Label-Free Raman Spectroscopy Reveals Metabolic Signatures Associated with MGMT Promoter Methylation Status in...

Glioblastoma (GBM) response to Temozolomide (TMZ) critically depends on O⁶-methylguanine-DNA methyltransferase (MGMT) promoter methylation. Clinically, the unmethylated MGMT subtype─representing the predominant GBM population─universally develops TMZ resistance, driving an urgent need for novel therapeutics. While metabolic reprogramming represents a potential therapeutic target, current methylation detection methods cannot provide targetable metabolic information. To elucidate methylation-associated metabolic signatures while mitigating complex biological background noise, we established a stepwise validation framework using CRISPR-engineered cell models. Raman spectroscopy (RS) analyzed CRISPR-dCas9-edited GBM cell lines to extract pure metabolic fingerprints. In MGMT hypermethylated (MGMT-HYPER) GBMs, the saturated fatty acid (SFA) Raman peak was elevated. Conversely, MGMT hypomethylated (MGMT-HYPO) GBMs exhibited enhanced peaks for deoxyribonucleic acid (DNA), amino acids, and unsaturated fatty acids (UFAs). RS-identified metabolites were validated by liquid chromatography-mass spectrometry. Subsequently, uniform manifold approximation and projection (UMAP) effectively clustered HYPER/HYPO Raman spectra. A Raman classification model utilizing a core 13-feature signature from in vitro findings achieved a 94.5% area under the curve (AUC) in distinguishing methylation status across 62 clinical specimens. Correlation analysis demonstrated: (1) a positive correlation of SFA content (1444 cm^-1) with methylation levels in MGMT-HYPER tumors; (2) an inverse association of DNA levels (1093 cm^-1) with methylation in intermediate-methylation GBMs; and (3) a negative correlation between UFA content (1266 cm^-1) and methylation in unmethylated GBMs. This study confirms RS potential for assessing MGMT methylation status in tumors and elucidates metabolic signatures associated with differential methylation in GBMs, providing targets for metabolic intervention against TMZ resistance.