LeGO-3D: 3D imaging of lung metastases and vascularisation using light sheet fluorescence microscopy

Cancer metastasis involves a complex cascade of events, where cancer cells migrate from their site of origin to secondary sites via the lymphatic and circulatory system. During this process, some cancer subclones will successfully ‘seed’ at distant organs to generate lethal metastases. Here, we optimised a method for tracking cancer cells in metastatic breast cancer tumours and investigate their complex interplay with the lung vasculature using lentiviral-based optical barcoding (LeGO). Given the regional heterogeneity in lung tissue microenvironments as well as lobar asymmetry, we used light sheet microscopy to perform three-dimensional (3D) imaging of wholemount lung lobes. The results revealed that polychromatic metastases occurred less frequently than monochromatic metastases and were more likely to be located nearer to blood vessels in both spontaneous (i.e. mammary fat pad injections) and experimental (i.e. tail vein injections) mouse assays of metastasis. This 3D imaging and analytic pipeline can provide unique insights about metastatic heterogeneity and dynamics, and represents a new avenue for studying therapeutic response across large volumes of lung tissue.