A new method for the study of biophysical and morphological parameters in 3D cell cultures: Evaluation in LoVo spheroids treated with crizotinib

Azzurra Sargenti,Carola Cavallo,Simone Bonetti,Mattia Lauriola,Daniele Gazzola,Francesco Bacchi,Francesco Musmeci,Giuseppe Filardo,Martina Mazzeschi,Simone Pasqua,Spartaco Santi,Irina V Balalaeva

doi:10.1371/journal.pone.0252907

Azzurra Sargenti, Carola Cavallo + Show 10 more

Open Access

https://doi.org/10.1371/journal.pone.0252907

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Abstract

Three-dimensional (3D) culture systems like tumor spheroids represent useful in vitro models for drug screening and more broadly for cancer biology research, but the generation of uniform populations of spheroids remains challenging. The possibility to properly characterize spheroid properties would increase the reliability of these models. To address this issue different analysis were combined: i) a new device and relative analytical method for the accurate, simultaneous, and rapid measurement of mass density, weight, and size of spheroids, ii) confocal imaging, and iii) protein quantification, in a clinically relevant 3D model. The LoVo colon cancer cell line forming spheroids, treated with crizotinib (CZB) an ATP-competitive small-molecule inhibitor of the receptor tyrosine kinases, was employed to study and assess the correlation between biophysical and morphological parameters in both live and fixed cells. The new fluidic-based measurements allowed a robust phenotypical characterization of the spheroids structure, offering insights on the spheroids bulk and an accurate measurement of the tumor density. This analysis helps overcome the technical limits of the imaging that hardly penetrates the thickness of 3D structures. Accordingly, we were able to document that CZB treatment has an impact on mass density, which represents a key marker characterizing cancer cell treatment. Spheroid culture is the ultimate technology in drug discovery and the adoption of such precise measurement of the tumor characteristics can represent a key step forward for the accurate testing of treatment's potential in 3D in vitro models.

Highlights

Three-dimensional (3D) culture systems are widely recognized as an in vitro model with a close resemblance to solid tumors in vivo
These results support the potential of the new proposed methodology to investigate 3D models, with morphological characterization being supported by biophysical parameters correlation
These results were further supported by the protein quantification and the deep imaging analysis that showed protein level decrease in line with a reduced proliferation, a decrease of the number of nuclei and the spheroid volume, but a strong increase in nuclear density and cell-cell compactness, according to mass density data

Summary

Introduction

Three-dimensional (3D) culture systems are widely recognized as an in vitro model with a close resemblance to solid tumors in vivo. 3D models recapitulate the complex cellular organization of the tumor bulk Both cell–cell adhesions [4] and cell–matrix interactions, along with a de novo synthesis of extracellular matrix (ECM) proteins [5,6], contribute to the generation of nutrient and signal gradients typical of solid tumors, offering a clinically relevant study model [7,8,9,10,11]. For these reasons, spheroids represent a useful tool for drug screening [12,13] and more broadly for cancer biology research [5,14]. Spheroids are useful for toxicological studies, as concerning drug penetration and absorption, providing insights on drug diffusion [15] and exhibit better immuno-modulatory, proliferation, and activation abilities than 2D cultures [16]

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