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 marrow-derived multinucleated cells (MNCs) that effect tumour osteolysis. Osteoclasts are known to form from macrophages by both receptor activator for nuclear factor-kappaB (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+) 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)-alpha 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-alpha. These results indicate that TAMs in Ewing's sarcoma are capable of osteoclast differentiation by both RANKL-dependent and TNF-alpha-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.

Figure 1

(A) TRAP⁺ and (B) VNR⁺ MNCs formed after 14 days when Ewing's sarcoma-derived TAMs were cultured in the presence of RANKL and M-CSF. (C) Extensive lacunar resorption on a dentine slice after TAMs were cultured for 21 days in the presence of RANKL and M-CSF (Toluidine blue staining). (D) No resorption was seen on dentine in 21-day TAM cultures when RANKL was omitted (toluidine blue staining). Zoledronate-treated cultures showed a similar appearance. Bars=50 μm.

Figure 2

(A) TRAP⁺ and (B) VNR⁺ MNCs in a 14-day coculture of human PBMCs with Ewing's sarcoma-derived fibroblasts in the presence of M-CSF. (C) Lacunar resorption pits formed on a dentine slice in a 21-day coculture of human PBMCs with Ewing's sarcoma-derived fibroblasts in similar conditions. Bars=50 μm.

Figure 3

Expression of RANKL, OPG and TRAIL mRNA by fibroblasts derived from Ewing's sarcoma and TC71 cells. Reverse transcription-polymerase chain reaction products were fractionated on agarose gel. Lane 1, positive control; lane 2, negative control; lane 3, TC71; lanes 4–5, Ewing's sarcoma-derived fibroblasts from two patients; lanes 6–8, normal bone stromal cells.

Figure 4

(A) % surface area (SA) resorption formed in human PBMC cultures incubated with M-CSF and TC71 conditioned medium relative to positive control (PBMC cultures with M-CSF and RANKL). Error bars denote s.e.m. (n=3). (B)The effect of OPG, RANK: Fc and neutralising antibodies to TNF-α on resorption in PBMC cultures incubated with M-CSF and 10% TC71 conditioned medium. The data represent the mean % surface area (SA) lacunar resorption relative to the positive control (PBMC cultures with M-CSF and RANKL). Error bars denote s.e.m. (n=6).