Watching the great migration

The trait of cancerous cells to metastasise is distinctive of some types of cancer. As such cell migration assays are vital research and diagnostic tool - here we learn of a new type of assay that can measure cell migration in real-time

Metastasis is one of the hallmarks of cancer. Cell migration is essential for metastasis and is a complex process involving focal adhesions, the actin cytoskeleton and cell polarity. Phosphoinositides are inositol lipids that can act as signalling molecules in the abovementioned processes. The regulation of phosphoinositides occurs through the actions of lipid kinases and phosphatases. Some, like PI3K, are well studied but the functions of many of these proteins remain unknown.

Significant effort is therefore expended by researchers to screen compound libraries for migration modulators. Investigators are seeking migration assays that offer key improvements over both the Boyden chamber based assays – which lack the ability to view cells in real-time during experiments – and scratch assays, where wound sizes can be inconsistent and results can be impacted by factors released from wounded cells and by damage imparted to the extracellular matrix.

The Oris Cell Migration Assays present certain advantages over the traditional migration assay including the ability to measure migration in real-time. Using siRNA and this assay new cell migration regulators from the phosphoinositide modulators group can be identified.

The assay utilises cell seeding stoppers made from a medical-grade silicone or a non-toxic biocompatible gel to restrict cell seeding to the outer annular regions of the wells. Removal of the stoppers or dissolution of the BCG reveals a 2mm diameter un-seeded region in the centre of each well, i.e., the detection zone, into which the seeded cells may then migrate (Figure 1). 

This unique assay format allows an unobstructed view of cell motility throughout the duration of the experiment and the ability to use multiple fluorescent stains for phenotypic analysis of cells. Data can be quantified using fluorescence microplate readers, inverted microscopes or imaging instruments (HCS/HCI imaging systems).

Formatted in 96 or 384-wells, the plates can be tissue culture treated or coated with basement membrane extract, collagen I, fibronectin or tricoated. With this technique significantly more information to be derived from each assay well (i.e. modulation of migration, cell morphology, cytostaining for subcellular structures, multiplexing of stains, toxicity, and viability) than other migrations assays. In this way, information can be collected regarding cell shape, velocity, distance and direction of migration.

Experimental example

A siRNA library targeting 90 known phosphoinositide modulators was custom made by Sigma-Aldrich while NTER transfection reagent and negative control siRNA and positive control Rac siRNA were obtained. DiI-C16 was obtained from Molecular Probes. MCF-10A cells were grown in DMEM/F12 containing L-glutamine, 5% horse serum, 20ng/ml EGF, 0.5?g/ml hydrocortisone, 100ng/ml cholera toxin, 10?g/ml insulin and pen/strep.  

The Oris Cell Migration Assay was obtained from AMSBIO. Assays were analysed on a POLARstar Omega multidetection microplate reader with CO2 incubator from BMG Labtech.  

MCF10A cells were seeded in six well plates at 120000 cells/well. The next day cells were transfected with 30nM siRNA per well using NTER transfection reagent according to manufacturer’s manual.  24 hours post-transfection cells were trypsinised, washed and counted. Cells were diluted down to 240000 cells/ml and were incubated with 2.5?M DiI at 37°C for 30 minutes. Unincorporated dye was removed by centrifugation and cells were suspended in 600?l medium. Three wells with stoppers and one well without stopper were seeded with 100ul cell suspension.

The following day, the inserts were removed and the medium was flicked out. Subsequently, 200?l phenol red free medium was added to each well. The mask was attached to the bottom of the plate and the plate was put in the pre-warmed and gassed plate reader. Fluorescence was measured with excitation at 544nm and emission at 590. Readings were taken at 20 minute intervals over a period of 24 hours using the bottom optic.

MCF10A cells were transfected with siRNA targeting 90 well known phosphoinositide modulators. 24 hours post-transfection, cells were incubated with 2.5?M DiI before being seeded in a 96 wells ORIS plate: three wells with stoppers and one well without stopper for each siRNA, in order to eliminate variation due to cell number differences.

The day after, the inserts were removed and the mask was attached to the bottom of the plate incubated in a CO2 microplate reader. Fluorescence in the detection zone was measured every 20 minutes during 24 hours (Figure 2).  

Data analysis was performed using the MARS analysis software developed by BMG Labtech. Wells without stoppers were used as cell density controls (Figure 3).

An area under the curve was calculated for each sample. For wells with stoppers, all were aligned to baseline at point 7 till 9. The area under the curve was calculated from this base point till the end of the assay for each curve. These values were divided by the area under the curve of the wells without stopper. These values were averaged for each triplicate and a standard deviation calculated.  

Out of the library for 90 genes, our screen initially identified 19 genes whose knockdown had an effect on migration. We were able to confirm that nine of these did cause migration effects (increase or decrease).

The migration assay was repeated for the 19 genes to select reproducible hits. Furthermore, mRNA for the respective genes were determined in each knockdown to eliminate off-target effects.

To minimise changes of siRNA off-target effects we obtained separate siRNA pools for six genes that contained siRNA with different target sequences than the original siRNA pools. Migration assays were done with these and confirmed that knockdown of any of these six genes caused an effect on cell migration.

Using the assay, we screened 90 phosphoinositides modulators to identify genes involved in cell migration. So far, we have identified six genes that, when knocked down, decrease migration.

This experimentation described demonstrates that this  technology provides an easy, robust, efficient and cost effective cell migration system for the high throughput screening of compounds. In future, use of Oris technology promises to accelerate and make easier discoveries in the cell migration and cell invasion fields.