Page 71 - 2021_10-Haematologica-web

CLL cells affect osteoblastogenesis/osteoclastogenesis
matched controls.8 However, the mechanisms underlying bone erosion in CLL patients have not been elucidated so far, and may be part of a complex set of events participat- ing in disease pathogenesis. Recent evidence demonstrat- ed that mutual interactions between stromal and leukemic cells facilitate survival and expansion of the neoplastic clone9-13 and contemporarily affect functions of microenvi- ronmental cells. We previously described a direct correla- tion between the levels of RANKL (nuclear factor-κB lig- and) expression on CLL cells and the degree of bone ero- sion in CLL patients.2
We suggested that leukemic cells, progressively infiltrat- ing bone marrow, may affect the differentiation of osteoblasts and osteoclasts, ultimately leading to alter- ations of physiological bone remodeling. RANKL, expressed by osteoblasts, is the major cytokine regulating osteoclast differentiation after binding to RANK+ mono- cytes. Osteolytic bone lesions are considered a hallmark of multiple myeloma and malignant plasma cells show high RANKL expression and secretion; in this disease, the inter- actions between malignant plasma cells, releasing RANKL, and the bone microenvironment progressively undermine the integrity of bone structures due to stimulation of osteoclast activity and subsequent excessive bone resorp- tion.14-16 RANKL is also expressed at high levels in malig- nant CLL cells but, at difference from multiple myeloma cells, CLL cells release it only in exceptional cases.17 Borge et al. detected high levels of plasma RANKL (800 pg/mL) in one CLL patient with lytic bone lesions and demon- strated that, in vitro, leukemic B cells released large amounts of RANKL (1,600 pg/mL) after stimulation with CpG.3 However, RANKL stimulation induces the produc- tion of pro-osteoclastic cytokines, such as tumor necrosis factor (TNF)α, interleukin (IL)-6 and IL-8 by CLL cells.17,18 Therefore, several growth factors may contribute to the microenvironment/leukemic B-cell crosstalk that governs bone homeostasis.
Based on these premises we investigated whether cytokines secreted by leukemic cells participate in bone alterations in CLL patients. Some evidence, reported here, indicates that released CLL cytokines prevent osteoblast- induced bone formation and contemporarily stimulate the expansion of osteoclast progenitors or of fully mature osteoclasts. Collectively, these observations suggest that the disruption of the physiological bone marrow niche may create favorable conditions for CLL-cell expansion. The demonstration that the survival/expansion of leukemic cells appears to be promoted by activated osteo- clasts is relevant to this notion.
Methods
This study was approved by the Ethics Committee of IRCCS Ospedale Policlinico San Martino (Genoa, Italy). Forty-five patients were included in the study: 19 had Binet stage A, 17 had stage B and 9 had stage C. MEC-1 cells, obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (German cell line collection) were used in selected experiments. Details of all the Methods are provided in the Online Supplementary File.
Osteoblast generation
Bone marrow stromal cells (BMSC), previously expanded,12 were osteogenically induced, co-cultured with CLL B cells (con- tact/transwell), CLL-cell conditioned media (CLL-cm) or CLL sera
(CLL-sr) for 5 more days (Online Supplementary Figure S1), detached and processed.
Messenger RNA extraction, reverse transcription and real-time quantitative polymerase chain reaction
Reverse transcription quantitative real-time polymerase chain reaction (RT-PCR) was used to assess the expression of GAPDH, osteopontin (OP), osteocalcin (OC), RUNX2, Dikkpof-1 (DKK-1), cathepsin K (Cat-K), NFATc-1 and matrix metalloproteinase 9 (MMP9).
Evaluation of matrix deposition
Alizarine staining was performed, using a commercial kit, on paraformahaldeyde-fixed cultures of osteo-induced BMSC, under differenttreatments.
Short-interfering RNA-mediated knockdown of specific cytokines in MEC-1 or CLL cells
All the short-interfering RNA (siRNA) used, (non-specific and specific for TNFα, IL-6, and IL-11) were obtained commercially.
Osteoclast generation
Monocytes purified from peripheral blood mononuclear cells of healthy donors or from CLL patients were induced to differentiate toward osteoclasts and then cultured under different conditions (Online Supplementary Figure S1): CLL-cm with/without neutraliz- ing anti-cytokine/receptor monoclonal antibodies, ibrutinib, deno- sumab, or from cytokine-silenced CLL cells.
TRAP staining and bone resorption assay
Tartrate-resistant acid phosphatase (TRAP) was detected using a commercial kit. Osteoclast activity was evaluated by sizing resorp- tion pits produced in an inorganic crystalline material.
Viability and cell cycle evaluation of CLL B cells co-cultured with osteoclasts
Osteoclasts derived at 14 days were co-cultured with CLL cells and apoptosis of leukemic cells was determined through DiOC6 staining19 or by propidium iodide cell cycle-phase distribution through multiparameter flow cytometric analysis.20 Ki-67 expres- sion was also determined by flow cytometry.
Immunohistochemical analyses of bone biopsies
B5-fixed, decalcified and paraffin-embedded tissue blocks were sectioned, de-paraffinized and rehydrated. Endogenous peroxidase was blocked with 5% H2O2; serial sections were immunostained for TRAP and CD79a.
Cytokine levels in cultured-cell media and murine CLL sera
Human TNFα levels in media from osteo-induced BMSC and CLL cells were determined using a Millipore Milliplex kit. We fur- ther determined human TNFα levels in sera of mice displaying documented bone loss after human CLL-cell engraftment.2
The amounts of TNFα in co-cultures of monocytes, differentiat- ing toward osteoclasts under various conditions – with monocyte colony-stimulating factor (MCSF)+RANKL, MCSF+RANKL+CLL- cm or MCSF+CLL-cm only – were assessed through a qualitative enzyme-linked immunosorbent assay and compared. The levels of TNFα, IL-11 or IL-6 in conditioned medium of CLL-cm, per se, or upon cytokine-knockdown, were determined using the same approach.
Gene expression profiling
Gene expression of B cells from CLL samples and normal
haematologica | 2021; 106(10)
2599