Distinct metabolic adaptations to resistance to CDK4/6 inhibitors in ER+ breast cancer identified using microfluidic mass spectrometry platform

Background: Cyclin-dependent kinase inhibitors, CDK4/6i, have been one of the most significant practice-changing advances in the treatment of estrogen receptor positive (ER+) breast cancer in the recent decade. Despite their clinical success, CDK4/6i resistance remains a major clinical challenge. Importantly, therapeutic response does not correlate with expression levels of CDK4, CDK6, or other canonical cell cycle proteins, suggesting that resistance arises from complex adaptive mechanisms. Methods: To address this challenge, we generated drug-tolerant persister cell populations (DTPs) from LCC2, LCC9 and T47D breast cancer cell lines to both palbociclib and abemaciclib. Transcriptomic profiling of these DTPs revealed significant alterations in the expression of metabolic enzymes, suggesting that metabolic reprogramming plays a central role in the survival and maintenance of CDK4/6i-tolerant cells. To further identify and quantify changes in metabolites and metabolic pathways, we analyzed these DTP populations using a dynamic sampling platform (DSP) integrating a sample introduction mechanism, a microfabricated processing device, and electrospray ionization mass spectrometry (ESI-MS) analysis. The device, manufactured via advanced microfabrication techniques, enables integration of real-time multiple processing and metabolic profiling. Full untargeted metabolomics data were evaluated using pathway level gene set enrichment analysis (GSEA). Results: Across resistant cell states, multiple metabolic pathways exhibited coordinated shifts associated with DTP-like adaptation. Palbociclib resistant LCC2-DTPs exhibit a distinct metabolic remodeling trajectory characterized by consistently elevated ubiquinone biosynthesis, an adaptation absent in abemaciclib-resistant LCC2-DTPs and other DTP sublines. This drug-specific metabolic signature suggests that palbociclib engages mitochondrial pathways in a unique manner, potentially influencing therapeutic resistance. On the other hand, pathways involving electron transport chain and fatty acid oxidation were upregulated in abemaciclib DTPs, suggesting differential metabolic regulation in response to each CDK4/6i. Conclusions: These findings highlight divergent metabolic consequences for palbociclib and abemaciclib in endocrine-resistant ER+ breast cancer and underscore the utility of microfluidic ESI-MS workflows for resolving drug-specific adaptive states relevant to therapeutic resistance and tumor progression.