Cargo and Functional Profile of Saliva-Derived Exosomes Reveal Biomarkers Specific for Head and Neck Cancer

Linda Hofmann; Valentin Medyany; Jasmin Ezić; Ramin Lotfi; Beate Niesler; Ralph Röth; Daphne Engelhardt; Simon Laban; Patrick J Schuler; Thomas K Hoffmann; Cornelia Brunner; Edwin K Jackson; Marie-Nicole Theodoraki

doi:10.3389/fmed.2022.904295

Cargo and Functional Profile of Saliva-Derived Exosomes Reveal Biomarkers Specific for Head and Neck Cancer

Front Med (Lausanne). 2022 Jul 11:9:904295. doi: 10.3389/fmed.2022.904295. eCollection 2022.

Authors

Affiliations

¹ Department of Otorhinolaryngology, Head and Neck Surgery, Ulm University Medical Center, Ulm, Germany.
² Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Services Baden-Württemberg-Hessen, Ulm, Germany.
³ Institute for Transfusion Medicine, University Hospital Ulm, Ulm, Germany.
⁴ nCounter Core Facility, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany.
⁵ Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States.

Abstract

Background: Exosomes contribute to immunosuppression in head and neck squamous cell carcinoma (HNSCC), a tumor entity which lacks specific tumor biomarkers. Plasma-derived exosomes from HNSCC patients correlate with clinical parameters and have potential as liquid biopsy. Here, we investigate the cargo and functional profile of saliva-derived exosomes from HNSCC patients and their potential as non-invasive biomarkers for disease detection and immunomodulation.

Methods: Exosomes were isolated from saliva of HNSCC patients (n = 21) and healthy donors (HD, n = 12) by differential ultracentrifugation. Surface values of immune checkpoints and tumor associated antigens on saliva-derived exosomes were analyzed by bead-based flow cytometry using CD63 capture. Upon co-incubation with saliva-derived exosomes, activity and proliferation of T cells were assessed by flow cytometry (CD69 expression, CFSE assay). Adenosine levels were measured by mass spectrometry after incubation of saliva-derived exosomes with exogenous ATP. miRNA profiling of saliva-derived exosomes was performed using the nCounter^® SPRINT system.

Results: Saliva-derived, CD63-captured exosomes from HNSCC patients carried high amounts of CD44v3, PDL1 and CD39. Compared to plasma, saliva was rich in tumor-derived, CD44v3⁺ exosomes and poor in hematopoietic cell-derived, CD45⁺ exosomes. CD8⁺ T cell activity was attenuated by saliva-derived exosomes from HNSCC patients, while proliferation of CD4⁺ T cells was not affected. Further, saliva-derived exosomes produced high levels of immunosuppressive adenosine. 62 HD- and 31 HNSCC-exclusive miRNAs were identified. Samples were grouped in "Healthy" and "Cancer" based on their saliva-derived exosomal miRNA profile, which was further found to be involved in RAS/MAPK, NF-κB complex, Smad2/3, and IFN-α signaling.

Conclusions: Saliva-derived exosomes from HNSCC patients were enriched in tumor-derived exosomes whose cargo and functional profile reflected an immunosuppressive TME. Surface values of CD44v3, PDL1 and CD39 on CD63-captured exosomes, adenosine production and the miRNA cargo of saliva-derived exosomes emerged as discriminators of disease and emphasized their potential as liquid biomarkers specific for HNSCC.

Keywords: exosomes; head and neck squamous cell carcinoma (HNSCC); liquid biopsy; miRNA; saliva.