Fig 2: NSCLC diagnosis and prediction of immunotherapy response with AuSERP. (a) Receiver operating characteristic (ROC) curves for NSCLC diagnosis based on single-EV analysis. (b) Scatter plot of fluorescence intensities of single-EV dual PD-1/PD-L1 mRNA biomarkers. Optimal cut-off values for NSCLC diagnosis based on single-EV PD-1/PD-L1 mRNA signals were obtained from the ROC curves in Figure 5(a). An individual is diagnosed with NSCLC if either the single-EV PD-1 mRNA signal is larger than 3.95 or if the single-EV PD-L1 mRNA signal is larger than 2.15, lending a diagnostic accuracy of 93.2%. (c) ROC curves for predictions of NSCLC patient responses to anti-PD-1/PD-L1 immunotherapy based on single-EV analysis. (d) Scatter plot of fluorescence intensities of single-EV dual PD-1/PD-L1 mRNA biomarkers. Optimal cut-off values for immunotherapy prediction based on single-EV PD-1/PD-L1 mRNA signals were obtained from the ROC curves in Figure 5(c). A patient is predicted as a responder if either the single-EV PD-1 mRNA signal is larger than 5.56 or if the single-EV PD-L1 mRNA signal is larger than 3.94, lending a prediction accuracy of 72.2%. 54 patients were evaluated (27 responders and 27 non-responders), along with 20 healthy donors

Fig 3: Measurements of PD-1/PD-L1 protein and mRNA biomarkers levels in single EVs from non-small cell lung cancer (NSCLC) patient serum with AuSERP. (a) Representative IHC staining image of PD-L1 protein in tissue biopsies. PD-L1 protein was stained brown (by Dako PD-L1 IHC 22C3 pharmDx), and cell nuclei were stained blue (by haematoxylin). (b) Heatmap of single-EV PD-1/PD-L1 protein and mRNA expression levels measured with AuSERP compared to the corresponding IHC score for PD-L1 expression in patient tissue (negative, positive, or unknown; detailed patient information is provided in Table S2). (c) Representative TIRF microscopy images and their corresponding histograms of single-EV PD-1/PD-L1 protein and mRNA biomarkers characterized with AuSERP. The images were cropped and enlarged from their original images, which are provided in Figure S11. (d) Box plots of quantitative fluorescence intensities of PD-1/PD-L1 protein and mRNA expression levels (*P < 0.05, **P < 0.01, ****P < 0.0001, Mann-Whitney U test). RFI, relative fluorescence intensity; a.u., arbitrary units. 54 patients were evaluated (27 responders and 27 non-responders), along with 20 healthy donors

Fig 4: Communication among tumor microenvironment components. A-B. Heatmap showing interactions between fibroblast subgroups and immune cells (A) or other stromal cells (B). The color represents the number of interactions (blue to red); n=8 tumors. C. Selected specific interactions between CAF subgroups and immune cells in tumors. The size indicates the p values, and the color indicates the mean values of the receptor/ligand pairs between two clusters. D. IHC staining and pseudofluorescence synthesized by multistaining registration for CD8, PD1, CD34, and COL1A1 in the tumor tissue from patient 6. The box indicates adjacent cells. E. Images of IHC staining and pseudofluorescence synthesized by multistaining registration for E-cadherin, CD163, periostin, and COL1A1 in the tumor tissue from patient 6. The box indicates adjacent cells. F. Immunofluorescence staining for COL1A1 and periostin in the fifth generation of CAFs. G. Crystal violet staining of M2 macrophages in response to MSCs and CAFs. Bar plot showing the average crystal violet-stained area in Transwells calculated using ImageJ software. The p value was calculated using the unpaired t test (P value = 0.0038). H. Western blot results showing a comparison of the periostin and GAPDH bands between MSCs and CAFs. I. Western blot results showing a comparison of the periostin and GAPDH bands between NC-MSCs and periostin-overexpressing MSCs. J. Crystal violet staining of M2 macrophages that were recruited in response to NC-MSCs and periostin-overexpressing MSCs. Bar plots showing the average crystal violet-stained area in the Transwells calculated using ImageJ software. The p value was calculated using the unpaired t test (P value = 0.0004).

Fig 5: The intrinsic function and regulation of palmitoylated PD-1 in cancer cells. Schematic model showing the roles of palmitoylation in promoting PD-1 expression and intrinsic mTOR signaling to facilitate tumor growth.