Figure 3. Platelet-Derived TGFβ Promotes Lung Metastasis.
(A) Concentration of TGFβ1 in washed platelets and platelet-rich plasma (PRP) from wild-type (WT), Pf4-cre+; TGFβ1fl/fl or Pf4-cre+; TGFβ1fl/+ mice measured by ELISA (n=2).
(B) Relative luciferase activity (RLU) in Ep5 cells stably expressing a luciferase reporter under the control of the SBE promoter and treated with buffer, platelets from wild-type (WT), Pf4-cre+; TGFβ1fl/fl, Pf4-cre+; TGFβ1fl/+, or with wild-type platelets + SB431542 (10μM) for 20h (n=3).
(C) Zymography for MMP-9 in the conditioned medium from Ep5 cells treated with buffer, platelets from wild-type (WT), Pf4-cre+; TGFβ1fl/fl or Pf4-cre+; TGFβ1fl/+ mice for 40h.
(D–E) Numbers of metastatic foci at the surface of lungs (2 largest lobes) 14 days after tail-vein injection of MC38GFP cells in wild-type (WT), Pf4-cre+; TGFβ1fl/fl or Pf4-cre+; TGFβ1fl/+ mice (n=7–9), and representative pictures of lungs (E).
(F) Numbers of metastatic foci at the surface of lungs (2 largest lobes) 14 days after tail-vein injection of MC38GFP cells pretreated with buffer (−), platelets from WT mice (WT) or platelets from Pf4-cre+; TGFβ1fl/fl (fl/fl) and injected into WT or Pf4-cre+; TGFβ1fl/fl mice (n=9–14).
(G) Numbers of tumor cells at the surface of lungs 3h, 21h and 48h after tail-vein injection of MC38GFP cells in wild-type (WT) or Pf4-cre+; TGFβ1fl/fl mice. Each point represents the mean ± SEM number of cells/view field (3X) (n=3–14). *p<0.05, **p<0.01 were determined by Student’s t-test.
(H) Percentage of intravascular and extravascular MC38GFP cells in lungs of wild-type (WT) or Pf4-cre+; TGFβ1fl/fl mice 3h, 21h and 48h after tail-vein injection of tumor cells (n=16–46 cells). **p<0.01 as determined by Fisher’s exact test.
(I–J) Confocal microcopy of lungs of wild-type (WT) or Pf4-cre+; TGFβ1fl/fl mice 3h and 48h after tail-vein injection of tumor cells for MC38GFP cells (green) and either blood vessels (H; PECAM-1 staining; red) or platelets (I; GP1bβ staining; red. Note platelet aggregates surrounding tumor cells.). Scale bar=50μm.
For panels A, B, D, and F bars represent the mean ± SEM, and *p<0.05, **p<0.01, ***p<0.001 vs WT or buffer were determined by one-way ANOVA followed by Tuckey’s post test.