Metastatic bone cancer is the most common cause of pain in patients with malignant tumors. Prolonged opioid treatment remains the primary method to treat pain in these patients. Sustained morphine exposure enhances both bone cancer-induced pain and bone loss in mice implanted with sarcoma cells. Sustained treatment of bone marrow cultures with morphine results in COX-2 dependent upregulation of RANKL and PGE2, and suppression of OPG. This results in increased osteoclastogenesis which was dependent on COX-2 and OPG/RANKL regulatory axis. Treatment with morphine does not induce any direct changes in osteoclasts or sarcoma cells. The in vitro data was validated in the animals where morphine induces an increase in the osteoclastogenesis and RANKL, and suppresses OPG. These data indicate that morphine enhances osteoclastogenesis by modulating the OPG/RANKL regulatory axis in osteoblasts through a COX-2 dependent mechanism.Prolonged opioid exposure induces an opioid-receptor dependent hyperalgesia in humans and in animals. Studying the direct effect of opioids on primary sensory neurons we demonstrate a modest increase in CGRP cellular content that was not opioid-receptor dependent. Although dynorphin A (2-13) and PGE2 enhanced the release of the neuropeptide, pretreatment with opioids does not influence the capsaicin or KCl evoked CGRP release. These date indicate that the neurochemical changes seen in vivo may be dependent on factors upregulated in the periphery and/or the CNS.It has been demonstrated that sensory neurons innervating the femur express markers of neuronal injury and the intramedullary region of the femur becomes devoid of nerve fibers as the tumor expands. In this study we demonstrate that the sarcoma cells generate high levels of ROS and release hydrogen peroxide into the surrounding space, which induces death and injury to both sensory neurons and glia. This death was prevented by the anti-oxidants BHA and catalase. The present study provides evidence that ROS released by cancer cells can directly lead to injury and death of sensory neurons. ROS induced injury may be one of the mechanism through which sensory neurons are injured in the murine bone cancer pain model.