The initiation and progression of human cancers is profoundly influenced by interaction with the adaptive and innate immune system. Moreover, activation of T cell immunosurveillance is the foundation of contemporary immunotherapy. However, the composition and organization of the tumour-immune microenvironment (TIME) remains incompletely understood. This webcast will describe an analysis of the TIME in multiple human tumours (e.g. melanoma, colorectal cancer) using newly developed high-plex 3D cyclic immunofluorescence (3D-CyCIF) imaging. The high spatial resolution of 3D imaging in comparison to common 2D “spatial proteomics” makes it possible to analyse individual juxtacrine signalling complexes and intracellular organelles. Precise 3D imaging of cell membranes also reveals that cells in tumours are tightly packed: T cells, for example, simultaneously interact with 4-10 adjacent cells. 3D-CyCIF is a public domain approach that can be immediately implemented on existing confocal and light sheet fluorescence microscopes (LSFM). Learn: Strengths and limitations of 3D spatial proteomics and how 2D and 3D approaches can be combined New insights into the tumour microenvironment that 3D imaging provides How my group can perform 3D-CyCIF and start to analyse 3D spatial profiling data