Glycoprotein Ibα‐Dependent Platelet Activation is Essential for Tumor Cell–Platelet Interaction and Experimental Metastasis

Figures

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  FIGURE 1. The role of GPIbα in metastasis. (A and B) Representative images (A) and the number of metastatic foci (B) in the lung of WT, Gp1ba−/− and Mpl−/− mice (n = 6 mice per group). (C) Platelet counts in WT, Gp1ba−/− and Mpl−/− mice (n = 6 mice per genotype). (D) Surface levels of GPIbα on wild‐type (WT), Gp1ba−/− , and Mpl−/− platelets analyzed by flow cytometry (n = 6 mice per genotype). MFI: mean fluorescence intensity. (E and F) Quantification plots of basal P‐selectin (E) and PS (F) levels on resting WT, Gp1ba−/− , and Mpl−/− platelets in PRP (WT and Mpl−/− , n = 7 mice; Gp1ba−/− , n = 6 mice). PS, phosphatidylserine. (G and H) Quantification plots of B16F10 cells‐induced P‐selectin (G) and PS (H) exposure on WT, Gp1ba−/− , and Mpl−/− platelets (n = 6 mice per group). One‐way ANOVA followed by Tukey's post hoc test in (B–H). Data are shown as mean ± SD (B–H). *p < 0.05, **p < 0.01, and ***p < 0.001. ns, not significant.

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  FIGURE 2. The GPIbα cytoplasmic tail is essential for tumor cell‐induced platelet activation. (A and B) Quantification plots of basal P‐selectin (A) and PS (B) levels on resting WT and 10aa−/− platelets in PRP (n = 12 mice per genotype). (C and D) Quantification plots of B16F10 cells‐induced P‐selectin (C) and PS (D) exposure on WT and 10aa −/− platelets (n = 6 mice per group). (E and F) Representative traces of B16F10 cell‐induced WT and 10aa−/− platelet aggregation (E) and the quantification of maximal aggregation rate (F) (n = 10 independent experiments). (G) Representative state‐of‐the‐art spinning disk intravital microscopy (SDIM) images (left) and quantification (right) of tumor cell‐induced thrombus fluorescence (red) of live WT and 10aa−/− lung 5 min after intravenous injection of B16F10 cells. ADU: analog‐to‐digital unit. Scale bar: 30 µm. (H) B16F10 cells induced platelet clearance in WT and 10aa−/− mice. Two‐tailed Student's t‐test in (A), (B), and (F). One‐way ANOVA followed by Tukey's post hoc test in (C) and (D). Mann–Whitney U‐test in panel (G). Two‐way ANOVA followed by Dunnett's post hoc test in (H). Data are shown as mean ± SD (A–D and F–H). *p < 0.05, **p < 0.01, and…

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  FIGURE 3. The GPIbα cytoplasmic tail deficiency inhibits the interaction of B16F10 cells with platelets and experimental metastasis. (A) The in vitro adhesion of WT and 10aa−/− platelets to B16F10 cells was analyzed by flow cytometry using an antiplatelet CD41 antibody (n = 6 independent experiments). (B and C) Representative confocal images (B) and quantification (C) of the interaction of GFP–B16F10 with WT and 10aa−/− platelets in vitro (n = 10 visual fields from 10 mice of each genotype). Scale bar: 25 µm. D‐F, Representative blot (D) and quantification of N‐cadherin (E) and vimentin (F) in B16F10 cells treated with WT and 10aa−/− platelets (n = 3 mice per genotype). (G) Representative images of B16F10 cell wound‐healing (left) and quantification plots (right) of the wound areas of B16F10 cells incubated with washed WT and 10aa−/− platelets (n = 9 visual fields from three mice of each genotype). Scale bar: 30 µm. (H and I) Representative images (left) and quantification plots (right) of invasion (H) and migration (I) of B16F10 cells incubated with WT and 10aa−/− platelets (n = 9 visual fields from three mice of each genotype). Scale bar: 30 µm. (J) Representative confocal images (left)…

