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MicroPOTS Analysis of Barrett's Esophageal Cell Line Models Identifies Proteomic Changes after Physiologic and Radiation Stress.

ABSTRACT: Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of R > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.

SUBMITTER: Weke K

PROVIDER: S-EPMC8155554 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe enrichment of Gram-negative bacteria of oral origin in the esophageal microbiome has been associated with the development of metaplasia. However, to date, no study has comprehensively assessed the relationships between the esophageal microbiome and the host.MethodsHere, we examine the esophageal microenvironment in gastro-esophageal reflux disease and metaplasia using multi-omics strategies targeting the microbiome and host transcriptome, followed by targeted culture, comparative genomics, and host-microbial interaction studies of bacterial signatures of interest.ResultsProfiling of the host transcriptome from esophageal mucosal biopsies revealed profound changes during metaplasia. Importantly, five biomarkers showed consistent longitudinal changes with disease progression from reflux disease to metaplasia. We showed for the first time that the esophageal microbiome is distinct from the salivary microbiome and the enrichment of Campylobacter species as a consistent signature in disease across two independent cohorts. Shape fitting and matrix correlation identified associations between the microbiome and host transcriptome profiles, with a novel co-exclusion relationship found between Campylobacter and napsin B aspartic peptidase. Targeted culture of Campylobacter species from the same cohort revealed a subset of isolates to have a higher capacity to survive within primary human macrophages. Comparative genomic analyses showed these isolates could be differentiated by specific genomic features, one of which was validated to be associated with intracellular fitness. Screening for these Campylobacter strain-specific signatures in shotgun metagenomics data from another cohort showed an increase in prevalence with disease progression. Comparative transcriptomic analyses of primary esophageal epithelial cells exposed to the Campylobacter isolates revealed expression changes within those infected with strains with high intracellular fitness that could explain the increased likelihood of disease progression.ConclusionsWe provide a comprehensive assessment of the esophageal microenvironment, identifying bacterial strain-specific signatures with high relevance to progression of metaplasia.

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MicroPOTS Analysis of Barrett's Esophageal Cell Line Models Identifies Proteomic Changes after Physiologic and Radiation Stress.

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