This study reveals that in vitro senescence induction methods significantly influence intracellular biomolecular profiles, showing both shared and unique metabolic responses. A novel bioanalytical pi....

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In recent years, mass spectrometry-based imaging techniques have improved at unprecedented speeds, particularly in spatial resolution, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) experiments can now routinely image molecular profiles of single cells in an untargeted fashion. With the introduction of MALDI-immunohistochemistry (IHC), multiplexed visualization of targeted proteins in their native tissue location has become accessible and joins the suite of multimodal imaging techniques that help unravel molecular complexities. However, MALDI-IHC has not been validated for use with cell cultures at single-cell level. Here, we introduce a workflow for combining MALDI-MSI and MALDI-IHC on single, isolated cells. Patient-derived cells from glioblastoma tumor samples were imaged, first with high-resolution MSI to obtain a lipid profile, followed by MALDI-IHC highlighting cell-specific protein markers. The multimodal imaging revealed cell type specific lipid profiles when comparing glioblastoma cells and neuronal cells. Furthermore, the initial MSI measurement and its sample preparation showed no significant differences in the subsequent MALDI-IHC ion intensities. Finally, an automated recognition model was created based on the MALDI-MSI data and was able to accurately classify cells into their respective cell type in agreement with the MALDI-IHC markers, with triglycerides, phosphatidylcholines, and sphingomyelins being the most important classifiers. These results show how MALDI-IHC can provide additional valuable molecular information on single-cell measurements, even after an initial MSI measurement without reduced efficacy. Investigation of heterogeneous single-cell samples has the potential of giving a unique insight into the dynamics of how cell-to-cell interaction drives intratumor heterogeneity, thus highlighting the perspective of this work.