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.
Highlights
Conventional 2D cell cultures are not capable of mimicking the complexity and heterogeneity of clinical tumors as in vivo tumors grow in a three-dimensional (3D) conformation with a specific organization and architecture that a 2D monolayer cell culture cannot reproduce
It is common opinion that in vitro 3D cultures could fill the gap between conventional 2D in vitro testing and animal models[12], and many researchers recommend the use of 3D cell cultures in drug screening programs as support for conventional 2D monolayer studies and before activating animal protocols[13,14]
Before starting the analysis it is worth introducing the notion of “equivalent diameter”, needed in the presence of a non-perfect sphericity, and defined as the diameter of a circle having the same area as the spheroid section being imaged[29]
Summary
Conventional 2D cell cultures are not capable of mimicking the complexity and heterogeneity of clinical tumors as in vivo tumors grow in a three-dimensional (3D) conformation with a specific organization and architecture that a 2D monolayer cell culture cannot reproduce. As reported in detail by Kimlin et al.7, 3D cell cultures possess several in vivo features of tumors such as cell-cell interaction[8], hypoxia[9], drug penetration[10], response and resistance[9], and production/deposition of extracellular matrix[7]. All of these factors shift growth dependence away from the phenotype of unrestrained proliferation which is dominant in standard 2D cultures. We validated a viability assay capable of providing rigorous data about cytotoxic effects on spheroids of up to 650 μ m in diameter
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