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

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. Researchers at Heidelberg University 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) (UCopt) and SEC. However, SEC yielded considerably fewer SEVs. Comparing the surface marker cargo, SEVs isolated by UCopt 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 UCopt 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.

Morphological and technical features of SEVs from HNC patients and HDs

Figure 2

(A) Representative TEM images of the SEVs and (B) NTA-detected size distribution histograms of one representative HNC patient and HD. (C) Particle concentration and (D) total protein content of the SEVs from HDs and HNC patients with early (I/II) and advanced (III/IV) stage tumors. (E) Protein content of EV-defining markers (tetraspanins, TSG101) and immunoregulatory markers (PD-L1, TGF-β1, CTLA-4, TRAIL). Original Western blot images are available in Suppl. Figure 5. A p-value below 0.05 was considered significant (*p < 0.05, **p < 0.01). n: number of patients/controls.

Tengler L, Tiedtke M, Schütz J, Bieback K, Uhlig S, Theodoraki MN, Nitschke K, Worst TS, Seiz E, Scherl C, Rotter N, Ludwig S. (2024) Optimization of extracellular vesicles preparation from saliva of head and neck cancer patients. Sci Rep 14(1):946. [article]

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