癌症生物学

Cell migration is a highly complex and dynamic biological process, essential in several physiological phenomena and pathologies including cancer dissemination and metastasis formation. Thus understanding single cell migration is highly relevant and requires a suitable image-based assay. Depending on the speed of the moving cells, one may require fast time-lapse microscopy, which is not always suitable for high-throughput screening. To overcome this, a quantitative and fixed single cell migration assay was developed based on the PhagoKinetic Tracks (PKT) procedure. Briefly, cells are seeded on top of a monolayer of carboxylated latex beads, and as cells migrate, they phagocytose these beads and leave behind a migratory track. These bead-free migratory tracks can be visualized using a standard bright field microscope and analysed for a multiparametric quantitative assessment of single cell migration (Naffar-Abu-Amara et al., 2008).

Here we describe a detailed and optimized protocol of the PKT assay, adaptable for both RNAi and drug screening (van Roosmalen et al., 2015). This protocol allows the user to study migratory behaviour at the single cell level, without fast and live-imaging microscopy.

This protocol is designed to quantify invadopodia formation and activity. Invadopodia are protrusive structures elaborated by cancer cells that mediate cell attachment and remodeling of the extracellular matrix. These structures contribute to the ability of cancer cells to invade and metastasize. In this protocol, both the presence of invadopodia and their activity is simultaneously assessed and quantified by a fluorescent microscopy-based assay.

Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay is also useful in screens for compounds that act as chemoattractants or inhibitors of chemotaxis for cells. The assay employs a permeable layer of support, usually a tissue-culture-treated microporous membrane, which is positioned between two compartments that mimic two different sets of microenvironments for cell survival/growth. Cells on one side of the membrane, when sensing chemoattractants placed on the other side of the compartment that diffuses through the membrane, can migrate through the pores in the membrane towards the source of the chemoattractants. Cells that migrate across the membrane can be quantified by fixing and counting. Human breast epithelial adenocarcinoma MD-231 cells grow relatively fast and are metastatic. The MB-231 cell line is used here to describe the procedures of an in vitro cell migration assay using the transwell apparatus.