Single-cell Raman and mass spectrometry analysis to probe cellular heterogeneity in tamoxifen uptake and metabolism.

Current drug discovery is limited by the lack of single-cell data on drug uptake, metabolism, and effects, as population-level methods obscure cellular heterogeneity. While single-cell RNA sequencing has revealed drug resistance mechanisms, it cannot simultaneously measure drug concentrations and cellular responses. Raman spectroscopy probes single-cell drug effects but lacks sensitivity for drug or its metabolite quantification, whereas single-cell mass spectrometry (MS) offers high sensitivity but consumes samples, preventing repeated measurements. Integrating Raman spectroscopy with MS enables simultaneous assessment of cellular states and drug metabolism. However, existing studies are limited by small sample sizes and single drug concentrations. We employ a combined single-cell Raman and mass spectrometry (Raman-MS) approach to investigate variability in drug uptake, metabolism, and effects in HepG2 liver cancer cells. The cells were exposed to three concentrations of tamoxifen, after which we quantified the heterogeneity in tamoxifen and its hepatotoxic metabolites. This validates the potential of single-cell analysis for advancing drug discovery and cancer research. Our results indicated that tamoxifen induces concentration-dependent metabolic changes in single liver cancer cells, as revealed by Raman spectroscopy and mass spectrometry. The findings highlight a potential threshold concentration beyond which cellular integrity is compromised, underscoring the importance of single-cell approaches for understanding drug uptake, metabolism, and therapeutic heterogeneity.