Ambient mass spectrometry imaging enables spatial metabolomics of optimal cutting temperature compound (OCT)-embedded tumors

Abstract 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 . However, to date, MSI of primary patient 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 compound (OCT), which introduces high-abundance polymeric interferents. Herein, we use nanospray desorption electrospray ionization (nano-DESI) to demonstrate the MSI of metabolites in OCT-embedded tissue. Metabolite coverage and sensitivity for prepared tissue mimetic homogenates embedded in OCT and an MSI-compatible material, carboxymethylcellulose (CMC), showed excellent agreement. We apply our ambient MSI workflow to detect changes in intratumoral methionine using a preclinical cancer mouse model undergoing adoptive T-cell therapy. Eight days after tumor incubation, 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 levels and elevated S-adenosylmethionine, relative to the control group. Overall, this work demonstrates spatial metabolomics on fresh-frozen OCT-embedded tissue, unlocking the wealth of information stored in primary tissue biobanks and consequently accelerating our understanding of cancer metabolism and treatment.