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Supplementary Figures and Figure legends, Supplementary Video legends and supplementary materials and methods

Crossing the blood–brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and endpoint in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (EC) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by cross-talk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it.Significance:Tracking single extravasating cancer cells using multimodal correlative microscopy uncovers a brain seeding mechanism involving endothelial remodeling driven by cancer cell–derived MMP9, which might enable the development of approaches to prevent brain metastasis.See related commentary by McCarty, p. 1167

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 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.

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.

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Suppl Video: Tumor cells (green) and monocytes (white) are seeded on a confluent endothelial monolayer (red) on a 3-D collagen matrix.

Tumor cells interact with blood constituents and these interactions promote metastasis. Selectins are vascular receptors facilitating interactions of tumor cells with platelets, leukocytes, and endothelium, but the role of endothelial E-selectin remains unclear. Here we show that E-selectin is a major receptor for monocyte recruitment to tumor cell–activated endothelium. Experimental and spontaneous lung metastasis using murine tumor cells, without E-selectin ligands, were attenuated in E-selectin–deficient mice. Tumor cell–derived CCL2 promoted endothelial activation, resulting in enhanced endothelial E-selectin expression. The recruitment of inflammatory monocytes to metastasizing tumor cells was dependent on the local endothelial activation and the presence of E-selectin. Monocytes promoted transendothelial migration of tumor cells through the induction of E-selectin–dependent endothelial retractions and a subsequent modulation of tight junctions through dephosphorylation of VE-cadherin. Thus, endothelial E-selectin shapes the tumor microenvironment through the recruitment, adhesion, and activation of monocytes that facilitate tumor cell extravasation and thereby metastasis. These findings provide evidence that endothelial E-selectin is a novel factor contributing to endothelial retraction required for efficient lung metastasis. Cancer Res; 76(18); 5302–12. ©2016 AACR.

Crossing the blood–brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and endpoint in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (EC) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by cross-talk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it. Significance: Tracking single extravasating cancer cells using multimodal correlative microscopy uncovers a brain seeding mechanism involving endothelial remodeling driven by cancer cell–derived MMP9, which might enable the development of approaches to prevent brain metastasis. See related commentary by McCarty, p. 1167

Immune checkpoint inhibition (ICI) has yielded remarkable results in prolonging survival of metastatic melanoma patients but only a subset of individuals treated respond to therapy. Success of ICI treatment appears to depend on the number of tumor-infiltrating effector T-cells, which are known to be influenced by activated eosinophils. To verify the co-occurrence of activated eosinophils and T-cells in melanoma, immunofluorescence was performed in 285 primary or metastatic tumor tissue specimens from 118 patients. Moreover, eosinophil counts and activity markers such as eosinophil cationic protein (ECP) and eosinophil peroxidase (EPX) were measured in the serum before therapy start and before the 4th infusion of ICI in 45 metastatic unresected melanoma patients. We observed a positive correlation between increased tumor-infiltrating eosinophils and T-cells associated with delayed melanoma progression. High baseline levels of eosinophil count, serum ECP and EPX were linked to prolonged progression-free survival in metastatic melanoma. Our data provide first indications that activated eosinophils are related to the T-cell-inflamed tumor microenvironment and could be considered as potential future prognostic biomarkers in melanoma.

The microenvironment of malignant melanomas defines the properties of tumor blood vessels and regulates infiltration and vascular dissemination of immune and cancer cells, respectively. Previous research in other cancer entities suggested the complement system as an essential part of the tumor microenvironment. Here, we confirm activation of the complement system in samples of melanoma patients and murine melanomas. We identified the tumor endothelium as the starting point of the complement cascade. Generation of complement-derived C5a promoted the recruitment of neutrophils. Upon contact with the vascular endothelium, neutrophils were further activated by complement membrane attack complexes (MACs). MAC-activated neutrophils release neutrophil extracellular traps (NETs). Close to the blood vessel wall, NETs opened the endothelial barrier as indicated by an enhanced vascular leakage. This facilitated the entrance of melanoma cells into the circulation and their systemic spread. Depletion of neutrophils or lack of MAC formation in complement component 6 (C6)–deficient animals protected the vascular endothelium and prevented vascular intravasation of melanoma cells. Our data suggest that inhibition of MAC-mediated neutrophil activation is a potent strategy to abolish hematogenous dissemination in melanoma.

