A Label-Free Approach to Identify Inhibitors of α4β7-Mediated Cell Adhesion to MadCAM

Abstract

Traditionally, cell adhesion assays are performed in a manual workstation format using fluorescence-based readouts. Herein, the authors describe a label-free homogeneous assay to identify inhibitors of α4β7 integrin-mediated cell adhesion to its ligand, the mucosal addressin cell adhesion molecule (MadCAM), using the SRU BIND platform. The biosensor is optically based and comprises a subwavelength polymer grating. The assay was validated using standard compounds and an α4 blocking antibody and correlated very closely with the manual assay format when running a battery of test compounds of varying potencies. Cell adhesion was strictly dependent on the presence of divalent cations where Mg2+ was greater than Ca2+ at promoting cell adhesion. This homogeneous and label-free format exhibited low variability with a calculated Z′ of 0.6. In addition to measuring α4β7-mediated 8866 cell adhesion to MadCAM, the authors also demonstrate that this platform can measure adhesion of Jurkat cells expressing α4β1 to the vascular cell adhesion molecule. Thus, the SRU BIND platform is widely applicable to measuring cell adhesion events mediated by other integrins binding to their receptors in an assay format that is amenable to high-throughput screening.

Label-free, live optical imaging of reprogrammed bipolar disorder patient-derived cells reveals a functional correlate of lithium responsiveness

“…The phenotype itself, ΔPWV, can be interpreted as a measure of adhesion of cells to the biosensor. 13 BD lithium nonresponder cells adhered less strongly than cells from bipolar patients who respond well to lithium. Interestingly, cells from control individuals without BD appeared to be intermediate between lithium-responsive and nonresponsive BD patient cells.…”

“…For example, the human 8866 B cell line endogenously expresses the α4β7 integrin, which is known to be a ligand for the extracellular matrix protein, MadCAM. 13 The addition of 8866 cells to MadCAM-coated biosensors elicits a strong ΔPWV response whereas a minimal response is measured when biosensors are coated with a control protein. Furthermore, the ΔPWV response of 8866 cells on MadCAM-coated sensors is blocked to completion by an α4-specific antibody, demonstrating that the ΔPWV signal is mediated by integrin–CAM interactions.…”

“…12 These changes can be measured and quantified with high sensitivity and serve the basis of functional cell-based assays for identifying compounds that can modulate specific ligand–receptor signaling pathways. 13,14 The BIND Scanner used in our experiments has sufficiently high spatial and temporal resolution that single cell morphologies can be segmented and a number of cellular phenotypes quantified, including cell migration, membrane ruffling, mitotic events, apoptosis, proliferation and immune cell activation. 14 This makes the platform particularly advantageous for characterizing disease-associated cellular phenotypes, which may be applied for high-throughput screening or diagnostic efforts.…”