profiling analysis, highlight IL-11 as a cytokine released by CLL cells (Online Supplementary Figure S4). An association between increased levels of IL-6 produced by CLL cells and worse clinical outcomes has been reported recently.25 The substantial amounts of TNFα, present in conditioned media derived from CLL cells cul- tured alone (basal conditions: Control), but significantly reduced in media from CLL/BMSC co-cultures (Online Supplementary Figure S5), suggest that TNFα, constitutively secreted by CLL cells and metabolically active, plays a role in suppressing osteoblast differentiation.
Enhancement of differentiation of allogeneic or autologous monocytes into osteoclasts by CLL-conditioned media
Next we investigated whether the presence of CLL-cm could affect osteoclastogenic differentiation of monocytes in vitro. Purified and pre-activated monocytes, from periph- eral blood of healthy donors or of CLL patients, were chal- lenged with CLL-cm, or medium only, for 7 days (Online Supplementary Figure S1). The number of osteoclasts, evalu- ated by counting the number of TRAP+ cells with three or more nuclei, increased significantly after the addition of CLL-cm (Figure 3A, C). Moreover osteoclasts derived under this condition appeared larger than those in control cultures, fully differentiated and characterized by multiple nuclei: a canonical ruffle border was sometimes evident (Figure 3B). The enhancement in osteoclast differentiation was also detectable when the same experiment was per- formed in an autologous setting (Figure 3D, E). A consid- erable reduction in the number of large multinucleated cells was however observed when monocytes were stimu- lated without RANKL in the induction phase (MCSF+CLL- cm only). Under this condition, a consistent number of tri- nucleated TRAP+ cells appeared drastically smaller, in com- parison to osteoclasts present in control cultures derived with exogenous RANKL. We were thus prompted to eval- uate the relative proportions of TRAP+ tri-nucleated small or large cells. Figure 4A and B show that, while pre-stimu- lation of monocytes with MCSF+RANKL, with or without CLL-cm, led to their complete maturation, inducing a high- er number of typical, multinucleated osteoclasts, the addi- tion of MCSF+CLL-cm alone failed to achieve the same effect, consistently impairing the generation of large osteo- clasts (Figure 4C and Online Supplementary Figure S6). Low levels of soluble RANKL, found in media derived from cul- tured CLL cells (n=10; data not shown), could thus promote initial osteoclast differentiation only. It is however evident that factors released by CLL cells synergize with RANKL, when exogenously added, thus causing a significant ampli- fication of osteoclastogenesis. This observation also appears to be sustained by the finding that mRNA expres- sion of cathepsin K, MMP9, and NFATc1, which are pivotal in osteoclast differentiation, is higher in monocytes stimu- lated with MCSF+RANKL and CLL-cm than in monocytes treated with MCSF+RANKL only (Figure 4D). A statistical-
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