A new pipeline for pathophysiological analysis of the mammary gland based on organoid transplantation and organ clearing

Recent developments in techniques for tissue clearing and size reduction have enabled optical imaging of whole organs and studying rare tumorigenic events occurring in vivo. The adult mammary gland provides a unique model to investigate physiological or pathological processes as morphogenesis or epithelial cell dissemination. Here, we established a new pipeline to study rare cellular events occurring in the mammary gland, by combining orthotopic transplantation of mammary organoids with the uDISCO organ size reduction and clearing method. This strategy allowed us to analyze the behavior of individually labeled cells in the regenerated mammary gland. As a proof of concept, we analyzed the localization of rare epithelial cells overexpressing eGFP fused with atypical protein kinase C iota (GFP-aPKCi+) in the normal mammary gland. Using this analytical pipeline, we were able to visualize epithelial aPKCi+ cells escaping from the normal mammary epithelium and disseminating into the surrounding stroma. This technical resource should benefit mammary development and tumor progression studies.