3D angiogenesis-on-a-chip coupled with single-cell RNA sequencing analysis reveals potential roles of autophagy in angiogenic sprouting

There have been the emerging interests in the role of autophagy in angiogenesis. Cellular heterogeneity of autophagy in sprouting endothelial cells (ECs) has been overlooked, and an EC sprouting model recapitulating this heterogeneity is absent in previous autophagy studies. To address this dearth, we present an advanced angiogenesis-on-a-chip to robustly reconstruct multiple 3D sprouts with distinguishable sprout endpoints. We monitored the spatial pattern of an autophagy activation marker LC3 on sprout endpoints and observed severe defects in angiogenic sprouting by autophagy inhibition after drug treatment or gene downregulation. Single-cell RNA sequencing (scRNA-seq) on sprouting ECs revealed reciprocal expression patterns of autophagy-related genes between stalk- and tip-like ECs in the leading positions of elongating sprouts, implying a model that autophagy contributes to tip-stalk specification. Our systematic strategy coupling angiogenesis-on-a-chip with scRNA-seq analysis improves understanding of the roles of autophagy in angiogenesis, which may contribute to designing effective anti-angiogenic therapeutics.