The absence of RANK expression in OS tumor

The absence of RANK expression in OS tumor cells observed in the present study is in marked contrast to the two reports that RANK is highly expressed in the majority of primary human OS [20,22]. Mori et al. (2007) reported RANK expression to be homogeneously expressed (in 100% of tumor cells) in 57% of OS biopsy specimenss and Bago-Horvath [22] reported moderate or strongly positive RANK expression in 69% of OS tumor samples. Given that the IHC scoring systems used in all studies were not normalized, one cannot make an unbiased comparison of RANK expression incidence and intensity levels observed in those two studies with the current study. However, it is important to point out distinct features of the present study with the two previously published studies of RANK expression in human OS to address the very high incidence observed in those studies. Firstly, both previously published reports relied on commercially available anti-RANK Epigenetics Compound Library and did not provide any evidence for the specificity and reproducibility of staining for IHC applications nor confirmation of RANK expression using independent methods or reagents. Secondly, the use of mAbs for expression analysis in the present study avoids the technical pitfalls of run-to-run reproducibility inherent in polyclonal antibodies. Thirdly, neither previously-published study documented the expected RANK staining within normal myeloid cells in the tumor mass or at the bone/tumor interface within any OS sample to document RANK expression in the expected cell types and provide context for the signal to noise for the reagents and methods used. The validation of the RANK antibodies used for IHC demonstrated concordant positive (and negative) signals between multiple independent methodologies and addressed sensitivity down to fewer than 1800 receptors/cell. Furthermore, in our analysis of OS samples, two independent anti-RANK mAbs, each recognizing distinct epitope binding sites demonstrated an identical positive and negative staining pattern, essentially cross-validating one another. Different alternatively-spliced variants of the human RANK gene have been described which alter exons 7, 8, or 9 encoding in either the transmembrane or cytoplasmic regions of RANK [32], potentially impacting IHC detection. However, the antibodies N-1H8 and N-2B10 bind to the extracellular portion of RANK common to each variant, thus the IHC results reported here would be inclusive of any rare, alternatively-spliced forms. Altogether, these specificity and sensitivity controls, along with the observed expression of RANK in the expected osteoclasts and related cells, substantiates the observation that OS tumor cells do not express RANK to any major degree. It would seem likely that technical limitations to these previously published studies may have accounted for the reportedly high incidence and high expression levels of RANK within OS tumors. Aside from distinct technical approaches used, the well-established RANKL and RANK expression patterns in the bone further substantiates the expression patterns defined in the current study of OS. RANKL and RANK play major roles in bone metabolism due to the critical role of this pathway in osteoclastogenesis [4]. That is, RANK is expressed with the hematopoietic myeloid compartment contributing to myeloid-derived osteoclasts and their precursors consistent with the observed compartmentalization of RANK expression within giant cells, osteoclasts and osteoclast precursors found within OS samples in the present study. Given that OS tumors may arise from a mesenchymal-osteoblast origin [1,2], it is unlikely that RANK would be expressed in the tumor cells while expression of RANKL in OS tumor cells is perhaps not surprising. In normal and pathologic bone, RANKL expression is confined to the cells of the osteoblast lineage, including osteocytes and has also been observed in certain tumor cells [33]. The observation herein of RANKL within reactive bone stroma in OS tumors as well as many OS cells themselves suggests that RANKL may stimulate osteoclast differentiation and activation potentially via multiple sources. Molyneux et al. [35] has reported that human and mouse OS with reduced expression of the gene encoding the regulatory subunit α (RIa) of PKA have high RANKL levels suggesting that pathways regulating RANKL expression may be dysfunctional in some OS. For any RANKL-positive OS, it remains unclear why RANKL is not expressed uniformly in all OS tumor cells as observed in the present study, suggesting that perhaps some element of local regulation observed in normal osteoblast-lineage cells (e.g. responsiveness to PTH1r) is retained by OS tumor cells. In mouse models of OS, pharmacologic inhibition of RANKL is protective against bone destruction and also decreases tumor burden [18–20]. This is likely related to osteoclast inhibition, as treatment with bisphosphonates lead to essentially similar responses [21]. The reduction in skeletal tumor burden observed with osteoclast inhibitors results from interruption of the vicious cycle in which decreased osteoclastic bone resorption and subsequent reduction in localized bone matrix and growth factors indirectly reduces tumor growth and survival. These pharmacology observations demonstrating similar anti-tumor activity of bisphosphonates and RANKL inhibitors are consistent with the absence of RANK on OS tumor cells and a contribution of RANK-positive osteoclasts associated with OS tumors (this study and Avnet et al. [10]) to the bone pathologies observed in OS and potential indirect feedback to the skeletal tumor. The observation that RANKL is expressed in OS cells themselves suggests that these tumors may mediate an osteoclastic response independently of (or in addition to) RANKL within the normal or tumor-reactive bone stroma. However, the absence of RANK expression in OS tumor cells indicates that an autocrine RANKL/RANK response in human OS tumor cells is unlikely to be operative. While anti-RANKL therapy may influence the bone microenvironment and may be protective against bone pathologies in OS, the lack of RANK expression in tumor cells suggests that this approach would not directly affect the tumor.