Objective To determine the signaling axis of β2-M-pCREB-AR and the expression of RANKL inPCa and bone stromal cells in clinical primary and bone metastatic PCa (PCa-met) tissuespecimens, by using multiplexed quantum dots immunohistochemistry (mQD-IHC).Materials and Methods To establish the feasibility of mQD-IHC staining protocol (Xing, et al.Nature Protocol, 2007), we focused our analysis on primary and bone metastatic clinical PCatissue specimens at the single cell level. We compared the expression of β2-M, pCREB, and AR inbenign, prostatic intraepithelial neoplasia (PIN), and cancerous regions of the primary PCa andRANKL expression in bone PCa-met. The status of EMT in the bone PGa-met cells wasdetermined by the expression of an epithelial cell adhesion protein (EpCAM) and vimentin(VM).