Unlabelled Spatial Metabolic Covariance Analysis (US-MCA) Reveals Tumor-Associated Reorganization of Interorgan Choline Metabolic Response Patterns in Lung Cancer

Abstract Tumor progression is increasingly recognized as a systemic disease that extends beyond local metabolic reprogramming and alters the metabolism of host organs. However, existing metabolomics approaches either lack spatial information or provide predominantly static abundance measurements, limiting assessment of coordinated, organ-specific metabolic responses in vivo. We combined whole-body airflow-assisted desorption electrospray ionization mass spectrometry (AFADESI-MSI) with a semi-quantitative, perturbation-based framework termed Unlabelled Spatial Metabolic Covariance Analysis (US-MCA) to characterize tumor-associated spatiotemporal covariation in metabolic response patterns across organs. Whole-body AFADESI-MSI depicted organ-specific metabolic profiles under physiological conditions and revealed heterogeneous metabolic alterations in host organs during tumor growth. Comparison with the D9-choline tracing experiment showed concordant response patterns for the subset of reliably detected choline-derived signals, while highlighting the limited isotopic coverage of downstream one-carbon metabolites. US-MCA indicated tumor-associated reorganization of choline-related response patterns across multiple organs. Coordinated changes in choline- and methionine-related signals in the tumor and liver were consistent with a potential association between hepatic metabolic adaptation and tumor metabolic demand. Cell co-culture experiments further showed methionine dependence of lung cancer cells under the tested conditions, but did not directly provide inter-organ metabolite transport.This study established US-MCA as an exploratory framework for assessing semi-quantitative, spatially resolved metabolic covariation after a defined perturbation. The results support tumor-associated reorganization of choline-related metabolic response patterns, but do not constitute measurements of biochemical mass flux, instantaneous reaction rates, or direct inter-organ metabolite transport.