3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained

TLDR

Spheroid tumor models with heterogeneous cell states show promise for anticancer strategy development, yet reproducibility issues in 3D model data are poorly documented. We used a novel open‑source software for automatic 3D colony image analysis, compared commercial 3D viability assays, and validated a cytotoxicity test for spheroids up to 650 µm diameter. Morphology parameters such as volume and shape introduce variability in spheroid response; pre‑selection of homogeneous spheroids and a validated cytotoxicity test reduce variability and provide reliable data.

Abstract

The potential of a spheroid tumor model composed of cells in different proliferative and metabolic states for the development of new anticancer strategies has been amply demonstrated. However, there is little or no information in the literature on the problems of reproducibility of data originating from experiments using 3D models. Our analyses, carried out using a novel open source software capable of performing an automatic image analysis of 3D tumor colonies, showed that a number of morphology parameters affect the response of large spheroids to treatment. In particular, we found that both spheroid volume and shape may be a source of variability. We also compared some commercially available viability assays specifically designed for 3D models. In conclusion, our data indicate the need for a pre-selection of tumor spheroids of homogeneous volume and shape to reduce data variability to a minimum before use in a cytotoxicity test. In addition, we identified and validated a cytotoxicity test capable of providing meaningful data on the damage induced in large tumor spheroids of up to diameter in 650 μm by different kinds of treatments.