3D imaging of pulmonary lymphatics in metastatic melanoma. (A) Lungs were collected from Prox1-eGFP mice 18 days after i.v. B16.F10-mCherry melanoma cell injections. (B) Lungs were fixed, cleared, and immunostained for visualization of melanoma metastases, the pulmonary lymphatic vasculature, and smooth muscle surrounding airways and arteries in 3D using light-sheet microscopy. Images show 3D maximum intensity projections of subpleural metastases above single z-slices from the same nodules showing their association with initial lymphatic vessels. (C) 3D maximum intensity projections and matched single z-slice images showing examples of cancer cell material inside bronchovascular cuff and collecting lymphatics. Images are representative of three independent experiments.