Multiplexed 3D Analysis of Cell Plasticity and Immune Niches in Melanoma

Abstract

SUMMARYHomeostasis of normal tissues and the emergence of diseases such as cancer are controlled by changes in the proportions and states of diverse cell types, cell-cell interactions, and acellular components of the tissue microenvironment1. Spatial omics using highly multiplexed tissue profiling2makes it possible to study these processesin situ, usually on thin, 4-5 micron thick sections (the standard histopathology format)3. Microscopy-based tissue imaging is commonly performed at a resolution sufficient to determine cell types but not detect the subtle morphological features associated with cytoskeletal reorganisation, juxtracrine signalling, or membrane trafficking4. Here we introduce a 3D imaging approach using existing instruments and reagents that is able to characterize a wide variety of organelles and structures at sub-micron scale while simultaneously quantifying millimetre-scale spatial features. We perform high-resolution 54-plex cyclic immunofluorescence (CyCIF) imaging3on sections of primary human melanoma thick enough (30-40 microns) to fully encompass two or more layers of intact cells. In pre-invasive melanomain situ5, 3D imaging of entire cell volumes showed that transformed melanocytic cells are plastic in state and participate in tightly localised niches of interferon signalling near sites of initial invasion into the underlying dermis. Below this layer, immune cells engaged in an unexpectedly diverse array of membrane-membrane interactions as well as looser “neighbourhood” associations6. These data provide new insight into the transitions occurring during early tumour formation and demonstrate the potential for phenotyping tissues at a level of detail previously restricted to cultured cells and organoids.