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Optimization of extracellular vesicles preparation from saliva of head and neck cancer patients.

ABSTRACT: Small extracellular vesicles from saliva (SEVs) have high potential as biomarkers in Head and Neck cancer (HNC). However, there is no common consensus on the ideal method for their isolation. This study compared different ultracentrifugation (UC) methods (durations and + /- additional purification) with size exclusion chromatography (SEC) and investigated the potential of SEVs as diagnostic biomarkers and their biological activity on NK and CD8⁺ T cells. SEVs from 19 HNC patients and 8 healthy donors (HDs) were thoroughly characterized. Transmission electron microscopy confirmed the isolation of vesicles by all methods. The average size determined via nanoparticle-tracking analysis was smaller for SEVs isolated by SEC than UC. The highest particle-to-protein yield was achieved by UC (3 h + 3 h) (UC_opt) and SEC. However, SEC yielded considerably fewer SEVs. Comparing the surface marker cargo, SEVs isolated by UC_opt from HNC patients carried more PD-L1, FasL, and TGF-β than SEVs from HDs. These levels correlated with tumor stage and HPV status. SEVs downregulated NKG2D expression on primary NK cells. HNC SEVs accelerated CD8⁺ T cell death compared to HD SEVs. This study suggests that UC_opt is preferable when isolation of a high particle-to-protein load is required. Especially PD-L1 and FasL on SEVs hold substantial potential as diagnostic biomarkers.

SUBMITTER: Tengler L

PROVIDER: S-EPMC10781729 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Optimization of extracellular vesicles preparation from saliva of head and neck cancer patients.

Tengler Luisa L Tiedtke Moritz M Schütz Julia J Bieback Karen K Uhlig Stefanie S Theodoraki Marie-Nicole MN Nitschke Katja K Worst Thomas Stefan TS Seiz Elena E Scherl Claudia C Rotter Nicole N Ludwig Sonja S

Scientific reports 20240110 1

Small extracellular vesicles from saliva (SEVs) have high potential as biomarkers in Head and Neck cancer (HNC). However, there is no common consensus on the ideal method for their isolation. This study compared different ultracentrifugation (UC) methods (durations and + /- additional purification) with size exclusion chromatography (SEC) and investigated the potential of SEVs as diagnostic biomarkers and their biological activity on NK and CD8<sup>+</sup> T cells. SEVs from 19 HNC patients and ...[more]

PMID: 38200037

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Project description:BackgroundTo analyse over time changes in stimulated whole saliva regarding total protein, Immunoglobulin A (IgA), and mucin type O-glycans (mostly MUC5B and MUC7) in head and neck cancer patients.Methods29 dentate patients (20 men and 9 women, 59 ± 8 years) treated with curative radiation therapy and chemotherapy for cancer of the head and neck region were included. The stimulated whole salivary secretion rate was determined and saliva collected at four time-points: at pretreatment, and at 6 months, 1 and 2 years post treatment. The total protein concentration was determined spectrophotometrically by using Bicinchoninic Acid assay and Immunoglobulin A (IgA) by using ELISA technique. Glycosylation pattern of salivary mucins was determined in samples collected pre- and post treatment by using LC/MS electrospray and mucin content quantified using SDS-AgPAGE gels and PAS staining.ResultsCompared with pretreatment, the total protein concentration was increased already at 6 months post treatment (p < 0.01), and continued to increase up to 2 years post treatment (p < 0.001). During that period no significant changes in IgA concentration was detected. At pretreatment, the output/min of both total protein and IgA was significantly higher than at all time-points post treatment. Saliva from the cancer patients showed a low abundance/no detectable MUC7, while the MUC5B level remained, compared to saliva from a healthy control. The glycomic analysis showed that the percentage of core 2 O-glycans was increased as core 1, 3 and 4 O-glycans were decreased. The level of sialylation was higher at 6 months post treatment, while sulfation was lower.ConclusionA decreased output per minute of proteins at decreased salivary secretion rate, as well as reduced sulfation of MUC5B at 6 months post treatment tended to correlate with the patients' experience of sticky saliva and oral dryness. At 2 years post treatment, the decreased amount of IgA combined with a lowered salivary secretion rate indicate a reduced oral defense with increased risk of oral infections.

Dataset Information

Optimization of extracellular vesicles preparation from saliva of head and neck cancer patients.

Publications

Optimization of extracellular vesicles preparation from saliva of head and neck cancer patients.

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