Characterization of the Metabolic State and Molecular Crowding in Breast Cancer Spheroids

Abstract

Spheroids are 3D cultures of cells specifically grown in order to create a single spherical cell mass, which can be used as a model for studying the behavior of tumors in a controlled environment. Despite being a simplistic model, it allows for the rapid creation of the samples and displays a more complex physiology when compared to 2D cultures [1]. The spheroids grown for this study are derived from two different breast adenocarcinoma cell lines, MCF7 and MDA-MB231, which display less and more invasive behavior, respectively. From 20,000 cells, our protocol provides spheres with a diameter of approximately 400 μm. The spheres were then transferred to a collagen Type-1 gel matrix in order to mimic the extracellular matrix. Once obtained and transferred in the collagen, the spheres were observed at different time points over 24 hours. We measured their single cell metabolic signature by analyzing the NADH lifetime with the Phasor-FLIM approach [2] and assessed their molecular crowding using the ACDAN probe [3] and analyzing its emission spectrum. The main advantage of these techniques is the minimal interaction with the system, since NADH Phasor-FLIM is a label-free approach and ACDAN does not directly interact with the cellular machinery. Furthermore, both techniques can be implemented in a 2-photon microscope, which allows for deep sample penetration and reduced phototoxicity. Our preliminary results show a significant difference in the above-mentioned properties between the cells located at the center and at the periphery of the sphere. Furthermore, we could observe the early stage of metastatic behavior of our tumor model and analyze the characteristics of the cells invading the collagen matrix. [1] Costa et al Biotechnol. Adv. (2016) [2] Stringari et al Sci. Rep. (2012) [3] Thoke et al Sci. Rep. (2017)