‘On the Spot’ Digital Pathology of Breast Cancer Based on Single-Cell Mass Spectrometry Imaging

[Image: see text] The molecular pathology of breast cancer is challenging due to the complex heterogeneity of cellular subtypes. The ability to directly identify and visualize cell subtype distribution at the single-cell level within a tissue section enables precise and rapid diagnosis and prognosis. Here, we applied mass spectrometry imaging (MSI) to acquire and visualize the molecular profiles at the single-cell and subcellular levels of 14 different breast cancer cell lines. We built a molecular library of genetically well-characterized cell lines. Multistep processing, including deep learning, resulted in a breast cancer subtype, the cancer’s hormone status, and a genotypic recognition model based on metabolic phenotypes with cross-validation rates of up to 97%. Moreover, we applied our single-cell-based recognition models to complex tissue samples, identifying cell subtypes in tissue context within seconds during measurement. These data demonstrate “on the spot” digital pathology at the single-cell level using MSI, and they provide a framework for fast and accurate high spatial resolution diagnostics and prognostics.