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

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.

Figure 1

Characterization of exosomes isolated from human saliva and plasma. (A) Total salivary protein and exosome enriched protein obtained from saliva of healthy donors (HD; n = 12) and head and neck squamous cell carcinoma (HNSCC) patients (n = 21). (B) The ratio between exosome enriched protein and total saliva protein was determined for each sample and average percentage is shown for HD and HNSCC patients. (C) Representative transmission electron microscopy (TEM) images of exosomes derived from saliva of one HNSCC patient (left) or HD (right). (D) Western blot for detection of exosomal markers CD63, CD9, TSG101 and the cellular marker Grp94 in saliva-derived exosome preparations. HeLa-cell-lysate was used as a positive control for Grp94. (E) Nanoparticle tracking analysis (NTA) of saliva-derived exosomes. The histogram shows a representative size distribution of saliva-derived vesicles with a median diameter of 106 nm. (F) Exosomes from saliva of HNSCC patients showed a higher median diameter (n = 20, 135 nm) compared to exosomes from saliva of HD (n = 10; median diameter 116 nm). (G) Concentration of detected vesicles/ml was analyzed by NTA and compared between HD (n = 10) and HNSCC patients (n = 19). (H) Western blot for detection of exosomal markers CD63, CD9, TSG101, the cellular marker Grp94 and contaminant apolipoprotein ApoA1 in plasma-derived exosome preparations. HeLa-cell-lysate and plasma were used as a positive control for Grp94 and ApoA1, respectively. (I) NTA of plasma-derived exosomes. The histogram shows a representative size distribution of plasma-derived vesicles with a median diameter of 87 nm. (J) Representative TEM image of HNSCC patient's plasma-derived exosomes. (A,F,G) Results are plotted as box-and-whisker blots representing the median value, the 25^th and 75^th quartiles and the range. P-values were determined by Mann–Whitney test, **p ≤ 0.01, ***p ≤ 0.001, ns, not significant.

Figure 2

Surface levels of different antigens on saliva-derived, CD63-captured exosomes. Exosomes isolated from saliva of HD (n = 12) and HNSCC patients (n = 22) were stained for (A) CD44v3, (B) PDL1, (C) CD39, (D) CD73, (E) FasL, (F) PDL2, (G) EpCAM and (H) OX40L using capture with CD63 antibodies and subsequent bead-based flow cytometry. Surface levels are shown as relative fluorescence intensity (RFI) compared to isotype controls. Saliva-derived exosomes from HNSCC patients showed significantly higher surface values of CD44v3 (A), PDL1 (B) and CD39 (C) compared to saliva-derived HD exosomes. Results are plotted as box-and-whisker blots representing the median value, the 25^th and 75^th quartiles and the range. P-values were determined by Mann-Whitney test, **p ≤ 0.01, ***p ≤ 0.001, ns, not significant.

Figure 3

Functional effects of saliva-derived exosomes on activated CD8⁺ T cells, proliferating CD4⁺ T cells and ATP metabolism. (A,B) Activated CD8⁺ T cells were co-incubated with saliva-derived exosomes from HD (n = 9) and HNSCC patients (n = 13) for 16 h. CD69 expression of T cells was measured by flow cytometry. (B) shows representative plots for each condition. (C,D) CD4⁺ T cells were co-incubated with saliva-derived exosomes from the same HD (n = 9) and HNSCC patients (n = 13) for 4 d. Proliferation was evaluated by CFSE assay and flow cytometry. (D) shows representative histograms for each condition. Filled histograms represent proliferated cells and framed histograms the unstimulated parental generation. For (A–D), plasma-derived exosomes obtained from HNSCC patients (n = 4) were included as positive controls. (E) 5'AMP and (F) adenosine levels were measured by mass spectrometry upon incubating saliva-derived exosomes of HD (n = 12) and HNSCC patients (n = 20) with exogenous ATP. Results in (A,C,E,F) are plotted as box-and-whisker blots representing the median value, the 25^th and 75^th quartiles and the range. P-values were determined by Mann–Whitney test, *p ≤ 0.05, ****p ≤ 0.0001, ns, not significant.

Figure 4

Tumor-derived exosomes (TEX) and non-TEX amounts in total exosome populations from saliva. Exosomes isolated from plasma and saliva of HD and HNSCC patients (n = 7) were stained for (A) CD44v3 (TEX) and (B) CD45 (non-TEX) using bead-based flow cytometry. Surface levels are shown as RFI compared to isotype controls. Saliva had significantly lower amounts of CD45⁺ exosomes compared to plasma (B). Results are plotted as box-and-whisker blots representing the median value, the 25^th and 75^th quartiles and the range. P-values were determined by Mann–Whitney test, *p ≤ 0.05.

Figure 5

miRNA profiling of saliva-derived exosomes. (A) Venn diagram of miRNAs detected in saliva-derived exosomes of HD (green, n = 7) and HNSCC patients (purple, n = 14), created with InteractiVenn (36). (B) Volcano plot showing the log2 expression ratio of 101 miRNAs overlapping between saliva-derived exosomes of HD and HNSCC patients. Each dot represents one miRNA. miRNAs at x > 0 are upregulated in HNSCC saliva-derived exosomes, while miRNAs at x < 0 are downregulated in HNSCC saliva-derived exosomes compared to HD. miRNAs in blue are significantly downregulated (p ≤ 0.05) in HNSCC saliva-derived exosomes compared to HD. (C) Waterfall plot of significant miRNAs from (B) showing the log2 fold change difference between HD and HNSCC saliva-derived exosomes. Red color indicates the negative logarithm of the p-value (the stronger the color, the more significant).

Figure 6

Clustering and pathway analysis of miRNAs from saliva-derived exosomes. (A) Unsupervised hierarchical clustering heatmap and (B) Uniform manifold approximation and projection (UMAP) visualizations of saliva-derived miRNAs which were HD- or HNSCC-exclusive or overlapped with significant difference between the two groups (total of 101 miRNAs). (C,D) Ingenuity pathway analysis (IPA) of the same miRNAs. (C) Shows diseases and biological functions most significantly associated with the identified miRNAs. Categories within a group are shown in increasing order of p-value. (D) Shows the highest ranked network to which identified miRNAs (blue symbols) contribute. Solid lines indicate direct interaction, dashed lines indicate indirect relation.