D₂O-Probed Raman Microspectroscopy Distinguishes the Metabolic Dynamics of Macromolecules in Organellar Anticancer Drug Response

To profile the metabolic dynamics responding to drugs at the single-cell/organelle resolution, rapid and economical mechanism-revealing methods are required. Here, we introduced D₂O-probed Raman microspectroscopy in combination with the multivariate curve resolution-alternating least squares (MCR-ALS or MCR) algorithm. Exploiting MCR to deconvolute each macromolecular component specifically, the method is able to track and distinguish changes in lipid and protein metabolic activities in a human cancer cell line (MCF-7) and in Saccharomyces cerevisiae, in response to the metabolism-inhibitory effect of rapamycin, which inhibits the mammalian/mechanistic target of rapamycin (mTOR) signaling. Under rapamycin, in the lipid bodies of cancer cells, metabolic activities of both protein and lipid are suppressed; in the nucleus, protein synthesis remains active, whereas lipid synthesis is inhibited; in the cytoplasm, syntheses of protein and lipid are both dose- and duration-dependent. Thus, rapamycin differentially influences protein and lipid synthesis in mTOR signaling. Moreover, the strong correlation between macromolecular-specific components of yeast and those in MCF-7 cytoplasm, nucleus, and lipid bodies revealed similarity in rapamycin response. Notably, highly metabolically active cancer cells after high-dosage rapamycin exposure (500 or 5000 × IC₅₀) were revealed, which escape detection by population-level cytotoxicity tests. Thus, by unveiling macromolecule-specific metabolic dynamics at the organelle level, the method is valuable to mechanism-based rapid screening and dissection of drug response.