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Combining Kinetic Ligand Binding and 3D Tumor Invasion Technologies to Assess Drug Residence Time and anti-Metastatic Effects of CXCR4 Inhibitors下載
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February 13, 2015
Metastasis, the spread of cancer cells from the original tumor to secondary locations within the body, is linked to approximately 90% of cancer deaths (Saxe, 2013). The expression of chemokine receptors, such as CXCR4 and CCR7, is tightly correlated with the metastatic properties of breast cancer cells. In vivo, neutralizing the interaction of CXCR4 and its known ligand, SDF1-α (CXCL12), significantly impaired the metastasis of breast cancer cells and cell migration (Muller et al., 2001). Traditionally, the discovery of novel agents has been guided by the affinity of the ligand for the receptor under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. However, awareness of the importance of binding kinetics has started to increase due to accumulating evidence (Swinney, 2004; Copeland et al., 2006; Tummino and Copeland, 2008; Zhang and Monsma, 2009) suggesting that the in vivo effectiveness of ligands may be attributed to the time a particular ligand resides at its receptor (Drug-Target Residence Time).
Similarly, appropriate in vitro cell models have also been lacking to accurately assess the ability of novel therapies to inhibit tumor invasion. Tumors in vivo exist as a three-dimensional (3D) mass of multiple cell types, including cancer and stromal cells (Mao et al., 2013). Therefore, incorporating a 3D spheroid-type cellular structure that includes co-cultured cell types forming a tumoroid, provides a more predictive model than the use of individual cancer cells cultured on the bottom of a well in traditional two-dimensional (2D) format.
Here we examine the drug-target residence time of various CXCR4 inhibitors using a direct, homogeneous ligand binding assay and CXCR4 expressing cell line in a kinetic format. This inhibitor panel was further tested in a 3D tumor invasion assay to determine whether there is a correlation between the molecule’s CXCR4 residence time and inhibition of the phenotypic effect of tumor invasion. MDA-MB-231 breast adenocarcinoma cells, known to be invasive, and metastasize to lung from primary mammary fat pad tumors (Kamath et al., 2001), were included, in addition to primary human dermal fibroblasts. The cells were aggregated into 3D structures using Corning Spheroid Microplates containing an Ultra Low Attachment surface. A novel cell imaging multi-mode reader was incorporated to provide PMT-based assessment of drug-target residence time, as well as automated imagebased monitoring of tumor invasion through a basement membrane matrix. Cellular analysis algorithms provided accurate quantification of changes to the original tumoroid structure, as well as invadopodia development. The combination presents an accurate, yet easy-to-use method to assess target-based and phenotypic effects of new, potential anti-metastatic drugs.
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