Abstract B75: High-throughput, bright-field imaging of 3D tumor spheroid growth and morphology for therapeutic and efficacy screening: A study using chemotherapeutic compounds

Background

3D tumor microtissues or spheroids are widely accepted as a more representative biological model in phenotypic drug discovery. Size and morphology are important determinants to evaluate the biological behavior of 3D spheroids, particularly in development of anti-cancer drugs where monitoring cell growth is a particularly critical endpoint. The increasing number of applications of 3D tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including high-throughput image analysis. In this study, we assessed the growth and morphology changes of HCT116 colorectal cancer spheroids in response to two chemotherapy drugs-gemcitabine and docetaxel- using a high throughput, bright-field spheroid imager (Cell3iMager, SCREEN Holdings Ltd., Inc.).

Methods

Tumor spheroids consisting of the HCT116 colorectal cancer cell line were aggregated in InSphero's GravityPLUS™ plate (a scaffold-free hanging drop platform). The aggregated spheroids were transferred into a GravityTRAP™ plate prior to dosing. Spheroids were dosed with anti-cancer drugs-gemcitabine and docetaxel (0.8-20nM) at day 0 and re-dosed at day 3. The plates were scanned and spheroid size and morphology were analyzed daily for 7 days. Endpoint analysis of ATP content was carried out at day 7 utilizing a luminescence-based ATP assay (CellTiter-Glo® 2.0 Assay, Promega)

Results

HCT116 spheroids were measured daily for 7 days to determine dose-response effects of gemcitabine and docetaxel treatment (0.8-20nM) on spheroid area (um²). A size increase was observed for the control and the lowest compound concentration treated spheroids whereas the spheroid growth was inhibited at the higher concentrations tested (>4nM). Among the tested compounds, HCT116 spheroids showed less sensitivity to docetaxel compared to gemcitabine. At day 7 the treatment with 20nM gemcitabine resulted in more than 50% reduction in tumor area (um2) which was positively correlated with the decrease in cell viability, determined by ATP content. Using HCT116 tumor microtissues cultured in InSphero's GravityTRAP™ plates, we demonstrate that the measurement of tumor size with the Cell3iMager could be a suitable endpoint compared to that of ATP content for drug induced cytotoxicity in tumor spheroids, without lysing cells or otherwise interfering with long term culture of spheroids.

Conclusions

Label-free measurement of changes in 3D microtissue size and morphology using the Cell3iMager enables efficient assessment of phenotypic endpoints without disrupting tumor growth, and can be used to focus selection of therapeutic targets and treatment strategies before costly and tedious testing in animal models.

Citation Format: Leena Mol Thuruthippallil, Jens M. Kelm, David Fluri. High-throughput, bright-field imaging of 3D tumor spheroid growth and morphology for therapeutic and efficacy screening: A study using chemotherapeutic compounds. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B75.