Figure 2.
Genetic deficiency or antibody-mediated blockade of GPVI impairs platelet–tumor cell interaction. Platelet adhesion to tumor cells was quantified based on the fluorescence detection of anti-GPIX–labeled platelets, as described in “Materials and methods.” (A-B) Washed WT or Gp6−/− mouse platelets (Plts) were allowed to adhere to MC38 colon and AT-3 breast cancer cells. The bar represents 20 µm. (C) Similar experiments were performed with WT mouse platelets treated with JAQ1 F(abʹ)2 or an irrelevant control F(abʹ)2 antibody. (D) Representative SEM images of WT and Gp6−/− mouse platelets adhering to MC38 tumors cells. Washed mouse platelets were mixed with MC38 colon cancer cells (100 platelets/cell), incubated for 1 hour at 37°C, and then analyzed by SEM. The bar represents 8 μm. Arrows indicate platelets (magenta) and tumor cells (black). (E-F) Washed human platelets were isolated from patients carrying the heterozygous (GP6+/−) or homozygous (GP6−/−) mutation in the GP6 gene or from healthy donors and coincubated with human HT29 colon and MDA-MB-231 breast cancer cells. Shown are representative images of GP6+/− and GP6−/− platelets adhering to HT29 and MDA-MB-231 cells. The bar represents 20 µm. (B-C,F) Quantification of the fluorescence signal corresponding to the number of mouse (B-C) and human (F) platelets adhering to tumor cells. (B-C) Mean ± standard deviation (SD); n = 4 mice per group; *P < .05, by Mann-Whitney test. (F) Mean ± SD; Ctrl (healthy donor), n = 3; GP6+/−, n = 2; and GP6−/−, n = 3. *P < .05; **P < .01, by unpaired Student t test. (G,H) Experimental design: thrombocytopenic mice were transfused with Cy3-conjugated anti-GPIX-labeled WT or Gp6−/− platelets (red) and CFSE-labeled MC38 tumor cells (green). One hour after injection, mice were euthanized, the lungs were collected, and the colocalization of MC38 tumor cells with platelets was determined by confocal microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The bar represents 20 µm. (H) Quantification of the number of tumor cells surrounded or not by platelets. Mean ± SD, n = 4 mice per group, tumor cells (TC) without Plts WT vs Gp6−/−, *P < .05; TC with Plts WT vs Gp6−/−; **P < .01, by 2-way analysis of variance with Bonferroni's multiple-comparison test.

Genetic deficiency or antibody-mediated blockade of GPVI impairs platelet–tumor cell interaction. Platelet adhesion to tumor cells was quantified based on the fluorescence detection of anti-GPIX–labeled platelets, as described in “Materials and methods.” (A-B) Washed WT or Gp6−/− mouse platelets (Plts) were allowed to adhere to MC38 colon and AT-3 breast cancer cells. The bar represents 20 µm. (C) Similar experiments were performed with WT mouse platelets treated with JAQ1 F(abʹ)2 or an irrelevant control F(abʹ)2 antibody. (D) Representative SEM images of WT and Gp6−/− mouse platelets adhering to MC38 tumors cells. Washed mouse platelets were mixed with MC38 colon cancer cells (100 platelets/cell), incubated for 1 hour at 37°C, and then analyzed by SEM. The bar represents 8 μm. Arrows indicate platelets (magenta) and tumor cells (black). (E-F) Washed human platelets were isolated from patients carrying the heterozygous (GP6+/−) or homozygous (GP6−/−) mutation in the GP6 gene or from healthy donors and coincubated with human HT29 colon and MDA-MB-231 breast cancer cells. Shown are representative images of GP6+/− and GP6−/− platelets adhering to HT29 and MDA-MB-231 cells. The bar represents 20 µm. (B-C,F) Quantification of the fluorescence signal corresponding to the number of mouse (B-C) and human (F) platelets adhering to tumor cells. (B-C) Mean ± standard deviation (SD); n = 4 mice per group; *P < .05, by Mann-Whitney test. (F) Mean ± SD; Ctrl (healthy donor), n = 3; GP6+/−, n = 2; and GP6−/−, n = 3. *P < .05; **P < .01, by unpaired Student t test. (G,H) Experimental design: thrombocytopenic mice were transfused with Cy3-conjugated anti-GPIX-labeled WT or Gp6−/− platelets (red) and CFSE-labeled MC38 tumor cells (green). One hour after injection, mice were euthanized, the lungs were collected, and the colocalization of MC38 tumor cells with platelets was determined by confocal microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The bar represents 20 µm. (H) Quantification of the number of tumor cells surrounded or not by platelets. Mean ± SD, n = 4 mice per group, tumor cells (TC) without Plts WT vs Gp6−/−, *P < .05; TC with Plts WT vs Gp6−/−; **P < .01, by 2-way analysis of variance with Bonferroni's multiple-comparison test.

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