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

Cytometry
of Reaction Rate Constant (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. Reliable CRRC requires
uniform exposure of cells to the reaction substrate followed by their
uniform imaging, which in turn, 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 multidrug
resistance (MDR) 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 a practical analytical tool.