Multimodal Mass Spectrometry Imaging with a Metal-Based Alignment Tool Enables Targeted Single-Cell Boronophenylalanine Uptake and Metabolic Profiling in Glioblastoma Tumors

Mass spectrometry imaging (MSI) is central to spatial multiomics; however, integrating multimodal MSI techniques and achieving accurate image coregistration remain major challenges. Here, we present an integrated multimodal MSI platform empowered by a novel metal-based alignment tool (MAT) for accurate coregistration across multimodal MSI. This platform combines laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) for single-cell elemental and isotope-tagged protein imaging with desorption electrospray ionization mass spectrometry (DESI-MS) for spatial metabolomics. Using this approach, we study the spatial distribution and metabolic fate of boronophenylalanine (BPA), a clinical boron carrier used in boron neutron capture therapy (BNCT), in an orthotopic U251 glioblastoma mouse model. Our data reveal a dual transport mechanism for BPA across both the blood-brain and blood-cerebrospinal fluid barriers. We observe pronounced intratumoral heterogeneity in boron uptake, spanning over a 2500-fold range, and identify six molecularly distinct tumor subpopulations, with the highest boron accumulation in proliferative and invasive phenotypes. Renal spatial metabolomics further show BPA clearance and biotransformation into tyrosine, 3-(4-boronophenyl)-2-hydroxypropanoic acid (BHA), and 3-(4-boronophenyl)-2-oxopropanoic acid (BOA), following the cortex-medulla-pelvis excretion pathway. This multimodal approach advances precision BNCT evaluation and offers a powerful tool for dissecting tumor heterogeneity and guiding personalized therapeutic strategies.