2.12. Orthotopic Murine Model of Metastatic OS

K7M2 murine osteosarcoma (OS) tumor cells (ATCC CRL-2836, ATCC, Manassas, VA) were donated by Dr. Kurt Weiss, MD (University of Pittsburgh, Pittsburgh, PA). Cells were transfected with the Promega (Madison, WI) luc2 reporter vector (luc-K7M2) as previously described (14) to allow for bioluminescent imaging of disease progression. Cells were determined to be mycoplasma free by polymerase chain reaction (PCR) and their identity confirmed via IDEXX BioResearch Case #6926–2018 (ID 3). For orthotopic inoculation, luc-K7M2 cells (1 × 10⁶) were injected intratibially into 15 male and female mice as previously described (30). Four weeks later, 12 of the 30 mice with the most similarly sized In Vivo Imaging System (IVIS)-confirmed primary tumors were randomized into three log-scale dose treatment groups. One week later, mice underwent survival surgical amputation of their tumor-bearing limbs. At 12 weeks post-inoculation, mice were humanely euthanized via cervical dislocation under anesthesia.