Temporal Dynamics of Tumor-Microenvironment Interaction and Treatment Responses Revealed through Time-Lapse Compartment-Specific Bioluminescence Imaging: Translational implications
Authors: Dhimolea Eugen , King Emily , Kastritis Efstathios , McMillin Douglas W. , Delmore Jake E. , Negri Joseph M. , Jacobs Hannah M. , Hayes Catriona A. , Sheffer Michal , Hu Yiguo , Sarkar Subhashis , Groen Richard W.J. , Bariteau Megan A. , Aftab Blake T. , Mitsiades Constantine S. 
Published: November 2019

Abstract
Most conventional methods to sensitively quantify tumor cell proliferation and viability in vitro involve processing of cells in ways that preclude continuation of the respective experiment or prevent the longitudinal collection of data. This common technical feature of conventional assays limits their ability to provide detailed insight into the kinetics of tumor cell responses to treatment(s). Additionally, these limitations hinder the use of these assays to monitor how the kinetics of treatment response can be altered by nonmalignant "accessory" cells of the tumor microenvironment (e.g. bone marrow stromal cells [BMSCs] for hematologic malignancies or bone metastases of solid tumors). To address these obstacles, we modified our previously developed tumor cell compartment-specific bioluminescence imaging (CS-BLI) platform (McMillin et al. Nat Med. 2010), to enable longitudinal assessment of tumor cell response to diverse experimental conditions; we cultured luciferase-expressing tumor cells, with or without stromal cells, in the presence of bioluminescent substrates, using optimized conditions which provide detectable bioluminescent signal even after several days of culture, while having no adverse effect on the viability of tumor or non-malignant cells in this system. This modified approach (time-lapse CSBLI, [TL-CSBLI]) preserved the linear correlation of bioluminescent signal with tumor cell viability. Furthermore, results obtained at the end of the experiment and during interim time-points are consistent with those generated using either non-time-lapse applications of CS-BLI or conventional techniques. We applied TL-CSBLI to delineate, in high-throughput manner, the temporal dynamics of the responses of tumor cells (e.g. multiple myeloma (MM) and other hematologic malignancies) to diverse treatments (e.g. conventional chemotherapeutics, glucocorticoids; proteasome inhibitors (PIs, bortezomib or carfilzomib), and kinase inhibitors). Using the time-lapse capabilities of this assay, we evaluated tumor cell responses in the presence vs. absence of stromal cells. We observed that the kinetics of tumor cell response to diverse therapeutic classes are heterogeneous, even within the same tumor type: for instance, tumor cells with pronounced responses at the end of drug incubation (e.g. 24, 48, 72, hrs after initiation of treatment with PIs, DNA-damaging chemotherapeutics, or dexamethasone respectively), can have different magnitude of responses at intermediate time points. This suggests that TL-CSBLI data can further stratify treatment-responsive tumor cells into those with early vs. late kinetics of response. We also observed that the kinetics of the proliferative / anti-apoptotic effect conferred by stromal cells on tumor cells are highly variable between different cell lines, even within the same tumor type. For instance, the time between initiation of coculture and maximum stimulation of tumor cell viability by stromal cells was variable between cell lines and did not correlate with the magnitude of stimulation by stromal cells. Importantly, TL-CSBLI identified that the response of diverse types of tumor cells to treatments can be delayed in the presence of stromal cells, compared to conventional tumor cell monocultures: this initial delay in treatment response of tumor cells in stromal co-cultures may be observed even in cases where similar cytoreductive responses are eventually observed at later time-points in both the presence and absence of stromal cells. This observation suggests that a more expansive definition of stroma-induced resistance to a given treatment may be warranted, to specifically incorporate the ability of stromal cells to delay the tumor cell response to such treatment. In summary, TL-CSBLI enables detailed characterization of the kinetics of tumor cell responses to diverse experimental conditions. Its use can provide insight into the underappreciated impact that cell-autonomous variations or stroma-induced changes in the kinetics of tumor cell response to a given anti-tumor therapy can have on determining its efficacy. This is particularly consequential for agents (e.g. PIs) which have clinical pharmacokinetic profiles associated with transient peak exposure.

Disclosures
McMillin: Axios Biosciences: Equity Ownership; DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Negri:DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Mitsiades:Johnson & Johnson: Research Funding; Amgen: Research Funding; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; DFCI: patent submission on stromal co-culture technologies Patents & Royalties.