Imaging‐Guided Live Single‐Cell Lipid Profiling of Leader and Follower Cells During Collective Migration of Triple‐Negative Breast Cancer Cells

ABSTRACT Leader and follower cells drive collective cancer cell migration through distinct and interactive roles, but their specific metabolic signatures remain largely unknown. Here, we developed and applied a workflow that integrates time‐lapse imaging, single‐cell tracking, microsampling and direct infusion high‐resolution mass spectrometry to profile the lipidomes of individually sampled leader and follower cells from highly migratory TNBC lines HCC1143 and HCC38. Approximately 120 metabolites were detected per cell at the MS ¹ level, and 70 compounds were assigned based on matched MS ² fragments, indicative of detected head‐group or side‐chain fragment ions. Distinct leader‐follower differences emerged in each cell line. HCC38 cells showed increased fatty acids (FA) in leader cells. In addition, alterations in several phosphatidylethanolamine (PE) and diacylglycerol (DG) species between leader and follower cell subpopulations were detected. Alternatively, leader and follower cells in HCC1143 exhibited changes in phosphatidylcholine (PC) species, with PC(34:1) confirmed by a fragment ions at m/z 184.0733. Overall, this study revealed the dynamic lipid metabolism in live migratory cancer cells, and highlights the potential of integrated dynamic imaging and single‐cell mass spectrometry for studying functional heterogeneity within leader‐follower cancer phenotypes.