Establishment of breast tumor spheroids: An emerging research tool

Abstract

Abstract Breast cancer research has historically relied on two model systems: two-dimensional cell culture systems and mouse models. These models, although very convenient, lack the functional phenotypic architecture of human tissue. Extrapolation of hormonal responses from models to human relevance also requires further investigation due to the anatomical, physiological, and biological differences. Three-dimensional spheroids provide alternative models as they better represent the architecture and maintain the histomorphological, functional, and micro-environmental features of the in vivo human tumor tissue. We established and characterized tumor spheroids in vitro from the breast cancer cell lines T47D and MCF7. The cells were seeded at densities of 5000 cells per well in low adhesive 96-well plates using specific growth media that sustains spheroid growth. After 10 days of culture, the spheroids were stained to verify spheroid viability using Calcein AM [green] and Ethidium homodimer-1 [red] (top row). Spheroids were stained with E-cadherin [yellow] and DAPI [blue] (bottom row) to verify the 3D architecture, and then imaged using a Leica spinning-disk confocal microscope. We noticed that MCF7 cells (left images) tended to form multiple spheroids in a single well, whereas the T47D cels (right images) formed single spheroids. Both spheroids expressed E-cadherin, demonstrating cell-to-cell adhesion and remained viable after 10 days of culture. More importantly the spheroids formed every time they were cultured in low-adhesive plates with appropriate growth media, demonstrating the effectiveness, reproducibility, and robustness of this in vitro model system. This article is protected by copyright. All rights reserved.