Optical Tissue Clearing and Small-Molecule Labeling of Paraffin-Embedded Breast Cancer and Axillary Lymph Node Human Tissue Samples.

Breast cancer is the most frequently diagnosed cancer in women, representing approximately 25% of all cancers in women worldwide. Both breast cancer research and histopathological diagnostics mainly show a two-dimensional planar view of the three-dimensional breast cancerous architecture. Recently, the application of optical tissue clearing, together with 3D microscopy, has been applied to visualize the complexity of whole tumor samples. Preliminary studies on whole-organ mouse mammary glands and tissues from human breast cancer patients subjected to optical tissue clearing and volumetric imaging have enabled the detection of previously unrecognized spatial cellular interactions and structural features within intact breast tissue. There is currently no standardized clearing workflow for breast and lymph node tissues. In this protocol, we optimized and validated the MASH (multiscale architectonic staining of human cortex) immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO)-like clearing and labeling pipeline for the investigation of formalin-fixed and paraffin-embedded (FFPE) breast tissue and lymph nodes obtained from breast cancer patients. This illustrates the application of the protocol in a new biological and clinical context, as human breast and lymph node tissues differ substantially from brain tissues in their composition, architecture, and optical properties. Whole FFPE tissue blocks are deparaffinized in liquid paraffin and xylene, bleached through methanol dehydration and a subsequent hydrogen peroxide incubation, and stained with a diverse set of small molecule dyes. As a next step, the tissues are delipidated and subjected to refractive index matching with ethyl cinnamate to reach optimal tissue transparency. Importantly, the applied dehydration and delipidation nicely preserve the morphology of the tissue, and the shrinkage is minimal. This allows reliable 3D imaging of large tissue samples within a timeframe of 10 days, providing clinicians and biomedical researchers with a more holistic view of the FFPE tissue sample and its spatial organization. Key features • Optimized, low-cost approach for optical tissue clearing and labeling of whole human breast and lymph node FFPE tissue samples ≥30 × 20 × 3 mm. • Prepared tissue samples can be assessed by means of advanced 3D volume microscope modalities, such as twophoton microscopy (TPM) or light-sheet fluorescence microscopy (LSFM). • The clearing protocol is compatible with a variety of small-molecule dyes, such as neutral red (cell body), eosin Y (cytoplasm, collagen), and methyl green (nucleus).