Abstract
Purpose :
We recently invented ligandomics and demonstrated its validity and utility for drug target discovery by applying the technology to anesthetized mice with or without diabetic retinopathy (DR), discovering secretogranin III (Scg3) as a disease-restricted angiogenic factor and developing novel anti-Scg3 therapy. However, a limitation is that in vivo ligandomics requiring intracardial perfusion can be applied only to vascular endothelial cells (ECs) but not to other cells. This study aims to develop ex vivo ligandomics with broad applicability to any isolated cells, including retinal cells, for drug target discovery.
Methods :
Single cells were isolated from retinas of diabetic and control mice and immunopanned to anti-CD31 (EC marker) and anti-CD90.2 (retinal ganglion cell (RGC) marker) mAbs immobilized on culture plates. Three rounds of ex vivo ligandomics profiling with phage cDNA libraries displaying cellular proteins were performed to enrich cell-binding ligands that were mapped by next-generation sequencing (NGS). Comparative data analysis for diabetic vs. healthy ECs or RGCs systematically mapped cell-specific disease-restricted ligands. To validate ex vivo ligandomics, binding of clonal phage displaying Scg3 (Scg3-Phage) or vascular endothelial growth factor (VEGF-Phage) to immunopanned cells was analyzed in the presence or absence of their cognate blockers.
Results :
Clonal phage binding assay confirmed that Scg3 selectively binds only to diabetic ECs but not other immunopanned cells. VEGF binds to both diabetic and healthy ECs and RGCs. These binding activity patterns are consistent with previous reports. Three rounds of ligandomics profiling enriched cell-binding ligands by 10.8- and 16.9-fold for healthy and diabetic immunopanned ECs, respectively. Similarly, ligandomics enriched 14.5- and 17.8-fold for ligands binding to healthy and diabetic immunopanned RGCs, respectively. NGS identified a list of enriched cell-binding ligands, including Scg3 that was preferentially enriched with a 5.4-fold increase in binding to diabetic vs. healthy ECs.
Conclusions :
Scg3- and VEGF-Phage binding activity patterns to immunopanned cells support the validity of ex vivo ligandomics. Ligandomics profiling globally mapped disease-selective ligands for immunopanned ECs and RGCs. This study confirmed that ex vivo ligandomics is broadly applicable to any type of isolated cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.