Combining Kinetic Ligand Binding and 3D Tumor Invasion Technologies to Assess Drug Residence Time and Anti-metastatic Effects of CXCR4 Inhibitors

Brad Larson and Leonie Rieger - Nicolas Pierre - Hilary Sherman

2015

BioTek Instruments, Inc., Winooski, VT - Cisbio US, Inc., Bedford, MA - Corning Incorporated, Life Sciences, Kennebunk, ME

Metastasis, the spread of cancer cells from the original tumor to secondary locations within the body, is linked to approximately 90% of cancer deaths1. 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 migration2. 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 ligandreceptor interaction. However, awareness of the importance of binding kinetics has started to increase due to accumulating evidence3, 4, 5, 6 suggesting that the in vivo effectiveness of ligands may be attributed to the time a particular ligand binds to 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 cells7. 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 tumors8, were included, in addition to primary human dermal fibroblasts. 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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