Noninvasive exosomal proteomic biosignatures, including cystatin SN, peroxiredoxin‐5, and glycoprotein VI, accurately predict chronic rhinosinusitis with nasal polyps

Abstract

BackgroundExosomes are secreted epithelial‐derived vesicles that contain a conserved protein array representative of their parent cell. Exosomes may be reproducibly and noninvasively purified from nasal mucus. The exosomal proteome can be quantified using SOMAscanTM, a highly multiplexed, aptamer‐based proteomic platform. The purpose of this study was to determine whether chronic rhinosinusitis with nasal polyps (CRSwNP) has a unique predictive exosomal proteomic biosignature.MethodsExosomes were isolated from whole mucus sampled from control and CRSwNP patients (n = 20 per group) by differential ultracentrifugation. The SOMAscanTM platform was used to simultaneously quantify 1310 biologically relevant human proteins. Matched tissue and whole mucus proteomes were also analyzed. Differential protein expression and discriminatory power were calculated using the unweighted pair group method with arithmetic‐mean and principal component analysis, respectively. Bioinformatic analysis was performed using Ingenuity Pathway, MetaCore, and GeneMANIA analyses.ResultsThe exosomal proteome demonstrated 123 significantly (p < 0.05) differentially regulated proteins in CRSwNP relative to control. Eighty of these proteins overlapped with the matched CRSwNP tissue proteome as compared with only 4 among matched whole mucus samples. Forty‐three significantly dysregulated pathway networks overlapped between the exosomal and tissue proteome in CRSwNP as compared with only 3 among matched whole mucus samples. The best‐performing protein set (cystatin‐SN, peroxiredoxin‐5, and glycoprotein VI) achieved an area under the curve (AUC) value of up to 99%.ConclusionOur data contribute a significant advance in the development of a reproducible, noninvasive, serial, and quantitative “liquid biopsy” for rhinosinusitis. The exosomal proteomic approach has revealed a unique biosignature associated with CRSwNP, which outperforms whole mucus sampling, and thus provides a method of noninvasive disease detection and proposes new potential therapeutic targets.

Unexpected effects of systemic steroids on the CRSwNP proteome: is protein upregulation more important than inhibition?

“…The SOMAscan assay has been well studied, demonstrating a median limit of detection in the picomolar range and a dynamic range across 7 orders of magnitude of protein concentration, as well as a median intraplate coefficient of variability between 3% and 4%. We have recently demonstrated that the SOMAscan is capable of handling the small sample size of exosomal material from patient mucus collection, with Western blot confirmation . The SOMAscan with 3 controls (included with the kit) and 1 buffer control without protein were run in parallel with the samples.…”

“…CRSwNP symptomatology is severe, often requiring frequent medical and surgical interventions to lower morbidity . Recent advances in our understanding of perturbations observed in the CRSwNP proteome have brought forth new potential diagnostic and therapeutic targets . These advances have come about through large‐scale efforts to analyze the transcriptome and proteome in health and disease, through direct tissue sampling and “liquid biopsy” of exosomes.…”

“…Thus, exosomal isolation is an effective minimally invasive substitute for tissue sampling, and can be more easily performed without the need for local anesthesia or disruption of the nasal mucosa. In contrast to whole‐mucus proteomic sampling methods, which are often poorly quantitatively representative of tissue protein levels, the exosomal proteome is much more reflective of the protein milieu of the host cell . This is thought to be in part due to protection of exosomal material from degradation by proteases and nucleases …”

Sample collection for laboratory‐based study of the nasal airway and sinuses: a research compendium

“…They further noted that the samples collected via polyurethane foam were stable for at least 1 month at 4°C or colder. Like other focal surface fluid collection techniques, foam sponges can be directed at different anatomic regions of interest, such as the internal nasal valve or ethmoid bulla, among others.…”

“…Consequently, exosomes may be used as a biomarker that shows great potential for noninvasive diagnostics and conducting “liquid biopsies.” As exosomes represent only a small fraction of the cell's secretome, exosome isolation techniques allow for further analysis of the exosome independent of other cell contents. Exosomes are isolated from nasal mucus, which is often gathered via focal surface fluid collection techniques, as described earlier (eg, polyvinyl acetate sponge application). Once isolated, exosomal samples may be used to study physiologic and pathophysiologic mechanisms, including sinonasal inflammation or malignancy.…”