Coculture of cancer cells with platelets increases their survival and metastasis by activating the TGFβ/Smad/PAI-1 and PI3K/AKT pathways

When cancer cells enter the bloodstream, they can interact with platelets to acquire stronger survival and metastatic abilities. To elucidate the underlying mechanisms, we cocultured metastatic melanoma and triple-negative breast cancer cells with species-homologous platelets. We found that cocultured cancer cells displayed higher viabilities in circulation, stronger capacities for cell migration, invasion, and colony formation in vitro, and more tumorigenesis and metastasis in mice. RNA sequencing analysis revealed that the level of serpin family E member 1 (SERPINE1) was significantly upregulated in cocultured cancer cells. Knockdown of SERPINE1 reversed the coculture-elevated survival and metastatic phenotypes of cancer cells. Mechanistic studies indicated that coculture with platelets activated the TGFβ/Smad pathway to induce SERPINE1 expression in cancer cells, which encodes plasminogen activator inhibitor 1 (PAI-1). PAI-1 then activated PI3K to increase the phosphorylation of AKTThr308 and Bad to elevate Bcl-2, which enhanced cell survival in circulation. Moreover, higher levels of PAI-1 were detected in metastatic tumors from melanoma and triple-negative breast cancer patients than in normal tissues, and high levels of PAI-1 were associated with a shorter overall survival time and worse disease progression in breast cancer. PAI-1 may act as a potential biomarker for detecting and treating metastatic tumor cells.


Fig. S2 .
Fig. S2.Coculture induced tumorigenesis and metastasis in orthotopic tumor models.(A) Representative image of the Transwell migration and invasion assays for human platelets, monocultured and cocultured 231-GFP cells.Scale bar, 200 μm.(B and C) MTT assays were used to compare cell proliferative ability between monocultured and cocultured B16F10-C3 and 231-GFP cells.(D and E) H&E staining images of lung tissues under the indicated conditions.Scale bar, 20 μm.(F and G) Images of the primary tumors inoculated with monocultured and cocultured B16F10-C3 (No. of mice = 6) and 231-GFP cells (No. of mice = 6 to 7).(H and I) Quantification of mouse body weight.The results represent the mean ± SEM.

Fig. S4 .
Fig. S4.Overexpression of PAI-1 promoted B16F10-C3 cell tumorigenesis and metastasis.(A) Representative image of the primary tumors after subcutaneous injection of B16F10-C3 cells into C57BL/6J mice (n = 6 to 7).(B and C) Quantification of the primary tumor formation rate and tumor weight of EV and OE-PAI-1 in B16F10-C3 cells.The results represent the mean ± SEM. (D) Quantified results of mouse body weight.(E) Quantified results of primary tumor volume.The results represent the mean ± SEM. (F) Representative images of metastatic tumors in the distant skin, lung, and iliac lymph nodes in the EV and OE-PAI-1 groups.The red arrowhead indicates metastatic tumors.Scale bars, 1 mm.(G) Quantification of the metastatic rate in different organs in mice in the EV and OE-PAI-1 groups.*P < 0.05 and ns, no significance.

Table S2
List of shRNA and siRNA.

Table S3
List of overexpression plasmids.

Table S4
Analysis of primary tumor formation of B16F10-C3 cells in orthotopic tumor models.

Table S5
Analysis of primary tumor formation of 231-GFP cells in orthotopic tumor models.

Table S6
Correlation of PAI-1 expression with clinicopathological features of melanoma patients.Age and tumor size were calculated by unpaired Student's t test. b