Fig 2: Characterization by ATR-FTIR and FTIRI spectra. The MCF-7, MDA-MB-231 and SK-BR3 breast cancer cell lines were characterized with their corresponding nanoprobe coupled to the respective surface protein (EpCAM, MUC-1 or HER-2) by ATR-FTIR. Blue line = cell line; red line = nanoprobe + cell line; black line = nanoprobe. The location of nanoprobes on the cell membrane was determined by Mapping image (FTIRI) were identified by IR score image as color-coding image red > yellow > blue (greater to smaller concentration of the nanoprobe, in which blue denotes scant or null presence).

Fig 3: Determination cell surface protein expression. With flow cytometry, fluorescence intensity was measured in three cell lines (MCF-7, MDA-MB-231 and SK-BR-3) to evaluate the expression of the corresponding surface proteins: (a) EpCAM, (b) MUC-1 and (c) HER-2.

Fig 4: Overview of sEVA.(A) EVs are labeled in solution with (i) the protein-reactive TFP dye (green) and (ii) biomarker-specific fluorescent antibodies [red, far red (shown in blue)]. (B) Following purification to remove unbound TFP and fluorescent antibodies, the EVs are (C) pipetted onto hydrophobic glass sides and (D and E) imaged and then analyzed. See fig. S1 for details on labeling and fig. S2 for the computational analysis pipeline. The image in (E) represents an example of AsPC-1 EV stained with TFP-AF488 (green) to label all EV KRASmut or P53mut (red channel) and EGFR, MUC1, and/or FG-P4OH (blue; scale bar, 1 μm).

Fig 5: sEVA analysis across multiple PDAC cell line–derived EVs.(A) Summary graph of the % EVs that stain for (i) KRASmut or P53mut (red); (ii) EGFR, MUC1, or FG-P4OH (blue); (iii) at least one of each of the former two marker panels (purple); or (iv) no marker (gray). The sum of EV was determined by pan-TFP labeling. Each column represents a different cell line or PDX line (see table S1 for characteristics of parental cells). (B) Data plotted in Venn diagrams to show EV distribution across cell lines. Note the often large fraction of EV negative for any marker.