Estimating cell diffusivity and cell proliferation rate by interpreting IncuCyte ZOOM™ assay data using the Fisher-Kolmogorov model.

Stuart T Johnston,Lisa K Chopin,D L Sean Mcelwain,Esha T Shah,Matthew J Simpson

doi:10.1186/s12918-015-0182-y

Stuart T Johnston, Lisa K Chopin + Show 3 more

Open Access

https://doi.org/10.1186/s12918-015-0182-y

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Abstract

BackgroundStandard methods for quantifying IncuCyte ZOOM™ assays involve measurements that quantify how rapidly the initially-vacant area becomes re-colonised with cells as a function of time. Unfortunately, these measurements give no insight into the details of the cellular-level mechanisms acting to close the initially-vacant area. We provide an alternative method enabling us to quantify the role of cell motility and cell proliferation separately. To achieve this we calibrate standard data available from IncuCyte ZOOM™ images to the solution of the Fisher-Kolmogorov model.ResultsThe Fisher-Kolmogorov model is a reaction-diffusion equation that has been used to describe collective cell spreading driven by cell migration, characterised by a cell diffusivity, D, and carrying capacity limited proliferation with proliferation rate, λ, and carrying capacity density, K. By analysing temporal changes in cell density in several subregions located well-behind the initial position of the leading edge we estimate λ and K. Given these estimates, we then apply automatic leading edge detection algorithms to the images produced by the IncuCyte ZOOM™ assay and match this data with a numerical solution of the Fisher-Kolmogorov equation to provide an estimate of D. We demonstrate this method by applying it to interpret a suite of IncuCyte ZOOM™ assays using PC-3 prostate cancer cells and obtain estimates of D, λ and K. Comparing estimates of D, λ and K for a control assay with estimates of D, λ and K for assays where epidermal growth factor (EGF) is applied in varying concentrations confirms that EGF enhances the rate of scratch closure and that this stimulation is driven by an increase in D and λ, whereas K is relatively unaffected by EGF.ConclusionsOur approach for estimating D, λ and K from an IncuCyte ZOOM™ assay provides more detail about cellular-level behaviour than standard methods for analysing these assays. In particular, our approach can be used to quantify the balance of cell migration and cell proliferation and, as we demonstrate, allow us to quantify how the addition of growth factors affects these processes individually.

Highlights

Standard methods for quantifying IncuCyte ZOOMTM assays involve measurements that quantify how rapidly the initially-vacant area becomes re-colonised with cells as a function of time
Traditional data extracted from IncuCyte ZOOMTM assays does not give us any indication of the relative roles of cell motility and cell proliferation. This additional information could be important in terms of understanding how a particular growth factor or a potential drug treatment affects collective spreading since it is possible that the addition of a growth factor or drug treatment could affect: (i) cell motility alone, (ii) cell proliferation alone, or (iii) both cell motility and cell proliferation, simultaneously. In this methodology article we describe an alternative method for interpreting IncuCyte ZOOMTM assay data using a continuum mathematical model
Comparing our estimates of K, λ and D for each assay with a different concentration of epidermal growth factor (EGF) provides us with information about how EGF affects cell proliferation and cell motility for the PC-3 cell line

Summary

Introduction

Standard methods for quantifying IncuCyte ZOOMTM assays involve measurements that quantify how rapidly the initially-vacant area becomes re-colonised with cells as a function of time. We provide an alternative method enabling us to quantify the role of cell motility and cell proliferation separately To achieve this we calibrate standard data available from IncuCyte ZOOMTM images to the solution of the Fisher-Kolmogorov model. A key limitation of scratch assays is the question of whether they are reproducible since the scratch can be made with various types of instruments and varying degrees of pressure, and the assay can be performed on several different types of substrates All of these variables have the potential to affect the results of the scratch assay. IncuCyte ZOOMTM assays have the advantage that the scratch is reproducibly created with a mechanical tool and live images are obtained without the need to interrupt the experiment for imaging purposes [12]

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