Integrated spectroscopic and morphological analyses reveal cellular shifts in gene-silenced melanoma CSCs

Intratumoral heterogeneity remains a major barrier to durable cancer therapies, largely driven by the persistence of cancer stem cells (CSCs). In this study, we employed an integrated, multi-scale approach to investigate how melanoma CSCs respond to siRNA-mediated silencing of three key regulatory genes: KLF4, SHH, and HIF1α. Using a combination of morphological, molecular, spectroscopic, and elemental analyses, we explored structural and biochemical consequences of gene knockdown. Gene silencing resulted in significant changes in cell shape and size, reduced F-actin organization, and decreased PFN1 expression, indicating a loss of stem-like properties. ATR-FTIR spectroscopy revealed shifts in biomolecular composition, notably a reduction in amide III intensity and an increase in lipid ester content. SEM-EDS point-based elemental analysis revealed relative differences in carbon and nitrogen levels between selected central and peripheral regions of silenced and control cells, at the micron-scale working depth, reflecting broader elemental distribution trends rather than precise subcellular compartmentalization. XPS analysis further confirmed these differences, providing additional insights into the elemental composition of the cellular surface. The integration of FTIR spectroscopy into this study highlights the potential of infrared spectroscopy as a powerful tool in cancer research. These findings demonstrate that targeting critical regulatory pathways induces cytoskeletal and biochemical remodelling in melanoma CSCs, offering a multi-dimensional perspective on cellular plasticity.