Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

Figure 1. Dependency of platelet activation by SW620 cells from the MACC1 expression. (A) Representative curves of platelet aggregation by light transmission measurement after contact with MACC1-positive SW620 Ctrl cells or the MACC1 KO variant. (B) ATP release from platelet dense granule induced by SW620 Ctrl and MACC1 KO cells. (C) Platelet adhesion to a cell layer of SW620 Ctrl or MACC1 KO cells exclude a diminished cell contact formation as the reason for lower platelet activation by the MACC1-positive cells. ** p < 0.01, *** p < 0.001.

Figure 2. The cell supernatant of SW620 cells interferes with platelet activation. (A) Representative traces showing platelet aggregation in response to TRAP-6 (black) and its inhibition by supernatant of SW620 MACC1 Ctrl cells (solid grey line) and a lower inhibition by MACC1 KO cells (dashed grey line), respectively. (B) Quantification of ATP release from resting platelets, platelets activated with TRAP-6, and co-incubated with supernatant of SW620 MACC1 Ctrl (s. MACC1 Ctrl) and MACC1 KO (s. MACC1 KO) cells, respectively. Data indicate a soluble inhibitory compound in the cell supernatant that is more potent in the SW620 Ctrl cells. *** p < 0.001.

Figure 3. MACC1 activity is directly related to IGFBP2 expression, which interferes with platelet activation. The downregulation of IGFBP2 in the MACC1 KO variant of SW620 cells was indicated at the mRNA level by qPCR (A) and confirmed at the protein level by ELISA (B). ELISA data confirm the lower expression of IGFBP2 by the MACC1 KO variant of SW620 cells, either in supernatant (s. MACC1 Ctrl vs. s.MACC1 KO, left columns) and at a lower level in the cell lysate (right columns). (C) IGF-I is costimulatory for platelet activation and accelerates the effect of 7.5 µM TRAP-6 to induce platelet aggregation. (D) IGF-I is associated with platelets and found in platelet plasma or supernatant after activation, but not in considerable amounts in supernatant of SW620 cells. (E) Recombinant IGFBP2 is able to block concentration-dependently the activity of TRAP-6 to induce platelet aggregation and (F), significantly, the platelet ATP release. *** p < 0.001; **** p < 0.0001.

Figure 4. IGFBP2 controls platelet activation by SW620 cells. (A) The shRNA-mediated knockdown of IGFBP2 in SW620 cells was confirmed by Western blot, compared to nontargeted knockdown IGFBP2 Ctrl, and quantified by IGFBP2 ELISA using cell supernatants (left columns) and cell lysates (right columns) (B). (C) The knockdown of IGFBP2 in (MACC1-positive) SW620 cells restored the activation potential to platelets in the aggregation assay, or in ATP release (D). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5. The impact of IGFBP2 as a functional downstream component of MACC1 on SW620 cell dynamics. (A,B) Detection of cell migratory dynamics in a 2D wound healing assay comparing the impact of MACC1 (A) and IGFBP2 (B) on migration and the effect of platelets to accelerate dynamic properties. (C) Analyzing cell invasion in a transmigration assay and the impact of MACC1 knockout or IGFBP2 knockdown. (D,E) Analyzing the impact of MACC1 KO or IGFBP2 KD on cell capability to induce coagulation indicates no differences in thrombin formation upon MACC1 KO (D) or IGFBP2 KD (E) when compared to the respective control cells. (F) Flow cytometry data confirm that the knockout of MACC1 in SW620 cells has no impact on tissue factor expression. * p < 0.05; ** p < 0.01, ns = non-significant.

Figure 6. IGFBP2 is the metastatic factor in vivo. After xenotransplantation of SW620 control or SW620 IGFBP2 KD cells, metastasis formation was inhibited by IGFBP2 knockdown, as shown by (A) human satellite DNA and (B) immunohistochemistry staining of human cytokeratin 19.