Tissue clearing and immunostaining to visualize the spatial organization of vasculature and tumor cells in mouse liver.

The liver has a complex and hierarchical segmental organization of arteries, portal veins, hepatic veins and lymphatic vessels. In-depth imaging of liver vasculature and malignancies could improve knowledge on tumor micro-environment, local tumor growth, invasion, as well as metastasis. Non-invasive imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI) and positron-emission transmission (PET) are routine for clinical imaging, but show inadequate resolution at cellular and subcellular level. In recent years, tissue clearing - a technique rendering tissues optically transparent allowing enhanced microscopy imaging - has made great advances. While mainly used in the neurobiology field, recently more studies have used clearing techniques for imaging other organ systems as well as tumor tissues. In this study, our aim was to develop a reproducible tissue clearing and immunostaining model for visualizing intrahepatic blood microvasculature and tumor cells in murine colorectal liver metastases. CLARITY and 3DISCO/iDISCO+ are two established clearing methods that have been shown to be compatible with immunolabelling, most often in neurobiology research. In this study, CLARITY unfortunately resulted in damaged tissue integrity of the murine liver lobes and no specific immunostaining. Using the 3DISCO/iDISCO+ method, liver samples were successfully rendered optically transparent. After which, successful immunostaining of the intrahepatic microvasculature using panendothelial cell antigen MECA-32 and colorectal cancer cells using epithelial cell adhesion molecule (EpCAM) was established. This approach for tumor micro-environment tissue clearing would be especially valuable for allowing visualization of spatial heterogeneity and complex interactions of tumor cells and their environment in future studies.