Cellular and humoral mechanisms of osteoclast formation in Ewing's sarcoma

Abstract

Cellular mechanisms that account for tumour osteolysis associated with Ewing's sarcoma are uncertain. Osteoclasts are marrowderived multinucleated cells (MNCs) that effect tumour osteolysis. Osteoclasts are known to form from macrophages by both receptor activator for nuclear factor-kB (RANK) ligand (RANKL)-dependent and -independent mechanisms. In this study, our aim has been to determine whether tumour-associated macrophages (TAMs) isolated from Ewing's sarcoma are capable of differentiating into osteoclasts and to characterise the cellular and humoral mechanisms whereby this occurs. Tumour-associated macrophages were isolated from two Ewing's sarcomas and cultured on both coverslips and dentine slices for up to 21 days with soluble RANKL and macrophage colony stimulating factor (M-CSF). Osteoclast formation from TAMs (CD14 þ ) was evidenced by the formation of tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR)-positive MNCs, which were capable of carrying out lacunar resorption. This osteoclast formation was inhibited by the addition of bisphosphonates. Both Ewing's sarcoma-derived fibroblasts and some bone stromal cells expressed RANKL and supported osteoclast formation by a contact-dependent mechanism. We also found that osteoclast differentiation occurred when Ewing's TAMs were cultured with tumour necrosis factor (TNF)-a in the presence of M-CSF and that TC71 Ewing's sarcoma cells stimulated osteoclast formation through the release of a soluble factor, the action of which was abolished by an antibody to TNF-a. These results indicate that TAMs in Ewing's sarcoma are capable of osteoclast differentiation by both RANKL-dependent and TNF-a-dependent mechanisms and that Ewing's sarcoma cells produce osteoclastogenic factor(s). Our findings suggest that anti-resorptive and anti-osteoclastogenic therapies may be useful in inhibiting the osteolysis of Ewing's sarcoma.

Zoledronic Acid as a New Adjuvant Therapeutic Strategy for Ewing's Sarcoma Patients

“…Because osteosarcoma and ES cells are metabolically active bone cells (17,18), studies with bisphosphonates and more specifically nitrogen-containing bisphosphonates have shown selective uptake and "poisoning" of these cells (19)(20)(21)(22)(23). We have previously shown that zoledronic acid is able to limit tumor progression in a rat model of osteosarcoma, to prevent tumor relapse compared with chemotherapy alone and to prevent osteolytic lesions (10).…”

Osteoclast-like cells in soft tissue leiomyosarcomas

“…Single-strand complementary DNA (cDNA) was synthesized from 2.0 µg of total RNA using the SuperScript® First-Strand Synthesis System for semiquantitative reverse transcription polymerase chain reaction (RT-PCR; Invitrogen, Paisley, UK). cDNA was amplified by PCR using specific primers to RANKL, OPG, and TRAIL with GAPDH control as previously described [24,25]. PCR products were fractionated on 1% agarose gels, and gel pictures were scanned with Alpha-Imager 2200 (Alpha Innotech Corporation, USA).…”

Analysis of stromal cells in osteofibrous dysplasia and adamantinoma of long bones

“…This finding suggests that osteolysis in adamantinoma of long bones and osteofibrous dysplasia most likely occurs by RANKL-expressing mesenchymal stromal cells inducing macrophages in osteofibrous dysplasia and adamantinoma of long bones to undergo osteoclast differentiation, as occurs in other primary bone tumours. 15,28,29 There are close similarities in tumour location and radiological features between osteofibrous dysplasia and adamantinoma of long bones. Our findings show that there are similarities and differences in stromal cell expression of epithelial and osteoblast markers in these lesions.…”