Functional Screening with a Live Cell Imaging-Based Random Cell Migration Assay

Abstract

Cell migration, essential in cancer progression, is a complex process comprising a number of spatiotemporally regulated and well-coordinated mechanisms. In order to study (random) cell migration in the context of responses to various external cues (such as growth factors) or intrinsic cell signaling, a number of different tools and approaches have been developed. In order to unravel the key pathways and players involved in the regulation of (cancer) cell migration, a systematical mapping of the players/pathways is required. For this purpose, we developed a cell migration assay based on automatic high-throughput microscopy screen. This approach allows for screening of hundreds of genes, e.g., those encoding various kinases and phosphatases but can also be used for screening of drugs libraries. Moreover, we have developed an automatic analysis pipeline comprising of (a) automatic data acquisition (movie) and (b) automatic analysis of the acquired movies of the migrating cells. Here, we describe various facets of this approach. Since cell migration is essential in progression of cancer metastasis, we describe two examples of experiments performed on highly motile (metastatic) cancer cells.

Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant

“…Plates were then fixed using 4% formaldehyde and stored for later immunostaining. All data were converted and analyzed using custom-made ImagePro Plus (MediaCybernetics) macros (42). Cell migration was quantified by tracking of the GFP signal over time using a custom ImageJ-based macro.…”

Podosomes in space

“…4B). 115,116 Moreover, gap closure assays, commonly used for monolayer-forming cells such as primary endothelial or tumor cells, can also be utilized in set ups with macrophages. The creation of a gap by scratching the cell population induces directional movement of the cells without the addition of a chemoattractant (Fig.…”

Migration rather than proliferation transcriptomic signatures are strongly associated with breast cancer patient survival

“…Up to four positions per well were automatically defined and nuclei (stained with live Hoechst) were imaged overnight every 10 to 20 minutes using NIS controlling software (Nikon) and a CCD camera (Pixel size: 0.78 or 0.32 µm). The .nd2 files acquired from NIS were exported to .tiff files as mono image for each channel and then converted to .avi files and analyzed using custom made ImagePro Plus macros as previously described 33 . The mean speed of cell migration was quantified per time-lapse by tracking each nucleus separately over time.…”