Abstract 186: Advances in 3D cell culture analysis: Utilizing novel flow cytometry technology for rapid analysis of intact spheroids

Abstract Cancer cells grown in two dimensional (2D) monolayers are a convenient model to use for basic research and in generating lead candidates in the drug discovery process. However, cells grown in 2D cultures lack many of the structural, biological, and functional attributes of solid tumors, which contributes to the high clinical failure rates for biologic and small molecule drugs. Multicellular three-dimensional (3D) tumor spheroids are advanced models which more faithfully mimic the physiological tumor milieu and is more predictive of therapeutic efficacy than traditional monolayer cultures. However, the inability to generate and to analyze large numbers of intact spheroids with a simplified workflow remains a barrier in the adoption of 3D models for a wide range of applications. To overcome these barriers, we used the Velocyt® large particle flow cytometer (Bennubio) to evaluate spheroid health in a well-established small molecule spheroid killing assay. The Velocyt technology rapidly and gently analyzes particles from 3 to 300 microns and comes with a suite of novel informatics tools purpose built for large particle analysis. To exemplify the use of large particle flow cytometry for the analysis of intact spheroids we generated uniformed-sized spheroids (~180 microns) from the H1650 human lung and HCT116 human colon cancer cell lines using microwell technology. Two to three days after spheroid generation, the spheroids were treated with increasing concentrations of staurosporine and other compounds. Spheroids were harvested at one to three days post treatment (depending on drug treatment) and stained with fluorescent cell viability and apoptotic dyes. Intact spheroids were analyzed for fluorescence and size using the Velocyt flow cytometer and between one and two thousand spheroids were assessed for each treatment in approximately three minutes. In untreated control spheroids, fluorescence was detected in a subpopulation of spheroids consistent with expected low levels of background apoptosis. In treated spheroids, an increase in the mean fluorescence intensity (MFI) of each particle indicated an increase in the number of non-viable and apoptotic cells in each spheroid. MFI increased nearly 6-fold with high concentrations of staurosporine compared to control spheroids, and dead cells were detected in nearly 100% of spheroids that were administered the highest staurosporine concentration. Using the size-based measurement parameters of the Velocyt, we also showed a decrease in overall spheroid size concurrent with increased numbers of spheroid fragments and single cells with increasing drug concentration. These data highlight that the use of 3D cell models combined with the Velocyt large particle flow cytometer is a powerful tool to help advance basic and translational cancer research. Citation Format: John P. O'Rourke, Mathew Saunders, Valerie Sanchez. Advances in 3D cell culture analysis: Utilizing novel flow cytometry technology for rapid analysis of intact spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 186.