Abstract
Wound healing assay performed with automated microscopy is widely used in drug testing, cancer cell analysis, and similar approaches. It is easy to perform, and the results are reproducible. However, it is usually used as a semi-quantitative approach because of inefficient image segmentation in transmitted light microscopy. Recently, several algorithms for wound healing quantification were suggested, but none of them was tested on a large dataset. In the current study, we develop a pipeline allowing to achieve correct segmentation of the wound edges in >95% of pictures and extended statistical data processing to eliminate errors of cell culture artifacts. Using this tool, we collected data on wound healing dynamics of 10 cell lines with 10 min time resolution. We determine that the overall kinetics of wound healing is non-linear; however, all cell lines demonstrate linear wound closure dynamics in a 6-h window between the fifth and 12th hours after scratching. We next analyzed microtubule-inhibiting drugs’, nocodazole, vinorelbine, and Taxol, action on the kinetics of wound healing in the drug concentration-dependent way. Within this time window, the measurements of velocity of the cell edge allow the detection of statistically significant data when changes did not exceed 10–15%. All cell lines show decrease in the wound healing velocity at millimolar concentrations of microtubule inhibitors. However, dose-dependent response was cell line specific and drug specific. Cell motility was completely inhibited (edge velocity decreased 100%), while in others, it decreased only slightly (not more than 50%). Nanomolar doses (10–100 nM) of microtubule inhibitors in some cases even elevated cell motility. We speculate that anti-microtubule drugs might have specific effects on cell motility not related to the inhibition of the dynamic instability of microtubules.
Highlights
Cell migration is essential for many physiological processes including embryonic development, wound repair, angiogenesis, and tumor metastasis (Bindschadler and McGrath, 2007; Friedl and Gilmour, 2009)
The overall conclusion from the studies on the effect of MT inhibitors is that the dynamic microtubules are required for cell motility, and in certain cases, it is even not related to the ability of drugs to inhibit cell division (Ngan et al, 2001; Ganguly et al, 2010)
We found that a dose-dependent response to MT inhibitors becomes significant only for some cultures and always at concentrations exceeding minimal mitostatic ones, and the effect of micromolar concentrations of these drugs is very different between cell lines examined
Summary
Cell migration is essential for many physiological processes including embryonic development, wound repair, angiogenesis, and tumor metastasis (Bindschadler and McGrath, 2007; Friedl and Gilmour, 2009). Cell migration largely depends on the actin cytoskeleton (Ridley et al, 2003), and directional migration of fibroblasts and tumor cells depends on microtubules (Liao et al, 1995). The low concentrations of MT inhibitors are sufficient only for the inhibition of dynamic instability of MTs and do not induce their depolymerization; they effectively inhibited certain cell migration (Liao et al, 1995; Vacca et al, 1999; Yang et al, 2010). The overall conclusion from the studies on the effect of MT inhibitors is that the dynamic microtubules are required for cell motility, and in certain cases, it is even not related to the ability of drugs to inhibit cell division (Ngan et al, 2001; Ganguly et al, 2010). MTs are thought to be involved in cell motility through the regulation of Rho GTPases via several pathways (Kaverina and Straube, 2011), facilitating focal adhesion turnover (Kaverina et al, 1999) and providing directional delivery of different cargoes like Golgi-derived vesicles, endosomes, etc., by kinesins to the lamellae (Schmidt et al, 2009; Kaverina and Straube, 2011; Fife et al, 2014)
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