Abstract 32: Development of a three-dimensional spheroid model system to more accurately evaluate schedule dependent mechanistic differences between metronomic and conventional dosing of topotecan
Authors: Davis Joshua , Ghosh Taraswi Mitra , Jasper Lani , Eggert Matthew , Warner Jeff , Cummings Brian , Arnold Robert 
Published: August 2019

Alternative treatment schedules, such as low-dose, high-frequency (metronomic; METRO) of some chemotherapeutics enhance efficacy and reduce toxicity in some cancers when compared to chemotherapy given conventionally (CONV). Previously, we demonstrated that low-dose, prolonged exposure to topotecan (30-day infusion 0.1 mg/kg/day via Alzet pump) improved survival and reduced tumor volume ~120%, compared to topotecan given CONV (4 mg/kg, every 4 days) in xenograft model of aggressive prostate cancer in athymic mice. Yet, detailed examination of the mechanisms underlying this enhanced activity and optimization of treatment schedules is limited with existing 2-dimension (2D) cell culture models that do not adequately reflect in vivo reality. Therefore, we developed and applied a 3D spheroid model that allows for the evaluation of longer drug exposures. We used PC-3-Luc-GFP, an aggressive, metastatic castration-resistant prostate cancer that expresses luciferase (Luc) and green fluorescent (GFP) that support bioluminescent and fluorescence imaging. Cell growth and cytotoxicity studies were performed using 2D or 3D models. Briefly, 3D spheroids were formed by seeding cells in a poly-HEMA coated 96-well plate with 2.5% recombinant basement membrane followed by centrifugation (5 min at 1000 g) to encourage spheroid generation. The 2D cell cultures reached confluency in 72-96 hr. While we previously demonstrated enhanced activity in vivo, the 2D model showed no benefit (improved potency) over this time frame, ie, METRO vs CONV dosing (logIC50 2.278 (177nM), 95% CI [1.781-2.776] vs logIC50 2.248 (189 nM) 95% CI [1.723-2.773]. Attempts to reduce seeding density to increase study duration resulted in poor and inconsistent cell growth and greater variability. 3D spheroids yielded reproducible round spheroids of similar size (~800 μm) that grew for up to 28 days, thus permitting evaluation of long treatment exposures. Tumor stromal cells are also known to alter tumor pathology, drug delivery and responsiveness to therapy. Therefore, 3D spheroids with PC-3-Luc-GFP and macrophages and endothelial cells in different cell proportions (1:1:1, 1:2:1, etc.,) were examined. Growth of 3D spheroids was altered based on cell type and cell proportion. Further, differences in rates of growth and cellular morphology were observed for tumor activated vs wild macrophages. Overall, we developed a high-throughput, liquid overlay, scaffold-based 3D co-culture spheroid that can be kept for 28 days. This model will permit evaluation of single agent and concomitant therapy over prolonged periods better mimicking clinical practice. Further, this model will allow for more detailed molecular analysis and ability to discern the effect of tumor microenvironment on alternate treatment schedules.

Citation Format: Joshua Davis, Taraswi Mitra Ghosh, Lani Jasper, Matthew Eggert, Jeff Warner, Brian Cummings, Robert Arnold. Development of a three-dimensional spheroid model system to more accurately evaluate schedule dependent mechanistic differences between metronomic and conventional dosing of topotecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 32.