Jose Ramon Robador

Cancer-related venous thromboembolisms (VTE) are associated with metastasis and reduced survival in patients with urothelial cancer of the bladder. Although previous reports suggest the contribution of tissue factor and podoplanin, the mechanistic linkage between VTE and bladder cancer cell–derived molecules is unknown. Therefore, we compared distinct procoagulant pathways in four different cell lines. In vitro findings were further confirmed by microfluidic experiments mimicking the pathophysiology of tumor blood vessels and in tissue samples of patients with bladder cancer by transcriptome analysis and immunohistology. In vitro and microfluidic experiments identified bladder cancer–derived VEGF-A as highly procoagulant because it promoted the release of von Willebrand factor (VWF) from endothelial cells and thus platelet aggregation. In tissue sections from patients with bladder cancer, we found that VWF-mediated blood vessel occlusions were associated with a poor outcome. Transcriptome data further indicate that elevated expression levels of enzymes modulating VEGF-A availability were significantly connected to a decreased survival in patients with bladder cancer. In comparison with previously postulated molecular players, we identified tumor cell–derived VEGF-A and endothelial VWF as procoagulant mediators in bladder cancer. Therapeutic strategies that prevent the VEGF-A–mediated release of VWF may reduce tumor-associated hypercoagulation and metastasis in patients with bladder cancer.Implications:We identified the VEGF-A–mediated release of VWF from endothelial cells to be associated with bladder cancer progression.

Cancer-related venous thromboembolisms (VTE) are associated with metastasis and reduced survival in patients with urothelial cancer of the bladder. Although previous reports suggest the contribution of tissue factor and podoplanin, the mechanistic linkage between VTE and bladder cancer cell–derived molecules is unknown. Therefore, we compared distinct procoagulant pathways in four different cell lines. In vitro findings were further confirmed by microfluidic experiments mimicking the pathophysiology of tumor blood vessels and in tissue samples of patients with bladder cancer by transcriptome analysis and immunohistology. In vitro and microfluidic experiments identified bladder cancer–derived VEGF-A as highly procoagulant because it promoted the release of von Willebrand factor (VWF) from endothelial cells and thus platelet aggregation. In tissue sections from patients with bladder cancer, we found that VWF-mediated blood vessel occlusions were associated with a poor outcome. Transcriptome data further indicate that elevated expression levels of enzymes modulating VEGF-A availability were significantly connected to a decreased survival in patients with bladder cancer. In comparison with previously postulated molecular players, we identified tumor cell–derived VEGF-A and endothelial VWF as procoagulant mediators in bladder cancer. Therapeutic strategies that prevent the VEGF-A–mediated release of VWF may reduce tumor-associated hypercoagulation and metastasis in patients with bladder cancer.Implications:We identified the VEGF-A–mediated release of VWF from endothelial cells to be associated with bladder cancer progression.

S1: Schematic overview of the potential cross talk between bladder cancer cells, the endothelium and the coagulation system. S2: Expression of procoagulant factors and ability of T24 cells to produce thrombin. S3: UCB cells trigger angiogenesis. S4: VWF mediated blood vessel occlusion in peritumoral tissue. Supplementary Table 1. Correlation of the overall patients' survival with the expression of the indicated genes. Supplementary Video 1. Supernatants of UROtsa cells failed to induce endothelial activation.Supplementary Video 2. Supernatants of RT4 cells induced endothelial activation.

Supernatants of RT4 cells induced endothelial activation. Calcein blue labeled HUVECs (blue) were perfused with calcein red-orange labeled platelets (red) resuspended in cell culture medium conditioned by RT4 cells.

Supernatants of RT4 cells induced endothelial activation. Calcein blue labeled HUVECs (blue) were perfused with calcein red-orange labeled platelets (red) resuspended in cell culture medium conditioned by RT4 cells.

