Pixel-based AFM-IR uncovers nanoscale lipid remodeling in MPNST cells

Malignant peripheral nerve sheath tumors (MPNST) exhibit pronounced alterations in lipid organization that contribute to tumor aggressiveness and resistance to radiotherapy. In this work, we combine atomic force microscopy-infrared spectroscopy (AFM-IR) and fluorescence imaging to investigate nanoscale lipid remodeling in Schwann and MPNST cells exposed to cannabidiol (CBD) and ionizing radiation, while introducing a new semiquantitative strategy for AFM-IR image analysis. Conventional band-based AFM-IR spectroscopy was first employed to identify characteristic biochemical signatures in the perinuclear region, revealing CBD- and irradiation-dependent modifications of phospholipids (1260 cm^-1-1240 cm^-1) and cholesteryl esters, monitored via the ester carbonyl band at 1740 cm^-1. These spectral changes provided a biochemical basis for further nanoscale analysis, but were restricted to intensity-based interpretation. To overcome this limitation, we introduce, for the first time, a pixel-based AFM-IR semi-quantification framework that converts nanospectroscopic maps into statistically robust biochemical metrics. High-resolution AFM-IR images were processed to extract pixel-resolved ester-specific signals, enabling semi-quantitative determination of both the average cholesteryl ester signal intensity and the nanoscale surface area occupied by ester-rich domains. Statistical evaluation using ANOVA with Tukey's post-hoc test allowed direct comparison of lipid redistribution across experimental conditions. Application of this framework revealed distinct nanoscale patterns of cholesteryl ester remodeling in Schwann versus MPNST cells under CBD and irradiation, including pronounced spatial reorganization that was not evident from spectral intensities alone. Importantly, the AFM-IR-derived spatial metrics were independently validated by fluorescence lipid droplet staining, demonstrating similar trends between nanoscale infrared measurements and cellular lipid abundance. In parallel, AFM-IR analysis of the Amide I and II regions uncovered CBD-dependent modulation of protein secondary structure, highlighting differential responses between normal and malignant cells. Overall, this study establishes a transferable, pixel-based AFM-IR analysis strategy for nanoscale biochemical semi-quantification and demonstrates its utility in resolving lipid organization and remodeling in complex biological systems.