Analysis of small extracellular vesicles from plasma and saliva of head and neck cancer patients with calibrated spectral flow cytometry
Background
Small extracellular vesicles (sEVs) emerged as promising biomarkers for solid tumors, including head and neck cancer. Current bead-based flow cytometry approaches analyze the bulk sEV sample instead of single vesicles. sEV populations in biofluids are very heterogenous and represent a mixture released from different cell types. Thus, single vesicle analysis is desired for more specific diagnostic approaches but is challenging due to the small EV size and requires high sensitivity instruments. This project aimed to establish single vesicle surface stainings for the quantitative and standardized analysis of sEVs from biofluids of head and neck cancer patients.
Methods
Platelet-poor plasma from head and neck cancer patients was prepared from citrate blood by double centrifugation. Saliva was collected using Salivettes. sEVs were isolated by ultrafiltration and size exclusion chromatography (plasma) or ultracentrifugation (saliva) and thoroughly characterized according to the MISEV guidelines (EV-Track EV200068, EV210344). Calibrations, staining protocol establishments and measurements were performed on spectral flow cytometer Aurora.
Results
Flow cytometer settings (side scatter and fluorescence detector gains, trigger threshold) for optimal sEV measurement were determined using Nanosphere size standards. Flow rate, light scatter and fluorescence were calibrated using reference material (Apogee, Rosetta, MESF beads) to enable quantification of sEV concentration, size, and fluorescence intensity of the biological samples. Serial dilutions were performed to determine the optimal sample dilution without swarm detection. Panels for measurement of total sEVs as well as tumor-derived sEVs were established on isolated sEVs and pure biofluids. Respective antibodies were titrated and appropriate controls (unstained, isotype, detergent) as well as a post-staining clean up were included.
Conclusion
We will use our established panels for our future sEV-based biomarker studies in plasma and saliva for the identification and analysis of tumor-specific changes. They can further be applied by other researchers and are comparable interlaboratory due to the calibrated procedure.
Keywords
Single vesicle flow cytometry, calibration, HNSCC, biomarker
Funding/Acknowledgments
LH received project start-up funding from the Hertha-Nathorff-program of Ulm University. MNT received funding from the Deutsche Forschungsgemeinschaft (DFG), research fellowship #TH 2172/1-1. We would like to thank the Core Facility Immune Monitoring of the Medical Faculty at Ulm University for providing instrumentation (Cytek Aurora) funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – project number 514808265.
Authors
Linda Hofmann1 (Corresponding Author: linda.hofmann[at]uniklinik-ulm[dot]de), Kathrina Payer1, Lutz Schütt1, Thomas K Hoffmann1, Cornelia Brunner1,2, Marie-Nicole Theodoraki1,3