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Description

Cell migration is central to a variety of functions such as wound healing, cell differentiation, embryonic development and the metastasis of tumours. A better understanding of the mechanism by which cells migrate may lead to the development of novel therapeutic strategies in particular for cancer research where rated metastasis and tumour invasion appear as the main applications of cell migration assays.

In the present screening application, the label-free DHM imaging provide an informative and fast detection method of active compounds inhibiting cell proliferation/migration using both time-lapse and end point measurements. Moreover, active compounds can be categorized according to their potency through the generation of dose-responses but also according to the phenotype generated. In addition cytotoxic measurement is obtained without further post-processing or analysis using the average OPD information.

In conclusion, our methodological approach allows easy and cost-effective characterization of hits for their ability to perturb cell migration and simultaneously to gather valuable information related to cell phenotypic changes induced by the effect of the chemical compounds.

Material and methods

• Cell: HeLa cells were plated in Oris^TM-Pro 96-well plates (Platypus Technologies). Briefly, silicon-based stoppers provide a temporary physical barrier preventing cells adherence to the center of the well generating an annular monolayer of cells with a central cell-free area (exclusion zone) into which cell movement can occur
• Toxic compounds used for cell treatments: Cells were treated for 40 h with increasing concentrations of Cytochalasin D (n=3). This compound is a cell permeable potent inhibitor of the polymerization and the elongation of actin resulting in cell cycle arrest (at the G1-S transition) through the activation of p53-dependent pathways.
• DHM solution: Image acquisition and analysis: using a 10x/0.22 NA objective, 25 images per well  were recorded at the speed of about 2 images/second leading to a total time of 20 minutes for acquiring 2400 images of an entire 96 well plate. Time-points were acquired each hour for 40 h. Confluency, the readout for migration, was measured by simple thresholding of the images and the average OPD, related to cytotoxicity was measured using the average DHM signal.

Results

An entire plate has been imaged in time-lapse mode for the different assay conditions. The EC50 value calculated from the dose response curve generated at the end point 40h for the Cytochalasin D is in agreement with previously reported data. High content temporal and spatial information both have been easily generated with our label-free DHM imaging approach. Chemical compounds can be evaluated and quantified for their ability to prevent cell migration. Moreover, during the time-lapse measurements, phenotypic changes of cells can be recorded giving additional valuable information of the compounds action during time at a given concentration.

Our method is suitable for large scale screening at single compound concentration and focused high content analysis of selected molecules during hits-validation or hits-to-leads process.

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