Spheroid-Based Approach to Assess the Tissue Relevance of Analysis of Dispersed-Settled Tissue Cells by Cytometry of the Reaction Rate Constant

Abstract

File main pdf file (CRRC of dispersed-settled spheroidal cells.pdf) describes experimental results and their interpretation for a study of tissue relevance of analyses of dispersed-settled tissues cells by Cytometry of Reaction Rate Constant (CRRC). CRRC uses time-lapse fluorescence microscopy to measure a rate constant of a catalytic reaction in individual cells and, thus, facilitate accurate size determination for cell subpopulations with distinct efficiencies of this reaction. Practical CRRC requires that a tissue sample be disintegrated into a suspension of dispersed cells and these cells settle on the support surface before being analyzed by CRRC. We call such cells "dispersed-settled" to distinguish them from cells cultured as a monolayer. Studies of the dispersed-settled cells can be tissue-relevant only if the cells maintain their 3D tissue state during the multi-hour CRRC procedure. Here we propose an approach for assessing tissue relevance of the CRRC-based analysis of the dispersed-settled cells. Our approach utilizes cultured multicellular spheroids as a 3D cell model and cultured cell monolayers as a 2D cell model. The CRRC results of the dispersed-settled cells derived from spheroids are compared to those of spheroids and monolayers in order to find if the dispersed-settled cells are representative of the spheroids. To demonstrate its practical use, we applied this approach to a cellular reaction of multi-drug-resistance (MRD) transport which was followed by extrusion of a fluorescent substrate from the cells. The approach proved to be reliable and revealed long-term maintenance of MDR transport in the dispersed-settled cells obtained from cultured ovarian cancer spheroids. Accordingly, CRRC can be used to determine accurately the size of a cell subpopulation with an elevated level of MDR transport in tumor samples, which makes CRRC a suitable method for the development of MDR-based predictors of chemoresistance. The proposed spheroid-based approach for validation of CRRC is applicable to other types of cellular reactions, and, thus, will be an indispensable tool for transforming CRRC from an experimental technique into practical analytical tool. Additional (zip) files contain supporting images, kinetic traces, and histograms. Their detailed descriptions are provided in the main pdf file.

Multi-Drug-Resistance in Drug-Naive and Drug-Exposed Ovarian Cancer Cell Lines Responds Differently to Cell Culture Dimensionality

“…CRRC results of the drug-naïve A2780S cell line are shown in Figure 1A; they have been adopted from our recently published paper (10). The monolayer histogram (grey line) was found to be unimodal, suggesting a single population of cells.…”

“…Detailed description of experimental techniques of CRRC for cell monolayers and cells in intact spheroids can be found elsewhere (10). Briefly, monolayers and spheroids were cultured in DMEM medium supplemented with bovine serum and antibiotics under standard cell culture conditions.…”

“…spheroids or tissue samples) and allowed to settle on the surface (10). However, if CRRC is based on confocal microscopy, it can also be applied to intact cell clusters (10). Thus, CRRC is uniquely capable of accurately measuring MDR activity in both 2D and 3D models, making it suitable for addressing our question of how MDR activity of single cells differs from that of aggregated cells in (i) drug-naïve and (ii) drug-exposed tumor cells.…”

Multi‑drug‑resistance efflux in cisplatin‑naive and cisplatin‑exposed A2780 ovarian cancer cells responds differently to cell culture dimensionality

“…Small spheroids facilitate free MDR efflux from approximately 70% of spheroid cells and also minimize intraspheroidal gradients of oxygen, pH, etc. (9,10). Second, since the first order rate constant is defined by the dynamics rather than the absolute values of fluorescence signal, the CRRC method is robust to variations in substrate level or signal attenuation potentially associated with cell clustering or uneven illumination.…”

“…In this study, drug-naïve and drug-exposed tumor cells were modeled by the parental and cisplatin-resistant variants of the A2780 ovarian cancer (OC) cell line (A2780S and A2780CP, respectively) (11,12). Analytical advantages of A2780 cells include their inducible MDR capacity (13), ability to form small compact spheroids (14), and extensive use in the development of CRRC (8,9).…”