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

Abstract

BackgroundExosomes 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.MethodsExosomes 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.ResultsSaliva-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.ConclusionsSaliva-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.

INHBA is Enriched in HPV-negative Oropharyngeal Squamous Cell Carcinoma and Promotes Cancer Progression

“…miRNAs were extensively researched in HNSCC and shown to hold a prognostic value ( 47 ). We previously showed that miRNA as exosomal cargo correlates with clinical parameters in HNSCC ( 48, 49 ) and can alter biological processes like EMT ( 50 ). In the current study, three putative miRNAs, miR-106a, miR-20b, and miR-9, were outlined as repressors of INHBA expression, prompting their HPV-dependent upregulation in OPSCC prognostically favorable.…”

Comparison of plasma- and saliva-derived exosomal miRNA profiles reveals diagnostic potential in head and neck cancer

“…One possible explanation is that the presence and functionality of exosomal miRNAs depend on their origin and the environment in which they are expressed. Exosomal miRNAs from saliva provided Frontiers in Cell and Developmental Biology frontiersin.org better information regarding clinical parameters, which might be due to their proximity to the HNSCC site and the higher presence of tumor-exosomes compared to exosomes of other origin (e.g., immune cells) (Hofmann et al, 2022). This makes saliva an attractive source of eventually more specific tumor biomarkers, while changes in the immune system or the presence of lymph node metastasis might be better reflected in plasma-compared to saliva-derived exosomes.…”

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

“…Due to the particular exposure of saliva to the head and neck region, salivary small EVs (SEVs) have an exceptionally high proportion of TEVs from local HNCs (i.e. oral cavity, oropharynx) compared to small EVs from plasma 4 .…”

“…Previous studies have established that the cargo of small EVs from HNC plasma (particularly in advanced stages) suppresses anti-tumor immunity, contributing to immune escape 2 , 13 – 15 . Whereas several studies have previously examined the diagnostic and functional potential of small EVs from the plasma of HNC patients, limited research studies have investigated SEVs and mainly focused on the value of RNAs in SEVs as liquid biomarkers for HNC 4 , 16 – 18 or other cancer subtypes 18 . To account for this, we assessed the abundance of immunomodulatory proteins and performed functional experiments comparing SEVs from different isolation techniques regarding the regulation of NKG2D receptor expression on primary NK cells and apoptosis in primary CD8 + T cells.…”