Ibandronate therapy has a significant effect on reducing tumor-induced bone destruction at day 10 and day 14 post-tumor injection as assessed by high-resolution faxitron analysis. Sham animals, (A) show no radiographically apparent bone destruction at day 10, whereas sarcoma + vehicle (B) animals show a transition from the radio-opaque bone tissue to a radio-lucent appearance by day 10. Sarcoma + ibandronate (C) animals present a markedly reduced pattern and extent of bone destruction as sarcoma + vehicle animals. Sham-injected mouse femurs (F) present a net maintenance of trabecular bone while 2472 sarcoma-injected femurs (G) display osteolytic resorption of trabecular structure at the distal diaphysis of the femur when analyzed with microCT imaging at day 10 post-tumor injection. Treatment with ibandronate significantly attenuates the loss of trabecular bone when viewed with microCT imaging (G) at day 10 post-tumor injection. Scale bars: A–H; .4mm. AU: .4 OR 4?

Neurochemical changes associated with central sensitization are attenuated by administration of ibandronate. Representative confocal images of c-Fos expressing neurons of the spinal cord in sarcoma + vehicle (A) and sarcoma + ibandronate (B) mice. Following a normally non-noxious palpation of tumor-bearing limbs, sarcoma + vehicle mice showed an increased expression of c-Fos protein in neurons within the deep laminae. In sarcoma animals that received ibandronate therapy, there was a significant reduction of this increased expression of c-Fos protein. Representative confocal images of prodynorphin expression (DYN) in the dorsal horn of the spinal cord in sarcoma + vehicle (C) and sarcoma + ibandronate (D) mice. Sarcoma + vehicle mice displayed an increase in prodynorphin-IR neurons in deep laminae of the ipsilateral spinal cord (C), whereas ibandronate therapy significantly attenuated the increase in prodynorphin expression (D). Scale bar: A, B 150mcm; insets in C,D 200mcm.

Ibandronate therapy attenuates bone cancer pain-related behaviors. Ibandronate treatment (100 µg/kg at days 7, 8 and 9 post sham or sarcoma injection, IV) attenuated both ongoing and movement-evoked bone cancer pain behaviors throughout the progression of the disease. The time spent guarding and number of spontaneous flinches of the sarcoma injected limb over a 2-minute observation period was used as a measure of ongoing pain. This standardized 2-minute observation of the animals behavior was performed at 7, 8, 9, 10, 12 and 14 days post sham or sarcoma injection (A,B). Parameters of movement-evoked pain included quantification of time spent guarding and the number of flinches over a 2-minute observation period following a normally non-noxious palpation of the sham or sarcoma-injected femur (C, D). Note that ibandronate treatment (large triangle at days 7, 8 and 9) post-tumor injection (closed square) significantly reduced ongoing and palpation-evoked pain behaviors on days 8, 9, 10, 12 and 14 as compared to sarcoma + vehicle (open square). Acute morphine sulfate (small open triangle; 10 mg/kg subcutaneously) also significantly reduced both ongoing and movement-evoked guarding and flinching behaviors at day 10 post-tumor injection (Figure 1A, B), as compared to sarcoma + vehicle mice. At 7, 8, 9, 10, 12 and 14 days post injection, sham + vehicle (open circle) are significantly different from sarcoma + vehicle. Note that a one-time single dose (300µg/kg IV) was as effective as a loading dose in reducing ongoing (E) and movement-evoked (F) guarding behavior. Sarcoma + ibandronate was significantly different from sham + vehicle for ongoing and movement-evoked pain behaviors at all time points. Error bars represent S.E.M. * = P<0.05 vs. sarcoma + vehicle; + = P<0.05 vs. sham + vehicle.

Hematoxylin and eosin-stained histological comparison of tumor burden and necrosis in sham, sarcoma + vehicle and sarcoma + ibandronate treated femurs day 14 post-tumor injection. Sham-injected mouse femurs (A) present neither visibly detectable newly formed bone or bone destruction when assessed after hematoxylin and eosin staining (H&E). 2472 sarcomainjected femurs display primarily osteolytic characteristics and replacement of the intramedullary space with tumor cells and limited necrosis (B) where tumor cell apoptosis has occurred. Sarcoma + ibandronate femurs present complete replacement of the intramedullary space with tumor cells and extensive necrosis of the tumor mass at day 14 post-injection. High power images of sham (D), sarcoma + vehicle (E) and sarcoma + ibandronate (F) intramedullary space. A–C; Scale bar = 4mm, D–F; Scale bar = 0.25mm. T= tumor; H= hematopoeitic cells; N= necrosis.