Xenograft and Intra-bone Myeloma Models

Animal studies were approved by the Committee on Animal Research and Ethics of Tianjin Medical University, and all protocols conformed to the Guidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Research. 4–6 weeks old female NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice were used to establish the xenograft and intra-bone injection models as previously reported^{38 (link),39} . The mice were bred and maintained under specific pathogen-free conditions at the animal facilities of Tianjin Medical University. These mice were maintained in 12 h light/dark cycle, and the housing temperature and humidity were 21.5–24.5 °C and 45–65%, respectively. Mice were given standard laboratory chow diet and water ad libitum. For Xenograft model, MM cells (3 × 10⁶ cells/mouse) were injected subcutaneously into NSG mice. After 3 weeks, mice were treated with BTZ (0.5 mg/kg) (n = 12) every three days or BTZ + Romidepsin (1 mg/kg) (n = 12) every two days. Mice were weighted and tumors were measured every 3 days. After treatment 24 days, for xenografts experiments, mice were sacrificed and the tumor xenografts were collected for immunohistochemistry (IHC) and apoptosis analysis. The tumor volumes of all tumor-bearing mice involved in this study were controlled within 2500 mm³, which is in accordance with the permission from the ethics committee of Tianjin Medical University. The maximal tumor size/burden was not exceeded. For intra-bone injection experiments, BR MM.1 S (5 × 10⁵/mouse) were injected into the femurs of NSD mice. Mice without myeloma cells served as controls (No MM). The frequency and dose of drug injection in mice are consistent with the subcutaneous experiment. Mice femur were subjected to microCT scan with a Skyscan 1172 microtomograph, and mouse femurs were subjected to histological evaluations, where shows representative microCT reconstructions of mouse femurs to show osteolytic lesion area and less cortical perforations.