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  FIGURE 4. The GPIbα cytoplasmic tail supports tumor cell‐induced platelet activation and interaction by regulating PKC activity. (A) Representative blot of PKC substrate phosphorylation in resting WT and 10aa‐/‐ platelets and B16F10‐treated WT and 10aa‐/‐ platelets, and quantification of densitometry in the blots (n = 5 mice per genotype). (B and C) B16F10 cell‐induced P‐selectin (B) and PS (C) exposure on washed WT and 10aa‐/‐ platelets (n = 6 independent experiments). (D) The in vitro adhesion of WT and 10aa‐/‐ platelets to B16F10 cells was analyzed by flow cytometry using an antiplatelet CD41 antibody (n = 5 independent experiments). (E) Representative confocal images and quantification of the interaction of GFP–B16F10 with WT and 10aa‐/‐ platelets in vitro (n = 5 visual fields from five mice of each genotype). Scale bar: 25 µm. One‐way ANOVA followed by Tukey's post hoc test in (A–E). Data are shown as mean ± SD (A–E). *p < 0.05, **p < 0.01, and ***p < 0.001. ns, not significant.

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  FIGURE 5. The effect of PKC inhibitors on platelet‐induced B16F10 cell migratory capability and metastasis. (A) Western blotting of N‐cadherin, vimentin, and GAPDH in B16F10 cells treated with WT and 10aa‐/‐ platelets (n = 5 mice per genotype). (B) Wound‐healing assay of the B16F10 cells incubated with WT and 10aa‐/‐ platelets (pretreated by DMSO, Gö6983, Gö6976, or PMA) (n = 10 visual fields from five mice of each genotype). (C and D) Migration (C) and invasion (D) of B16F10 cells incubated with washed WT and 10aa‐/‐ platelets (pretreated by DMSO, Gö6983, Gö6976, or PMA) (n = 10 visual fields from five mice of each genotype). (E) Representative SDIM images and quantification of B16F10‐induced thrombus fluorescence or B16F10 cell area fluorescence in vivo of WT mice (pretreated by Gö6983 and Gö6976) 6 h after intravenous injection of B16F10 cells (n = 18 visual fields from three mice of each genotype). Scale bar: 30 µm. (F) The effect of Gö6983 and Gö6976 on tumor pulmonary metastasis in WT mice (n = 5 mice per group). One‐way ANOVA followed by Tukey's post hoc test in (A) and (C)–(F). Two‐way ANOVA followed by Bonferroni's post hoc test in (B). Data are shown as mean ± SD (A–D and F) and…

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  FIGURE 6. The role of PKCα in tumor‐platelet activation and metastasis. (A and B) B16F10 cell‐induced P‐selectin (A) and PS (B) exposure on washed WT and pkcα ‐/‐ platelets (n = 5 mice per group). PS, phosphatidylserine. (C) The in vitro adhesion of WT and pkcα ‐/‐ platelets to B16F10 cells was analyzed by flow cytometry using an antiplatelet CD41 antibody (n = 5 mice per group). (D) Western blotting of N‐cadherin, vimentin, and GAPDH in B16F10 cells treated with WT and pkcα ‐/‐ platelets (n = 5 independent experiments). (E and F) Relative mRNA expression levels of N‐cadherin (E) and vimentin (F) in B16F10 cells treated with WT and pkcα‐/‐ platelets. (n = 3 independent experiments). (G–I) Wound‐healing (G) and transwell assays (H and I) for B16F10 cells after treated with washed WT and pkcα ‐/‐ platelets (n = 9 visual fields from three mice of each genotype). (J) Representative SDIM images (left) of B16F10‐platelet aggregates in the liver of WT and pkcα ‐/‐ mice 6 h after intravenous injection of B16F10 cells. Quantification (right) of WT or pkcα ‐/‐ B16F10‐induced thrombus fluorescence or B16F10 cell area fluorescence in vivo 6 h after B16F10 cell intravenous injection (n = 21 visual…

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  FIGURE 7. Schematic diagram of the role of platelet GPIbα in metastasis. GPIbα primes and potentiates tumor cell‐induced platelet activation through associating with 14‐3‐3ζ and increasing PKCα activity. GPIbα‐dependent PKCα and platelet activation are required for tumor cell–platelet interaction and hematogenous metastasis. The inhibition of PKCα‐dependent platelet activation by pharmacologic inhibitors or genetic ablation of PKCα attenuates tumor cell–platelet interaction and hematogenous metastasis. Created by Adobe Illustrator.