Fig 2: The anti-CD63 functionalized EPAC for monitoring phenotypic changes of EVs from melanoma patient–derived cell lines in response to BRAF inhibitor treatment.EVs released from (A) LM-MEL-64 cells without treatment were used as a control and followed for 30 days. EVs derived from (B) LM-MEL-33, (C) SK-MEL-28, (D) LM-MEL-64, and (E) LM-MEL-35 cell lines were collected before (day 0), during (days 3 to 30), and after treatment (days 33 and 39). (A to E) Average biomarker signals are represented by red (MCSP), blue (MCAM), green (ErbB3), and yellow (LNGFR). LM-MEL-35 cell line is BRAF wild type but NRAS mutant, and the other three cell lines are BRAF mutant. Data in (A) to (E) are represented as means ± standard deviation, where error bars represent standard deviation of three separate experiments. (F to J) Clustering of EV populations before, during, and after treatment via LDA of SERS signals.

Fig 3: The anti-MCSP functionalized EPAC for phenotyping of plasma EVs from melanoma patients.(A) EV phenotypes of 15 melanoma samples (P1 to P15) and 12 healthy controls (H1 to H12). P1, P4, P7, and P9 are from the same patient but different time points, as are P5 and P10. (B) Representative false-color SERS spectral images and (C) corresponding average SERS spectra from patient and normal samples (P1, P8, and H1). For (A) and (B), the biomarker signals are represented by red (MCSP), blue (MCAM), green (ErbB3), and yellow (LNGFR). Data in (A) are represented as means ± standard deviation, where error bars represent standard deviation of three separate experiments. Scale bars, 10 µm.

Fig 4: Proliferation markers and role of YAP. (A,B) Anti-BrdU staining (A, control; B, #255 textile, black asterisks indicate cross sections of fibers.). In the control, proliferating cells appear only at the bottom. Proliferating cells appear at fiber surfaces in B (black arrows). (C,D) Anti-MCSP immunostaining (C, control; D, #255 textile, white asterisks indicate cross sections of fibers). MCSP-positive cells appear on fibers (white arrows). (E,F) Anti-YAP immunostaining (E, control; F, #255 textile). YAP-positive cells appear on fibers (red arrows). (G–J) Effects of YAP siRNA (#255 textile). (G,H) H&E staining. (G) Control shows thick epidermis and stratum corneum. (H) YAP siRNA application disturbed structure formation. (I,J) Anti-YAP immunostaining. (I) YAP is expressed on fibers in control (red arrows). (J) Very little YAP expression was observed in YAP siRNA-applied sample. Black asterisks indicate cross sections of fibers. Bars = 50 µm.

Fig 5: 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.