Figure 2.
Platelets upregulate tumor PD-L1 through EGF/EGFR pathway. (A) Heatmap showing mean concentration of indicated cytokines in conditioned medium from MDA-MB-468 monoculture, platelet, and MDA-MB-468 cell coculture (with platelets); cocultures treated with either aspirin or ticagrelor. N = 3 replicates. (B) Fold change in PD-L1 cell-surface expression measured by flow cytometry in response to indicated concentration of EGF relative to vehicle control for MDA-MB-468 cells. N = 4. (C) Fold change in PD-L1 cell-surface expression measured by flow cytometry in response to indicated concentrations of recombinant pro-EGF and EGF relative to vehicle control for MDA-MB-468 cells. N = 4 to 8. (D) EGFR levels of MCF7, A549, and MDA-MB-468 cells were measured by flow cytometry. Isotype control (IgG2b, κ) from each cell line was used as the negative control. (E) Left: representative western blot images of total EGFR and pEGFR (Tyr1068) of MDA-MB-468 cells. Right: graph of western blot quantification of pEGFR/total EGFR ratio for all donors (see supplemental Figure 3G for full blots and detailed information). N = 8. (F) Flow cytometric analysis of PD-L1 level of MDA-MB-468 cells cultured alone, cocultured with platelets, or cocultured with platelets and 3 μg/mL cetuximab. N = 6. (G) Flow cytometric analysis of PD-L1 levels of MDA-MB-468 cells cultured alone, cocultured with platelets, or cocultured with platelets and 2 μg/mL EGF-neutralizing antibody. N = 7. (H) Flow cytometric analysis of PD-L1 levels of MDA-MB-468 cells treated with CRP-induced activated platelet releasate with or without 2 μg/mL EGF neutralizing antibody. N = 5. (I) T-cell cytotoxicity efficiency in MDA-MB-468 cells cocultured with platelets or in the presence of 3 μg/mL cetuximab. N = 6 to 8. (J) Spearman’s rank correlation analysis for EGFR and PD-L1 (CD274) in The Cancer Genome Atlas breast and lung cancer data sets. Each line represents a separate experiment using platelets isolated from a different healthy donor. Paired t tests were used for testing data with 2 groups. One-way analysis of variance with Tukey correction was used for testing data with 3 or more groups. ns, not significant. ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001. -, tumor monoculture; +, tumor cocultured with platelets.

Platelets upregulate tumor PD-L1 through EGF/EGFR pathway. (A) Heatmap showing mean concentration of indicated cytokines in conditioned medium from MDA-MB-468 monoculture, platelet, and MDA-MB-468 cell coculture (with platelets); cocultures treated with either aspirin or ticagrelor. N = 3 replicates. (B) Fold change in PD-L1 cell-surface expression measured by flow cytometry in response to indicated concentration of EGF relative to vehicle control for MDA-MB-468 cells. N = 4. (C) Fold change in PD-L1 cell-surface expression measured by flow cytometry in response to indicated concentrations of recombinant pro-EGF and EGF relative to vehicle control for MDA-MB-468 cells. N = 4 to 8. (D) EGFR levels of MCF7, A549, and MDA-MB-468 cells were measured by flow cytometry. Isotype control (IgG2b, κ) from each cell line was used as the negative control. (E) Left: representative western blot images of total EGFR and pEGFR (Tyr1068) of MDA-MB-468 cells. Right: graph of western blot quantification of pEGFR/total EGFR ratio for all donors (see supplemental Figure 3G for full blots and detailed information). N = 8. (F) Flow cytometric analysis of PD-L1 level of MDA-MB-468 cells cultured alone, cocultured with platelets, or cocultured with platelets and 3 μg/mL cetuximab. N = 6. (G) Flow cytometric analysis of PD-L1 levels of MDA-MB-468 cells cultured alone, cocultured with platelets, or cocultured with platelets and 2 μg/mL EGF-neutralizing antibody. N = 7. (H) Flow cytometric analysis of PD-L1 levels of MDA-MB-468 cells treated with CRP-induced activated platelet releasate with or without 2 μg/mL EGF neutralizing antibody. N = 5. (I) T-cell cytotoxicity efficiency in MDA-MB-468 cells cocultured with platelets or in the presence of 3 μg/mL cetuximab. N = 6 to 8. (J) Spearman’s rank correlation analysis for EGFR and PD-L1 (CD274) in The Cancer Genome Atlas breast and lung cancer data sets. Each line represents a separate experiment using platelets isolated from a different healthy donor. Paired t tests were used for testing data with 2 groups. One-way analysis of variance with Tukey correction was used for testing data with 3 or more groups. ns, not significant. ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001. -, tumor monoculture; +, tumor cocultured with platelets.

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