BackgroundAn increased incidence of thrombotic complications associated with an increased mortality rate has been observed under immune checkpoint inhibition (ICI). Recent investigations on the coagulation pathways have highlighted the direct role of key coagulatory proteins and platelets in cancer initiation, angiogenesis and progression. The aim of this study was to evaluate the prognostic value of von Willebrand factor (vWF) and its regulatory enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), D-dimers and platelets in a cohort of patients with metastatic melanoma receiving ICI.MethodsIn a prospective cohort of 83 patients with metastatic melanoma, we measured the systemic levels of vWF-antigen (vWF:Ag), ADAMTS13 activity, D-dimers and platelets, before the beginning of the treatment (baseline), and 6, 12 and 24 weeks after. In parallel, we collected standard biological parameters used in clinical routine to monitor melanoma response (lactate deshydrogenase (LDH), S100). The impact of neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) on overall survival (OS) in patients receiving ICI was assessed. Univariable and multivariable Cox proportional models were then used to investigate any potential association of these parameters to clinical progression (progression-free survival (PFS) and OS). Baseline values and variations over therapy course were compared between primary responders and resistant patients.ResultsPatients with melanoma present with dysregulated levels of vWF:Ag, ADAMTS13 activity, D-dimers, LDH, S100 and CRP at the beginning of treatment. With a median clinical follow-up of 26 months, vWF:Ag interrogated as a continuous variable was significantly associated with PFS in univariate and multivariate analysis (HR=1.04; p=0.007). Lower values of vWF:Ag at baseline were observed in the primary responders group (median: 29.4 µg/mL vs 32.9 µg/mL; p=0.048) when compared with primary resistant patients. As for OS, we found an association with D-dimers and ADAMTS13 activity in univariate analysis and vWF:Ag in univariate and multivariate analysis including v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and Eastern Cooperative Oncology Group (ECOG) performance status. Follow-up over the course of treatment depicts different evolution profiles for vWF:Ag between the primary response and resistance groups.ConclusionsIn this prospective cohort, coagulatory parameters such as ADAMTS13 activity and D-dimers are associated with OS but baseline vWF:Ag levels appeared as the only parameter associated with response and OS to ICI. This highlights a potential role of vWF as a biomarker to monitor ICI response of patients with malignant melanoma.

Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients’ survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression. publishedVersion

Background The prognosis of patients with brain metastases (BM) is poor despite advances in our understanding of the underlying pathophysiology. The high incidence of thrombotic complications defines tumor progression and the high mortality rate. We, therefore, postulated that von Willebrand factor (VWF) promotes BM via its ability to induce platelet aggregation and thrombosis. Methods We measured the abundance of VWF in the blood and intravascular platelet aggregates of patients with BM, and determined the specific contribution of endothelial and platelet-derived VWF using in vitro models and microfluidics. The relevance for the brain metastatic cascade in vivo was demonstrated in ret transgenic mice, which spontaneously develop BM, and by the intracardiac injection of melanoma cells. Results Higher levels of plasma VWF in patients with BM were associated with enhanced intraluminal VWF fiber formation and platelet aggregation in the metastatic tissue and peritumoral regions. Platelet activation triggered the formation of VWF multimers, promoting platelet aggregation and activation, in turn enhancing tumor invasiveness. The absence of VWF in platelets, or the blocking of platelet activation, abolished platelet aggregation, and reduced tumor cell transmigration. Anticoagulation and platelet inhibition consistently reduced the number of BM in preclinical animal models. Conclusions Our data indicate that platelet-derived VWF is involved in cerebral clot formation and in metastatic growth of melanoma in the brain. Targeting platelet activation with low-molecular-weight heparins represents a promising therapeutic approach to prevent melanoma BM.

Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.