Multi-Modality Imaging of Multiple Myeloma Bone Disease In Orthotopic Xenograft Models

Abstract 5014

Pre-clinical evaluation of novel therapies for MM is an essential investigational aspect often challenged by the limited availability of animal models of diffuse disease that allow rapid appraisal of tumor progression. An ideal model requires an accurate reproduction of the clinical features of MM coupled to simple metrics for gauging tumor progression. Taking advantage of enhanced molecular optical reporters and micro-computed tomography (μCT), we established orthotopic xenograft models of diffuse MM that satisfy these conditions. To such end, we labeled two well-characterized human MM cell lines, JJN-3 and RPMI-8226, with an enhanced firefly luciferase and mCherry before implantation into immunocompromised NOD/SCID/IL2rγ^-/- mice. Intravenous injection of 1×10⁶ dual-labeled JJN-3 or RPMI-8226 cells resulted in their orthotopic engraftment and the generation of models of diffuse MM that reproduce the pathologic features of human disease, including homing of tumor cells to the bone marrow, development of osteolytic lesions, paraproteinemia, and physical manifestations including cachexia and paralysis. Interestingly, the two cell lines generated models of diffuse disease characterized by differentially aggressive growth. JJN-3 cells caused hind limb paralysis in all animals within ~4 weeks following implantation, whereas less-aggressive RPMI-8226 cells caused the same symptoms within ~6-7 weeks. The severity of the symptoms paralleled the growth characteristics of each cell line in vitro, whereby JJN-3 cells exhibited a shorter doubling time (20-24 hours) than RPMI-8226 cells (36-42 hours). High-sensitivity, real-time bioluminescence imaging (BLI) of live tumor-bearing hosts revealed MM cells in the axial skeleton (calvariae, thoracic and lumbar vertebrae, sterna), pelves and hind limbs as early as one week (JJN-3) or two weeks (RPMI-8226) after cell injection. BLI also revealed exponential tumor growth in both models, which mirrored the accumulation of secreted human paraproteins in the plasma of the hosts (~30 mg/L of kappa light chains in animals implanted with JJN-3 cells or ~15 mg/L of lambda light chains in animals implanted with RPMI-8226 cells). To further pinpoint the anatomical localization of the MM cells we examined tumor dissemination by fluorescence imaging (FLI) during necropsy. FLI analyses indicated pervasive involvement of calvariae, thoracic and lumbar vertebrae, sterna and ribs, pelves, tibiae, and femora, corroborating data obtained by BLI. Since MM remains the most common adult cancer with skeletal involvement and given that the majority of the complications in advanced disease stem from lytic bone lesions, we examined the extent of osteolysis associated with disease progression in our models by μCT. μCT revealed pronounced osteolytic lesions in both models which corresponded with the same anatomical sites observed by BLI and FLI. Widespread lytic bone lesions were readily observed in animals bearing JJN-3-derived tumors whereas animals implanted with RPMI-8226 cells developed less prominent osteolysis, consistent with a less destructive disease progression. Histopathology analyses further validated tumor homing to the bone marrow where we observed complete marrow replacement (JJN-3) or discrete medullary tumors (RPMI-8226), once more consistent with the aggressiveness of each model. Lastly, drug efficacy studies using bortezomib verified the preclinical value of our models. Animals engrafted with either JJN-3 or RPMI-8226 cells showed a response to bortezomib as early as four weeks after cell injection (2 weeks after initiation of treatment), and tumor progression was delayed in a dose-dependent manner in animals receiving bortezomib by up to 3.6-fold (JJN-3) and 15.3-fold (RPMI-8226) by the end of the experiment (5 or 7 weeks for JJN-3, and RPMI-8226, respectively). Collectively, our data indicate that our models accurately reproduce the pathological features of human MM, and coupled to our optimized multi-pronged imaging approach, they constitute powerful tools for assessment and validation of novel anti-MM therapies.