This article introduces a novel technique called three-dimensional cyclic immunofluorescence (3D CyCIF) for high-plex, high-resolution imaging of cell states and immune niches within human tumor tissues. The authors demonstrate that traditional thin tissue sections, typically 5 µm thick, result in fragmented cells and inaccurate phenotyping, whereas sections eightfold to tenfold thicker (30–40 µm) enable accurate morphological assessment of intact cells, organelles, and cell-to-cell contacts. Using 3D CyCIF on melanoma samples, the study reveals detailed tumor microarchitecture, including immune cell migration, organelle structures, and varied membrane-to-membrane interactions (classified as type I, II, or III) that represent different functional contacts between tumor and immune cells. The findings emphasize that 3D imaging is crucial for observing phenotypic plasticity in tumor cells and identifying complex, localized inflammatory and immune signaling neighborhoods that are often obscured in two-dimensional analysis.
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