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.

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 (BC) 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 BC cells that homed in the brain, extravasated, and successfully established a brain macrometastasis. Those BM-initiating breast cancer cells (BMICs) were mainly originating from a slow-cycling subpopulation that included only 16-20% 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 vs. non-BMICs was performed, which revealed up-regulation 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 BC, NDRG1 expression was heterogeneous, and high NDRG1 expression was associated with shorter metastasis-free survival. In conclusion, our data identifies temporary slow-cycling BC cells as the dominant source of brain and other metastases and demonstrates that this can lead to better understanding of BMICrelevant pathways, including potential new approaches to prevent BM in patients. ImplicationsCancer 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.