PNNL

Abstract

Human disease states are biomolecularly multifaceted and can span across phenotypic states, therefore it is important to understand diseases on all levels, across cell types, and within and across microanatomical tissue compartments. In order to obtain an accurate and representative view of the molecular landscape within human lungs, this fragile tissue must be inflated and embedded to maintain spatial fidelity of the location of molecules and minimize molecular degradation for molecular imaging experiments. Here, we evaluated multiple inflation and embedding matrices (agarose, 5% carboxymethyl cellulose, and hydroxypropyl methylcellulose polyvinylpyrrolidone) and determined effective tissue preparation protocols for performing bulk and spatial omics measurements as well as in vitro models with precision cut lung slices on the same lung tissue. Through the evaluation of the bulk proteomics, lipidomics, metabolomics, and spatial metabolomics results, we determined an optimal method for agarose inflated lungs for use in multi-modality experiments. Mass spectrometry imaging methods were optimized to boost the number of annotatable molecules in agarose inflated lung samples. This optimized protocol permitted the observation of unique lipid distributions within several airway regions in the lung tissue block. Laser capture microdissection (LCM) of these airway regions followed by high-resolution proteomic analysis allowed us to begin linking the lipidome with the proteome in a spatially resolved manner, where we observed proteins with high abundance specifically localized to the airway regions. Using this optimized method, we demonstrate the versatility of the inflation method as we start to reveal how the metabolome, lipidome, and proteome are connected spatially in human lungs and across disease states through a variety of different experiments.