Fig. 1: Direct platelet-tumor cell interactions result in PD-L1 expression on the platelet surface.

a Representative immunofluorescence staining of PD-L1 (red)and the platelet marker CD41 (green) in four different NSCLC tumor cell lines (A549, NCI-H23, NCI-H460, NCI-H226) co-incubated with human platelets (n = 3 biological replicates). Scale bars 200 μm. b Immunofluorescence microscopy of NCI-H460 cells interacting with human platelets derived from a healthy donor (PD-L1: red, CD41: green) (n = 3). Scale bar left 20 µm, right 10 µm. c Quantitative analysis of the PD-L1+ platelets per field of view (FoV), analyzed by immunofluorescence microscopy (n = 9 FoV (small symbols) were analyzed out of a total of n = 3 independent experiments (large symbols)). Horizontal lines represent means. d Percentage of PD-L1+ tumor cells per FoV (n = 3). Data are mean ± SEM. e Correlation of % PD-L1+ platelets/FoV vs. % PD-L1 tumor cells/FoV (n = 3). Correlation was determined by simple linear regression analysis. f Flow cytometry gating strategy for the quantification of PD-L1+ tumor cells (upper) and platelets (lower) after co-incubation. g, h Surface expression of PD-L1 and CD62P on control platelets (PLT) and platelets after co-incubation with A549, NCI-H23, NCI-H226, NCI-H460 cells (n = 4). Data are mean ± SEM. Statistical significance was calculated by two-tailed Student’s t test. i Phase-contrast image of A549 tumor cells after 41 min coculture with platelets (ratio 1:1000). Overlaid migration tracks were color-coded based on their mean velocity. j Image sequence depicting tumor cell interaction and protrusion of a single platelet followed by detachment derived from zoom-in area indicated in (i). k Percentage of stable platelet–tumor cell contacts lasting from contact initiation until the end of the observation period (total observation time: 41 min). l Contact duration of platelet–tumor cell interactions. Data derived from the analysis of n = 75 platelets out of one independent experiment. Boxes represent median and 25th to 75th percentiles, whiskers are minimum to maximum. m Scheme of vectors expressing PD-L1-GFP and FLAG-GFP used for transfection. n Immunofluorescence stainings of GFP of A549 cells transfected with FLAG-GFP or PD-L1-GFP (n = 3). Scale bar 50 µm. o Western blot analysis for PD-L1 in untreated and transfected A549 cells (n = 2). Vinculin was used as loading control. Presentation of full scan blots are provided in the Source data file. p, q Representative immunofluorescence microscopy of untreated, FLAG-GFP and PD-L1-GFP-transfected A549 cells interacting with platelets (n = 3). Tumor cells and platelets were stained for GFP (upper) and PD-L1 (lower). Scale bar 50 µm. r, s Flow-cytometry-based quantification of GFP (l) and PD-L1 (m) on platelet surfaces after co-incubation with untreated and transfected A549 cells (n = 3). t Expression of PD-L1 on platelets pre-treated with 100 µM cycloheximide after co-incubation with PD-L1-GFP-transfected A549 cells (n = 3). r–t Data are mean ± SEM. c, d, r–t Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test. Source data are provided as a Source Data file.