Microscopy and Plate Reader–based Methods for Monitoring the Interaction of Platelets and Tumor Cells in vitro

Abstract

Platelets and their activation status play an essential role in cancer metastasis. Therefore, the anti-metastatic potential of antiplatelet drugs has been investigated for many years. However, the initial screening of these antiplatelet drugs to determine which agents can inhibit the interactions of platelets and tumor cells is very limited due to reliance upon expensive, time-consuming, and low-throughput animal experiments for screening. In vitro models of the platelet–tumor cell interaction can be a useful tool to rapidly screen multiple antiplatelet drugs and compare their ability to disrupt platelet–tumor cell interactions, while also identifying optimal concentrations to move forward for in vivo validation. Hence, we adopted methods used in platelet activation research to isolate and label platelets before mixing them with tumor cells (MDA-MB-231-RFP cells) in vitro in a static co-culture model. Platelets were isolated from other blood components by centrifugation, followed by fluorescent labeling using the dye CMFDA (CellTrackerTM Green). Labeling platelets allows microscopic observation of the introduced platelets with tumor cells grown in cell culture dishes. These methods have facilitated the study of platelet–tumor cell interactions in tissue culture. Here, we provide details of the methods we have used for platelet isolation from humans and mice and their staining for further interaction with tumor cells by microscopy and plate reader–based quantification. Moreover, we show the utility of this assay by demonstrating decreased platelet–tumor cell interactions in the presence of the T-Prostanoid receptor (TPr) inhibitor ifetroban. The methods described here will aid in the rapid discovery of antiplatelet agents, which have potential as anti-metastatic agents as well.Key features• Analysis of platelet–tumor cell binding dynamics.• In vitro methods developed for measuring platelet–tumor cell binding to enable rapid testing of antiplatelet and other compounds.• Complementary analysis of platelet–tumor cell binding by imaging and fluorimetry-based readings.• Representative results screening the effect of the antiplatelet drug, ifetroban, on platelet–tumor cell binding using the protocol.• Validation results were presented with both a TPr agonist and ifetroban (antagonist).Graphical overviewRepresentative overview of the process to isolate and label platelets, incubate platelets and tumor cells in the presence of antiplatelet agents, and image and/or quantify platelet–tumor cell interactions.