Vibrational Spectroscopy Unveils Distinct Cell Cycle Features of Cancer Stem Cells in Melanoma

Cancer stem cells (CSCs) play a central role in melanoma growth, resistance to treatment, and relapse, however, their dynamic regulatory behavior remains poorly understood. Vibrational spectroscopy offers a unique, label-free approach to investigate cellular heterogeneity at the molecular level. Here, we explored the biochemical and regulatory dynamics of CSCs identified by using a time-course design, integrating infrared and Raman spectroscopies with cell cycle analysis and immunocytochemistry targeting the checkpoint proteins p16 and p21. CSCs, non-cancer stem cells (NCSCs), and bulk CHL-1 melanoma cells were monitored at 11, 24, 48, and 72 hours. CSCs showed a steady S-phase with an early rise in p16 followed by a subsequent increase in p21 expression, indicating a dynamic state of cell cycle checkpoints. In contrast, NCSCs and CHL-1 cells showed more transient p16/p21 expression and CHL-1 exhibited a marked p16 increase at 24 hours. Spectroscopic analysis revealed that CSCs exhibited distinct vibrational profiles, predominantly in the nucleic acid-, protein- and lipid-associated regions. These differences were further supported by principal component and hierarchical clustering analyses, which consistently distinguished CSCs from NCSCs. Our findings underline the potential of vibrational spectroscopy to sensitively detect CSC-specific regulatory patterns and support its use in detecting new therapeutic targets in melanoma.