Fig 2: Surface values of immune checkpoints on exosomes isolated from plasma of HNSCC patients.Exosomes from n = 17 HNSCC patients obtained at defined time-points were captured using biotinylated CD63-antibodies and stained with fluorochrome-conjugated antibodies against PD-L1 (a), CTLA-4 (b), OX40 (c) and OX40-L (d). Surface values as determined by on-bead flow cytometry are shown as RFI compared to an appropriate isotype control. Results are plotted as box-and-whisker blots representing the median value, the 25th and 75th quartiles and the range. Wilcoxon related samples tests were applied to each compare pre-OP, post-OP, end (C)RT and follow-up. Mann–Whitney test was applied to compare recurrence with follow-up. * or # and ** or ## correspond to p = 0.05 and p = 0.01, respectively. Asterisks and values above boxes indicate the significance compared to pre-OP. Hashtags above boxes indicate the significance compared to follow-up. c RT (chemo)radiotherapy

Fig 3: Anti-CD63 functionalized EPAC specificity.The specificity was studied using EV samples released from SK-MEL-28 and MCF7 cell lines, as well as control experiments including (++) EV-free cell culture medium, (-+) without the CD63 capture antibody, and (+-) with nontarget CD45 detection antibodies on SERS nanotags. (A) The expressions of MCSP, MCAM, ErbB3, and LNGFR in SK-MEL-28 and MCF7 cells were detected by flow cytometry. (B) Representative false-color SERS spectral images, (C) average SERS spectra obtained from corresponding SERS mapping datasets, and (D) average SERS intensities at 1075 cm-1 (red, MCSP), 1375 cm-1 (blue, MCAM), 1335 cm-1 (green, ErbB3), and 1000 cm-1 (yellow, LNGFR). Data in (D) are represented as means ± standard deviation, where error bars represent standard deviation of three separate experiments. Scale bars, 10 µm.

Fig 4: Selectivity of the QCM-D immuno-sensing approach toward CD63-postive exosomes in human serum. (A) Example frequency and (B) dissipation profiles from target surfaces (anti-CD63). (C) Bar chart comparing net frequency change and (D) net dissipation change between target and control surfaces (control-isotype) against 1 × 109 ESPs/mL spiked in increasing concentrations of human serum. Average QCM-D data taken from three independent experiments. Student t-test (two-tailed) compared responses from the control and target sensor (***p < 0.001). (E) AFM micrographs comparing control and target surfaces post-ESP incubation in 75% v/v human serum. Scale bar: 200 nm. Color scale (height): 100 nm).

Fig 5: 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 25th and 75th quartiles and the range. P-values were determined by Mann–Whitney test, **p = 0.01, ***p = 0.001, ns, not significant.