Supplementary Figure 4 shows IVIS results.

Supplementary Figure 5 shows cell cycle analysis.

Supplementary Figure 3 shows PKH26 intensity and depth of the cell in relation to the successful establishment of a metastasis. Further, the brain metastatic cascade for PKH26 low (slow cycling) and PKH26 (high) (ultra slow cycling) cells is illustrated.

Supplementary Table 1 shows entire gene list.

Supplementary Figure 7 shows correlation of NDRG1 expression with tumor growth in mouse and human samples.

Supplementary Figure 6 shows NDRG1 expression in several cell lines.

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

Supplementary Figure 2 cell index of PKH26 stained cells, the overlap of PKH26 loss and 2 other methods of cell division analysis as well as the reversion of a slow cycling cell population to a mixed cycling population.

Supplementary Figure 1 A-C shows loss of PKH26 over time. Supplementary Figure 1 D shows an additional example of a slow cycling cells with remaining PKH26 staining in vivo

Clinically relevant brain metastases (BMs) 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 in this process are not well understood. Here, in vivo multiphoton laser-scanning microscopy 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-term arrest. An extensive clot embedded ∼20% of brain-arrested cancer cells, and those were more likely to successfully extravasate and form a macrometastasis. Mechanistically, the generation of tissue factor-mediated thrombin 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 the formation of brain macrometastases. 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 formation of BMs. In conclusion, our data show that plasmatic coagulation is activated early by intravascular tumor cells in the brain with subsequent clot formation, which led us to discover a novel and specific mechanism that is crucial for brain colonization. Direct or indirect thrombin and VWF inhibitors emerge as promising drug candidates for trials on prevention of BMs.

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.

Abstract Specific biological properties of those circulating cancer cells that are the origin of brain metastases (BM) are not well understood. Here, single circulating breast cancer cells were fate-tracked during all steps of the brain metastatic cascade in mice after intracardial injection over weeks. A novel in vivo two-photon microscopy methodology was developed that allowed to determine the specific cellular and molecular features of breast cancer cells that homed in the brain, extravasated, and successfully established a brain macrometastasis. Those BM-initiating breast cancer cells (BMIC) were mainly originating from a slow-cycling subpopulation that included only 16% to 20% of all circulating cancer cells. BMICs showed enrichment of various markers of cellular stemness. As a proof of principle for the principal usefulness of this approach, expression profiling of BMICs versus non-BMICs was performed, which revealed upregulation of NDRG1 in the slow-cycling BMIC subpopulation in one BM model. Here, BM development was completely suppressed when NDRG1 expression was downregulated. In accordance, in primary human breast cancer, NDRG1 expression was heterogeneous, and high NDRG1 expression was associated with shorter metastasis-free survival. In conclusion, our data identify temporary slow-cycling breast cancer cells as the dominant source of brain and other metastases and demonstrates that this can lead to better understanding of BMIC-relevant pathways, including potential new approaches to prevent BM in patients. Implications: Cancer cells responsible for successful brain metastasis outgrowth are slow cycling and harbor stemness features. The molecular characteristics of these metastasis-initiating cells can be studied using intravital microscopy technology.

Gliomas are one of the cancers most at risk of causing life-threatening venous thromboemboli (VTE).Previously, we discovered that gliomas with mutations in isocitrate dehydrogenase (IDH1mut) are much less likely to contain intratumoral microthrombi, and are much less likely to cause peripheral VTE, compared to IDH1 wild-type (IDH1wt) gliomas.We also found that IDH1mut gliomas have methylation-associated suppression of Tissue Factor (TF), a key initiator of thrombosis.Furthermore, prior studies have shown that TF directly increases the malignancy of many cancers through multiple transmembrane signaling pathways.In the current study, we further explored the significance of TF in IDH1wt and IDH1mut gliomas.Three IDH1wt patient derived cell lines (GBM6, GBM12, GBM43) had high levels of TF protein and TF procoagulant activity in vitro, whereas 3 lines of patient-derived endogenous IDH1mut glioma cells (TB09, GBM164, BT142) had very low TF expression and activity.Exogenous treatment with the product of IDH1mut enzyme, D-2-hydroxyglutarate, suppressed the procoagulant activity of TF by up to 28% in IDH1wt cells.Likewise, induced expression of IDH1mut suppressed TF activity by 36% in IDH1wt cells.Thus far, in an ongoing prospective assessment of TF activity in the systemic circulation of glioma patients, 7/32 (22%) IDH1wt patients have had highly active circulating TF (defined as >2.0 pg/mL factor Xa generation), whereas 0/15 (0%) IDH1mut gliomas did-a pattern already trending toward significance (P=0.08).Among the 3 IDH1wt glioma cell lines, there was a direct correlation between TF expression and transwell migration, and TF knockdown with shRNA decreased migration by 93% (P=0.024).TF knockdown also reduced the average growth rate of orthotopic IDH1wt patient-derived glioma xenografts by 92%, as measured with bioluminescence imaging (P=0.03).These findings further indicate that suppression of TF expression and activity may be a critical component of the less thrombogenic, and less malignant, IDH1mut phenotype.