Increased Osteocyte Apoptosis in Multiple Myeloma Patients: A Potential Role in Bone Remodeling Alterations Related to Osteolytic Bone Lesions.
Authors: Giuliani Nicola , Ferretti Marzia , Abeltino Manuela , Mancini Cristina , Martella Eugenia , Palma Benedetta Dalla , Bolzoni Marina , Sgobba Valentina , Storti Paola , Rizzoli Vittorio , Palumbo Carla 
Published: October 2019

Abstract
Abstract 830
Osteocytes are non-proliferative differentiated cells of osteogenic lineage located in the bony lacuna/canalicular system of bone and that have been recently hypothesized to regulate local bone remodeling in part through the cell death and apoptosis. A reduction of osteocyte viability has been demonstrated in osteoporotic bone related to estrogen deficiency and glucorticoid administration. As known, multiple myeloma (MM)–induced osteolysis and/or osteoporosis are characterized by severely imbalanced and uncoupled bone remodeling due to increased osteoclast recruitment and suppressed osteoblast differentiation that occur in close contact with MM cells infiltration. Actually, the potential involvement of osteocytes in bone remodeling alterations occurring in MM patients is not known and it has been investigated in the present study.
Firstly we performed histological analysis on bone biopsies obtained from iliac crest in a cohort of 34 patients with MM at the diagnosis (ISS I-III, mean age±SD: 72±10), 52% of them with osteolytic bone lesions at the skeletal survey and 10 patients with monoclonal gammopathy of uncertain significance (MGUS) (mean age± SD: 71±14). Sex-aged matched subjects without hematological malignancies or osteoporosis or metabolic bone disease (n°=10) were also analyzed. Histological analyses were performed on Toluidine blu and Gomori's three-chromic stained sections observed under a Light Microscope (LM). On a total of 500 osteocyte lacunae X section we evaluated both the number of viable osteocytes and the number of dead/degenerated/apoptotic osteocytes together with the number of empty lacunae. We found a significant reduction in the percentage of viable osteocytes in MM patients as compared to healthy controls (% of viable osteocytes: mean ± SD: 25±10% vs. 36±9%; p= 0.003) whereas the difference between MM and MGUS did not reach a statistical significance (25±10% vs. 30.5±16%; p= 0.2). Consistently the number of death osteocytes and empty lacunae was significantly increased in MM vs. healthy subjects (mean ± SD/500 lacunae: 371.3± 52 vs. 315±46; p=0.006) but not as compared to MGUS (371.3±52 vs 337±81; p=0.2). As regard the skeletal involvement in MM patients we found that the percent of viable osteocytes was significantly lower in osteolytic vs. non-osteolytic patients (19±9% vs. 30±7%; p= 0.01) as well as the number of death osteocytes and empty lacunae was higher in osteolytic vs. non-osteolytic patients (mean ± SD/500 lacunae: 394±41 vs. 315±55; p=0.04). Following, in order to verify the occurrence of apoptosis both LM and Transmission Electron Microscopy (TEM) observations were also performed. Under LM, TUNEL analysis showed higher presence of apoptotic cells in those specimens obtained from MM patients in comparison with those obtained from healthy controls. Moreover an increase in the number of apoptotic osteocytes was observed in MM with bone lesions as compared to those without osteolysis. These observations were further confirmed in vitro by ultrastructural analysis on mono-layers of human preosteocyte cells (HOB-01-C1) incubated with or without conditioned media (CM) taken from human myeloma cell lines (HMCLs) KMS12 and JJN3 or co-cultured with them. TEM observations, showed cells in apoptosis with apoptotic bodies and degenerated non-apoptotic cells in preosteocytes treated with HMCL-CM or co-cultured with HMCLs as compared to non-treated cells. Interestingly, we also found that CM of preosteocytes co-cultured with HMCLs but not those of non co-cultured cells significantly increased CD14+ -derived osteoclastogenesis evaluated by tartrate-resistant acid phosphatase (TRAP) staining and pit-forming assay.
In conclusion our data demonstrate that MM bone is characterized by a reduction of viable osteocytes and an increase of osteocyte apoptosis in relation to the presence of bone lesions that may represent a triggering event to the increase of osteoclast recruitment in MM patients.

Disclosures:
No relevant conflicts of interest to declare.