Spatially Resolved Metabolomics of Optimal Cutting Temperature (OCT) Compound-Embedded Tumors

Mass spectrometry imaging (MSI) is emerging as a powerful tool for uncovering the distribution of metabolites in the tumor microenvironment and studying tumor metabolism in vivo. To date, MSI of biobanked tissues contextualized by patient data has been limited to peptides, proteins, and glycans-with few examples for metabolites. This is because most biobanked fresh-frozen tissue required for spatial metabolomics is embedded in optimal cutting temperature (OCT) compound to preserve structural features and mitigate thermal decay. However, OCT introduces abundant polyethylene glycol and polyvinyl alcohol interferents. Herein, we use nanospray desorption electrospray ionization (nano-DESI) to demonstrate MSI of metabolites in OCT-embedded tissue. Metabolite coverage and sensitivity for tissue mimetic homogenates embedded in OCT and an MSI-compatible material, carboxymethylcellulose (CMC), exhibited excellent agreement. We apply our ambient MSI workflow to study the impact of methionine-restriction in a preclinical mouse model undergoing adoptive T-cell therapy. After tumor incubation (8 days), lymphoma-bearing mice were maintained on a complete or methionine-restricted diet for 2 days. Nano-DESI MSI revealed a heterogeneous tumor microenvironment, with multiple methionine-cycle intermediates (S-adenosylmethionine, S-adenosylhomocysteine) and related metabolites, including known T-cell modulators (1-methylnicotinamide, polyamines) localizing to tumor subregions. Methionine-restricted tumors exhibited reduced methionine and elevated S-adenosylmethionine, relative to the control group. Overall, this work establishes the potential for spatial metabolomics of fresh-frozen OCT-embedded tumors, unlocking the wealth of information stored in primary tissue biobanks and consequently accelerating our understanding of cancer metabolism and treatment.