In Vitro and In Vivo Evidences of Osteocyte Involvement In Myeloma-Induced Osteolysis

Abstract 131

Multiple myeloma (MM)–induced osteolysis is characterized by severely imbalanced and uncoupled bone remodeling due to increased osteoclast recruitment and suppressed osteoblast differentiation. Osteocytes are located in the lacuna/canalicular system of bone and that have been recently hypothesized to regulate local bone remodeling through the cell death and apoptosis triggering osteoclast recruitment and formation. Actually, the potential involvement of osteocytes in MM-induced osteoclast formation and in the bone remodeling alterations occurring in MM patients is still unknown and was investigated in this study. Firstly we evaluated the effect of MM cells on osteocyte survival in a co-culture transwell system of human myeloma cell lines (JJN3, KMS12, XG-1) and the human pre-osteocytic cell line HOB-01. By Transmission Electron Microscopy (TEM) observations and TUNEL assay we showed the occurrence of either apoptotic cells or degenerated non-apoptotic cells in the monolayer of HOB-01 cells co-cultured with MM cells or treated with their conditioned media (CM) as compared to non-treated cells suggesting that MM cells may increase osteocyte cell death. Second, we investigated whether MM cells could affect the pro-osteoclastogenic profile and the osteoclastogenic properties of HOB-01 in co-culture. The microarray analysis, using Affymetrix GeneChip^® HG-U133Plus2.0 platform) identified 47 genes significantly modulated by MM cells in HOB-01, most of them up-regulated; among these, we identified the increased expression of the pro-osteoclastogenic genes IL11 and MMP1, a metalloproteinase involved in tumoral ostelysis. The up-regulation of both IL-11 and MMP-1 was then confirmed at both mRNA and protein level either by real time PCR and ELISA assay, respectively. Furthermore, the mRNA expression and the protein levels of the main pro-osteoclastogenic cytokines CCL3/MIP-1α and RANKL were also measured in the co-cultures showing that CCL3/MIP-1α levels were significantly higher than those observed either in HOB-01 or MM cells cultured alone whereas RANKL levels and RANKL/OPG ratio were unchanged. Analyzing separately HOB-01 and MM cells after the co-culture period, we demonstrated that increased levels of CCL3/MIP-1α were due to its up-regulation in MM cells. In line with the increase of the pro-osteoclastogenic cytokines observed in the co-cultures, we found that the CM of HOB-01 co-cultured with MM cells significantly increased CD14⁺-derived osteoclastogenesis in presence of RANKL as compared to the CM of HOB-01 or MM cells cultured alone. Interestingly this effect was completely blunted in presence of blocking antibody anti-CCL3/MIP-1α and in a lesser extent of anti-IL-11 and anti-MMP-1.

To translate into a clinical perspective such in vitro evidences, then we performed histological analysis on bone biopsies obtained from iliac crest of a cohort of 32 patients with MM at the diagnosis or relapsed (ISS I-III, mean age±SD: 72±10), 55% of them with osteolytic bone lesions and 10 patients with monoclonal gammopathy of uncertain significance (MGUS) (mean age± SD: 71±14). Ten sex-age matched healthy subjects without hematological malignancies or metabolic bone disease were also analyzed. Consistently with our in vitro observations, we found a significant reduction in the percentage of viable osteocytes with an increase of the number of death osteocytes/empty lacunae in MM patients as compared to healthy subjects (p=0.003) but not MGUS (p=0.4). Under Light Microscopy and TUNEL analysis we demonstrated that osteocyte cell death in MM patients was due, at least in part, to an increase of the osteocyte apoptosis. As regard the skeletal involvement in MM patients we showed significant lower percentage of viable osteocytes (p=0.05) and higher amount of death osteocytes and empty lacunae in osteolytic vs. non-osteolytic MM patients (p=0.05). Finally a significant negative correlation was observed in MM patients between the amount of viable osteocytes and that of osteoclasts [Spearman (r): -0.33, p=0.04].

Overall our data suggest a critical role of osteocytes in MM-induced osteolysis in MM patients showing that the interaction of MM cells with osteocytes trigger osteoclastogenesis through the increase of osteocyte apoptosis and the production of pro-osteoclastogenic cytokines.