By inhibiting osteoclast activity one possible option relies

By inhibiting osteoclast activity, one possible option relies to bisphosphonates, the synthetic analogs of pyrophosphate that induce osteoclast apoptosis, decrease osteoclast activation and function by inhibition of the mevalonate pathway [32]. Such therapeutic tools have been studied in primary bone tumor models [33,34] and are even used in the French clinical trial OS2006 for pediatric and adult osteosarcoma patients. However, these molecules exhibit secondary events especially renal toxicity and osteonecrosis of the jaw, and may interfere with bone growth when administered to young patients [35]. Therefore, another promising way to block osteoclast activation is to target RANKL, the pivotal cytokine that mediate osteoclast differentiation and activation [36]. Beyond physiological conditions, increased expression of RANKL has been observed in osteolytic malignancies, such as breast cancer and multiple myeloma [37–39]. A previous study demonstrated that the RANKL/OPG ratio was significantly increased in patients suffering from severe osteolysis associated with tumor or not [19]. Therefore, therapeutic strategies that used its decoy receptor OPG have emerged in osteolytic bone tumor pathologies. As an example, OPG was shown to inhibit cancer cell migration and bone metastasis through inhibition of the RANKL-induced effects in RANK-expressing cells from tumor origin [40]. OPG was also shown to inhibit tumor-induced osteoclastogenesis and bone tumor growth in osteopetrotic mice [41], to reduce bone cancer pain by the blockade of the ongoing osteoclast activity [42], to decrease the number and area of radiographically evident lytic bone lesions in a model of mouse colon adenocarcinoma [21], to exhibit beneficial effects in experimental models of myeloma [20,43] and to inhibit osteolytic lesions associated with prostate cancer [44]. In the present study, mice developing Ewing\'s sarcoma models were treated by OPG administered by non-viral gene therapy. In a first step, we confirmed that RANKL was indeed a good therapeutic target in ES, by analyzing its expression both in patients and also in the ES models used in the present study. RANKL expression was indeed found in ES microenvironment in both cases, confirming the results previously published by Taylor et al. in patient biopsies [10]. Moreover, we demonstrate in the present study that ES cells are directly producing RANKL. The availability of a xenograft model of ES enabled us to discriminate from human or murine origin of RANKL production. The glutathione peroxidase used were specific for each species and results clearly showed that the increased RANKL production in ES tumor model was due to direct synthesis by tumor cells. However, one interesting data is that some ES cell lines (such as TC-71) which express low levels of RANKL in vitro (Fig. 1A and B), show an elevated RANKL production when injected in the mouse and developing a tumor (Fig. 1C). Therefore, it suggests that during tumor development, the stroma may influence tumor cells to produce RANKL. These results constitute the rationale for the therapeutic use of OPG in ES. 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 cells 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.