Tissue Clearing for Lymph Nodes: 3D Mapping of Nerves and the Immune Microenvironment

ABSTRACT Lymph nodes (LNs) are central hubs of adaptive immunity and key relay sites for metastatic tumour cells. However, conventional two‐dimensional histology samples only a small fraction of each node, potentially missing micrometastases and multifocal involvement while obscuring long‐range spatial relationships among tumour deposits, immune populations, stromal structures, vasculature and nerves. Tissue clearing combined with light‐sheet fluorescence microscopy now enables whole‐mount imaging of intact LNs at near‐cellular resolution. Unlike general reviews of tissue clearing or three‐dimensional pathology, this review specifically considers the LN as a spatially organised neuro–immune–tumour organ. The major clearing‐method families and LN‐specific design considerations are summarised, including reagent penetration, fluorescence and epitope preservation, refractive‐index matching, labelling strategies and imaging optics. Quantitative workflows are then outlined for segmenting metastatic deposits and stromal compartments, skeletonising vascular and neural networks, and deriving volumetric and distance‐based metrics. These approaches enable assessment of metastatic burden, multifocality, compartmental localisation, and spatial relationships between tumours and the capsule, blood vessels, lymphatic sinuses, nerves and immune niches. On this basis, the neuro–tumour–immune niche is proposed as a conceptual framework for investigating how neural inputs, tumour cells, immune populations and stromal architecture are spatially coordinated during LN remodelling and metastatic progression. Finally, current research applications, technical limitations and future potential of cleared‐LN imaging are discussed as a complementary approach to conventional histopathology in experimental studies and, after prospective validation, in human sentinel and metastatic LNs.