Platelet-derived growth factor B (PDGFB) plays a crucial role in recruitment of PDGF receptor b-positive pericytes to blood vessels. The endothelium is an essential source of PDGFB in this process. Platelets constitute a major reservoir of PDGFB and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. Here, we show that tumor vascular function, as well as pericyte coverage is significantly impaired in mice with conditional knockout of PDGFB in platelets. A lack of PDGFB in platelets led to enhanced hypoxia and epithelial-to-mesenchymal transition in the primary tumors, elevated levels of circulating tumor cells, and increased spontaneous metastasis to the liver or lungs in two mouse models. These findings establish a previously unknown role for plateletderived PDGFB, whereby it promotes and maintains vascular integrity in the tumor microenvironment by contributing to the recruitment of pericytes.Significance: Conditional knockout of PDGFB in platelets demonstrates its previously unknown role in the maintenance of tumor vascular integrity and host protection against metastasis.

Platelets can promote several stages of the metastatic process and thus contribute to malignant progression. As an example, platelets promote invasive properties of tumor cells by induction of epithelial to mesenchymal transition (EMT). In this study, we show that tumor necrosis factor receptor‐associated factor (TRAF) family member‐associated NF‐κB activator (TANK)‐binding kinase 1 (TBK1) is a previously unknown mediator of platelet‐induced EMT in mammary carcinoma cells. Coculture of 2 mammary carcinoma cell lines, Ep5 from mice and MCF10A(MII) from humans, with isolated platelets induced morphologic as well as molecular changes characteristic of EMT, which was paralleled with activation of TBK1. TBK1 depletion using small interfering RNA impaired platelet‐induced EMT in both Ep5 and MCF10A(MII) cells. Furthermore, platelet‐induced activation of the NF‐κB subunit p65 was suppressed after TBK1 knockdown, demonstrating that TBK1 mediates platelet‐induced NF‐κB signaling and EMT. Using an in vivo metastasis assay, we found that depletion of TBK1 from mammary carcinoma cells during in vitro preconditioning with platelets subsequently suppressed the formation of lung metastases in mice. Altogether, these results suggest that TBK1 contributes to tumor invasiveness and may be a driver of metastatic spread in breast cancer.—Zhang, Y., Unnithan, R. V. M., Hamidi, A., Caja, L., Saupe, F., Moustakas, A., Cedervall, J., Olsson, A.‐K. TANK‐binding kinase 1 is a mediator of platelet‐induced EMT in mammary carcinoma cells. FASEB J. 33, 7822–7832 (2019). http://www.fasebj.org

<p>Data from endothelial cell qPCR array.</p>

<p>Supplementary Table 1 and Fig 1-8 including legends. Table S1: Observed and expected numbers of Pf4-Cre;Pdgfbfl/fl embryos at different developmental stages. Figure S1: Platelet-specific PDGFB knockout embryos display no obvious abnormalities. Figure S2: Platelet counts and proportion in peripheral blood from WT and pl-PDGFB KO mice. Figure S3: Generation of RIP1-Tag2 mice lacking PDGFB in platelets. Figure S4: Lack of platelet PDGFB does not affect the angiogenic switch or tumor growth in RT2 mice. Figure S5: Tumor vessels in MC38 colon carcinoma in mice with platelet-specific PDGFB ablation have impaired function and decreased pericyte coverage. Figure S6: Increased spontaneous lung metastasis in mice with platelet-specific PDGFB ablation in the HCmel12 melanoma model. Figure S7: Reduced pericyte coverage already in pre-tumorigenic tissue in mice lacking PDGFB in platelets. Figure S8: Expression of Cxcl3, Cxcl5, Cxcl7 and Cxcl15 in tumor tissue from Pf4-Cre- and Pf4-Cre+ mice.</p>

<p>Supplementary methods: Primer sequences</p>

<p>Antibody concentration and blocking conditions</p>

<p>Supplementary Table 1 and Fig 1-8 including legends. Table S1: Observed and expected numbers of Pf4-Cre;Pdgfbfl/fl embryos at different developmental stages. Figure S1: Platelet-specific PDGFB knockout embryos display no obvious abnormalities. Figure S2: Platelet counts and proportion in peripheral blood from WT and pl-PDGFB KO mice. Figure S3: Generation of RIP1-Tag2 mice lacking PDGFB in platelets. Figure S4: Lack of platelet PDGFB does not affect the angiogenic switch or tumor growth in RT2 mice. Figure S5: Tumor vessels in MC38 colon carcinoma in mice with platelet-specific PDGFB ablation have impaired function and decreased pericyte coverage. Figure S6: Increased spontaneous lung metastasis in mice with platelet-specific PDGFB ablation in the HCmel12 melanoma model. Figure S7: Reduced pericyte coverage already in pre-tumorigenic tissue in mice lacking PDGFB in platelets. Figure S8: Expression of Cxcl3, Cxcl5, Cxcl7 and Cxcl15 in tumor tissue from Pf4-Cre- and Pf4-Cre+ mice.</p>

<p>Supplementary methods: Primer sequences</p>