Novel Analytical Platform For Robust Identification of Cell Migration Inhibitors

Abstract

Wound healing assay is a simple and cost-effective in vitro assay for assessing therapeutic impacts on cell migration. its key limitation is the possible confoundment by other cellular phenotypes, causing misinterpretation of the experimental outcome. in this study, we attempted to address this problem by developing a simple analytical approach for scoring therapeutic influences on both cell migration and cell death, while normalizing the influence of cell growth using Mitomycin C pre-treatment. By carefully mapping the relationship between cell death and wound closure rate, contribution of cell death and cell migration on the observed wound closure delay can be quantitatively separated at all drug dosing. We showed that both intrinsic cell motility difference and extrinsic factors such as cell seeding density can significantly affect final interpretation of therapeutic impacts on cellular phenotypes. Such discrepancy can be rectified by using the actual wound closure time of each treatment condition for the calculation of phenotypic scores. finally, we demonstrated a screen for strong pharmaceutical inhibitors of cell migration in cholangiocarcinoma cell lines. our approach enables accurate scoring of both migrastatic and cytotoxic effects, and can be easily implemented for high-throughput drug screening.

Enriching the Arsenal of Pharmacological Tools against MICAL2

“…In the case of MRTF-A protein WHA is one of the most commonly used bioassays for evaluating the therapeutic impact on cell migration, mainly due to its simplicity in experimental setup and data processing. By scratching a cell monolayer to create a wound, one can consistently perform WHA across a large number of treatments [15].…”

“…The WHA, also known as the scratch assay, is an established two-dimensional (2D) technique that can be used to investigate collective migration and wound healing in vitro. This method was one of the first to be developed for the study of cell migration and measures the rate at which cells, in a cell monolayer, migrate to fill a cell-free gap [15]. It is a multistep procedure involving (1) growing a cell monolayer to confluence in a multiwell assay plate; (2) creating a "wound", a cell-free area in the monolayer, into which cells can subsequently migrate; (3) monitoring the recolonization of the cell-free gap to quantify cell motility.…”