Integrating multiplex immunofluorescent and mass spectrometry imaging to map myeloid heterogeneity in its metabolic and cellular context

Cells often adopt different phenotypes, dictated by tissue-specific or local signals such as cell-cell and cell-matrix contacts or molecular micro-environment. This holds in extremis for macrophages with their high phenotypic plasticity. Their broad range of functions, some even opposing, reflects their heterogeneity, and a multitude of subsets has been described in different tissues and diseases. Such micro-environmental imprint cannot be adequately studied by single-cell applications, as cells are detached from their context, while histology-based assessment lacks the phenotypic depth due to limitations in marker combination. Here, we present a novel, integrative approach in which 15-color multispectral imaging allows comprehensive cell classification based on multi-marker expression patterns, followed by downstream analysis pipelines to link their phenotypes to contextual, micro-environmental cues, such as their cellular (“community”) and metabolic (“local lipidome”) niches in complex tissue. The power of this approach is illustrated for myeloid subsets and associated lipid signatures in murine atherosclerotic plaque. [Display omitted] •Multispectral IF imaging reveals myeloid phenotypes and maps their tissue distribution•Integrating it with MS imaging unveils phenotype-associated micro-environmental niches•Plaque myeloid phenotypes strongly correlate with their cellular and molecular contexts•UV bleaching prior to IF staining strongly reduces the tissue’s autofluorescence In this paper, Goossens et al. introduce a novel approach that integrates 15-plex multispectral immunofluorescent microscopy with mass spectrometry imaging to reveal murine atherosclerotic plaque myeloid heterogeneity. It maps the phenotypes’ tissue localization while allowing their further histological characterization and correlations with their cellular and molecular micro-environmental contexts.