Compartment-Specific Bioluminescence Imaging Platform for the Open-Ended Identification of Novel Immunomodulatory Agents and High-Throughput Evaluation of Anti-Tumor Immune Function

Abstract 451

Given the heterogeneity of human cancers, to comprehensively interrogate the anti-tumor cytotoxicity of immune effector cells for the identification of novel treatments, high-throughput platforms are vital for screening the multitude of cell types and experimental conditions and drugs. Conventional assays for anti-tumor immune effector cell activity have limited high-throughput scalability (e.g. use of radioactivity for Cr release) or measure indirect markers (e.g. IFN-γ levels, LDH release). In addition, conventional cell viability assays (e.g. MTT, Alamar Blue, CellTiterGlo) are suboptimal because the background viability signal from immune cells confounds the selective readout of tumor cell viability. To address these limitations, we adapted the recently developed Compartment-Specific Bioluminescence Imaging (CS-BLI) technique (McMillin et al, Nature Med 2010) to study tumor-immune system interactions. We first applied CS-BLI to quantify the anti-tumor activity of IL-2 pre-stimulated PBMCs on luciferase (Luc)-expressing MM.1S multiple myeloma (MM) cells or KU812F leukemic cells. Immediate CS-BLI bioluminescence readout showed significant linear correlation with viable tumor cell number, both in the presence (r=0.9996, p<0.01) and absence (r=0.9994, p=0.01) of Luc-negative PBMCs. In 4hr co-cultures at increasing PBMC-to-tumor cell ratios were concordant with results obtained by Cr release assays under the same experimental conditions. Consistent with prior studies, CS-BLI confirmed that the anti-MM cytotoxicity of IL-2-stimulated (10 ng/mL, 24 hrs) healthy donor PBMCs was enhanced by PBMC pretreatment with lenalidomide (Len) or pomalidomide (Pom) (p<0.0001 for both compounds; 2-way ANOVA); or by independent pretreatment of both PBMCs and MM cells with either compound (p<0.0001 for both compounds). In contrast, Dex pretreatment of PBMCs attenuated their anti-MM activity (p=0.018). MM cytotoxicity was decreased by depletion of CD4+ (p<0.0001), CD8+ (p<0.0001), or CD56+ cells (p<0.0001), but the decrease was more pronounced after CD56+ cell depletion. Conversely, positively selected CD56+ cells had significantly higher anti-tumor activity than unselected PBMCs (p<0.0001). The tumor-selective nature of CS-BLI measurements allowed the evaluation the effect of stromal cells on the anti-tumor immune effector activity. Luc+ MM.1S target cells were co-cultured with IL-2-stimulated PBMCs from MM patients, both in the presence and absence of autologous bone marrow stromal cells (BMSCs). BMSCs from these matched donors significantly decrease the anti-tumor activity of IL-2-stimulated PBMCs over 4hrs. Len and Pom enhanced, while Dex decreased, the anti-tumor effect in the presence of stromal cells. We next utilized the CS-BLI platform to screen in an open-ended manner a library of compounds for immunostimulatory properties against MM cells. Compounds of an NCI drug library were pin-transferred (1μM final conc.) to plates with Luc+ MM.1S cells cultured alone or with IL-2-prestimulated PBMCs and tumor cell viability accessed. This screening allowed us to identify candidate immunomodulators, one of which, the depsipeptide Didemnin B, showed direct anti-tumor activity in the absence of PBMCs, but also enhanced the anti-MM cytotoxicity of PBMCs (or the NK cell line NKL). CS-BLI allows high-throughput screening of compounds not only on the basis of their direct cytotoxic activity in co-cultures, but also their ability to enhance anti-tumor immune responses. CS-BLI can be applied not only in oncology, but also other settings where agents with immuno-stimulatory (i.e. infection) or immunosuppressive (i.e. inflammation/autoimmunity and transplant immunology) properties are important. Our results therefore provide the framework to identify and validate novel pharmacologically or biologically active agents based on their ability to modulate the activity of immune effector cells. CS-BLI allows for rapid and specific evaluation of such agents in multiple conditions, including co-cultures with stromal cells. CS-BLI is a novel tool that can accelerate the identification and prioritization of novel immunomodulatory agents for clinical development