A previous study from our

A previous study from our group demonstrated that the truncated form of OPG (1-194) has greater activity than the complete form, given that proteoglycans present in the bone tumor microenvironment may bind to full length OPG, thereby limiting its bioavailability and bioactivity [31]. Therefore the truncated form of OPG was used in the present study. The transgene overexpression was confirmed both at the systemic and local level of production. Microscanner analysis confirmed the OPG biological activity at the bone level, by prevention of osteolytic lesions and preservation of cortical bone structure at the tumor-bone interface. In addition, inhibition of tumor progression had been observed in all the series studied, both in models induced by transplantation of tumor fragments or by tumor cell injection. In all cases, the inhibition of the mean tumor volume was not significant (between 20% and 30% inhibition), probably due to the heterogeneity of the model but when considering each animal taken individually, there is a clear tendency to inhibition by OPG treatment either on incidence or progression levels. Because OPG has no direct effect on ES cell proliferation, OPG induced diminution of tumor growth could be explained by an indirect inhibitory effect on RANKL mediated bone resorption. First, OPG is also able to bind another member of the TNF family, the TNF Related Apoptosis Inducing Ligand (TRAIL, TNFSF10)[45]. Several in vitro studies have even suggested that OPG could represent a protumoral factor for cancer cells, by inhibiting the pro-apoptosis activity of TRAIL [24–26]. This is why we searched for TRAIL expression and production in the ES microenvironment. We demonstrated that TRAIL is indeed detected in xenograft ES tumors induced by injection of corresponding human cells, but the ES sodium salt themselves do not express TRAIL as evidenced in 8ES cell lines out of 10. TRAIL protein level was also analyzed by immuno-histochemistry in human ES biopsies. Results showed that this cytokine is indeed present in the ES microenvironment, but mainly produced by immune cells rather than Es tumor cells. Therefore, TRAIL present in the microenvironment could interfere with OPG to modulate OPG inhibitory effect on RANKL activity [46]. In the same time, OPG expression was also studied, both in the ES cell lines and in human ES biopsies. We showed that ES cell lines do not express OPG at all, this result being contradictory with those of Taylor et al. [10]. No OPG expression could be also observed in patient biopsies. Therefore, another way to block RANKL activity should be proposed such as RANK-Fc or the humanized antibody anti-RANKL Denosumab [47]. However, because this fully humanized antibody cannot be used in preclinical studies in mice, we decided to test the soluble receptor OPG. Denosumab is able to specifically bind to RANKL blocking its activity, without any interference with other members of the TNF superfamily such as TRAIL or FasL [48]. At the clinical level, the use of Denosumab is already proposed for patients with malignancy associated osteolysis [49]. We could therefore propose to combine Denosumab with chemotherapy in the case of primary bone tumors, including Ewing\'s sarcoma. Another hypothesis is that osteoclast activation is not only driven by RANKL, but also by other cytokines such as TNF-α or M-CSF (Macrophage-Colony Stimulating Factor). Indeed, previous data reported that macrophages present in the ES tumor stroma microenvironment could differentiate into osteoclasts in the presence of M-CSF and TNF-α [50]. In complement, the presence of both cytokines in the microenvironment of ES xenograft models was evidenced by immuno-fluorescence and ELISA (not shown). Therefore, a combined treatment could be proposed, associating an anti-RANKL strategy with anti-TNF and anti-M-CSF ones.
Conclusions
Conflict of Interest statement
Acknowledgments This work was supported by a grant from Novartis Pharma (Rueil-Malmaison, France), by the “Fédération Nationale Enfants et Santé”, the “Société Française de lutte contre les Cancers et les leucémies de l\'Enfant et de l’Adolescent” and the “Institut National du Cancer” (Grant number R09018NN). The authors wish to thank G. Hamery and Y. Allain for their kind assistance at the animal facility care platform (Faculté de Médecine, Nantes, France) and J. Taurelle for his kind technical assistance (INSERM UMR957).