Compartment-Specific Bioluminescence Imaging (CS-BLI) High- Throughput Assays Provide Comparative Insights into the Impact of Osteoclasts Vs. Stromal Cells on Activity of Anti-Myeloma Therapeutics

Introduction: Osteoclasts (OCLs) have a central role in the pathophysiology of multiple myeloma (MM), because their excessive and unopposed activation leads to the characteristic bone lesions of this disease. However, unlike the well documented role of bone marrow stromal cells (BMSCs) in conferring resistance to diverse conventional anti- MM agents, the impact of OCLs in drug responsiveness of MM cells has not been studied extensively. This is due in part to limitations of conventional assays which are either not amenable to co-culture studies or not conducive to comprehensive high-throughput studies.

Methods/Results: To address this void in the MM field, we applied compartment specific bioluminescence imaging (CS-BLI) in vitro assay in the context of MM-osteoclast co-cultures. Specifically, in these co-cultures the MM cell compartment is engineered to stably express luciferase (Luc) and is co-cultured with Luc-negative primary mature osteoclasts generated after ex vivo differentiation of peripheral blood monocytes with MCSF and RANKL. In these co-cultures, the activity of luciferase in response to addition of substrate results in bioluminescence signal which is directly proportional to number of viable Luc+ cells only, thus allowing for selective and sensitive quantification of the viable MM tumor cell compartment. By applying the CS-BLI platform in the context of MM-OCL co-cultures and comparing them with results of MM-BMSC co-cultures, we observed that mature OCLs stimulate increase in the number of viable MM cells from stroma-responsive, but not in stroma-unresponsive, cell lines. Furthermore, stroma/OCL-responsive MM cell lines that are sensitive to glucocorticoids and anthracyclines (e.g. MM-1S) exhibit significant decrease in their response to these conventional anti-MM agents when co-cultured with OCLs, similar to the resistance conferred to these agents by BMSCs. In addition, novel anti-MM agents, such as the proteasome inhibitor bortezomib and hsp90 inhibitors, were equally active against MM cells in the presence vs. absence of OCLs at concentrations and durations of drug treatment that did not significantly affect the viability of OCLs, consistent with results obtained with similar co-cultures of MM cells with BMSCs. These results confirm that BMSCs are not the only cellular compartment of the BM microenvironment that can function to support MM cell proliferation, survival and drug resistance, and that OCLs can also function in that accessory role as well.

Conclusions: CS-BLI addresses key limitations that have precluded the highthroughput testing of novel agents in tumor-OCL co-cultures. Its comparative application in co-cultures of MM cells with OCL vs. BMSCs provides an opportunity to compare qualitatively and quantitatively the similarities or differences in patterns of drug resistance conferred by these accessory cell compartments. It also offers a powerful tool to identify new and, hopefully, more effective classes of drugs which are active in MM despite the effects of the BM microenvironment.