S1: Schematic overview of the potential cross talk between bladder cancer cells, the endothelium and the coagulation system. S2: Expression of procoagulant factors and ability of T24 cells to produce thrombin. S3: UCB cells trigger angiogenesis. S4: VWF mediated blood vessel occlusion in peritumoral tissue. Supplementary Table 1. Correlation of the overall patients' survival with the expression of the indicated genes. Supplementary Video 1. Supernatants of UROtsa cells failed to induce endothelial activation.Supplementary Video 2. Supernatants of RT4 cells induced endothelial activation.

Supernatants of UROtsa cells failed to induce endothelial activation. Calcein blue labeled HUVECs (blue) were perfused with calcein red-orange labeled platelets (red) resuspended in cell culture medium conditioned by UROtsa cells.

Supernatants of UROtsa cells failed to induce endothelial activation. Calcein blue labeled HUVECs (blue) were perfused with calcein red-orange labeled platelets (red) resuspended in cell culture medium conditioned by UROtsa cells.

Brain metastases (BM) represent a growing problem for breast cancer (BC) patients. Recent studies have demonstrated a strong impact of the BC molecular subtype on the incidence of BM development. This study explores the interaction between BC cells of different molecular subtypes and the blood–brain barrier (BBB). We compared the ability of BC cells of different molecular subtypes to overcome several steps (adhesion to the brain endothelium, disruption of the BBB, and invasion through the endothelial layer) during cerebral metastases formation, in vitro as well as in vivo. Further, the impact of these cells on the BBB was deciphered at the molecular level by transcriptome analysis of the triple-negative (TNBC) cells themselves as well as of hBMECs after cocultivation with BC cell secretomes. Compared to luminal BC cells, TNBC cells have a greater ability to influence the BBB in vitro and consequently develop BM in vivo. The brain-seeking subline and parental TNBC cells behaved similarly in terms of adhesion, whereas the first showed a stronger impact on the brain endothelium integrity and increased invasive ability. The comparative transcriptome revealed potential brain-metastatic-specific key regulators involved in the aforementioned processes, e.g., the angiogenesis-related factors TNXIP and CXCL1. In addition, the transcriptomes of the two TNBC cell lines strongly differed in certain angiogenesis-associated factors and in several genes related to cell migration and invasion. Based on the present study, we hypothesize that the tumor cell’s ability to disrupt the BBB via angiogenesis activation, together with increased cellular motility, is required for BC cells to overcome the BBB and develop brain metastases.

Clinically relevant brain metastases (BM) frequently form in cancer patients, with limited options for effective treatment. Circulating cancer cells must first permanently arrest in brain microvessels to colonize the brain, but the critical factors are not well understood. Here, in vivo multiphoton laser-scanning microscopy (MPLSM) of the entire brain metastatic cascade allowed unprecedented insights into how blood clot formation and von Willebrand factor (VWF) deposition determine the arrest of circulating cancer cells and subsequent brain colonization in mice. Clot formation in brain microvessels occurred frequently (>95%) and specifically at intravascularly arrested cancer cells, allowing their long-time arrest. An extensive clot embedded approximately 20% of brain-arrested cancer cells, and those were more likely to successfully extravasate and form a macrometastasis. Mechanistically, tissue factor-mediated thrombin generation by cancer cells accounted for local activation of plasmatic coagulation in the brain. Thrombin inhibition by treatment with low-molecular weight heparin or dabigatran and an anti-VWF antibody prevented clot formation, cancer cell arrest, extravasation, and brain macrometastasis formation. In contrast, tumor cells were not able to directly activate platelets, and antiplatelet treatments did reduce platelet dispositions at intravascular cancer cells but did not reduce overall BM formation. In conclusion, our data shows that plasmatic coagulation is activated early by intravascular tumor cells in the brain, with subsequent clot formation, discovering a novel and specific mechanism that is crucial for brain colonization. Direct or indirect thrombin and VWF inhibitors emerge as promising drug candidates for BM prevention trials.