bioRxiv preprint doi: https://doi.org/10.1101/183046; this version posted August 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Hemodynamic forces tune the arrest, adhesion and extravasation of circulating tumor cells Gautier Follain1-4, Naël Osmani1-4, Sofia Azevedo1-4, Guillaume Allio1-4, Luc Mercier1- 4, Matthia A. Karreman5, Gergely Solecki6, Nina Fekonja1-4, Claudia Hille7, Vincent Chabannes8, Guillaume Dollé8, Thibaut Metivet8, Christophe Prudhomme8, Bernhard Ruthensteiner9, André Kemmling10, Susanne Siemonsen11, Tanja Schneider11, Jens Fiehler11, Markus Glatzel12, Frank Winkler6, Yannick Schwab5, Klaus Pantel7, Sébastien Harlepp2,13-14, Jacky G. Goetz1-4* 1INSERM UMR_S1109, MN3T, Strasbourg, F-67200, France. 2Université de Strasbourg, Strasbourg, F-67000, France. 3LabEx Medalis, Université de Strasbourg, Strasbourg, F-67000, France. 4Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg, F- 67000, France. 5Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, 69117, Germany. 6Department of Neurooncology, University Hospital Heidelberg, Heidelberg, 69120, Germany and Clinical Cooperation Unit Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany. 7Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg, 20246, Germany. 8LabEx IRMIA, CEMOSIS, Université de Strasbourg, Strasbourg, F-67000 France. 9Zoologische Staatssammlung München, Munich, 81247, Germany. 10Department of Neuroradiology, University Medical Center Schleswig-Holstein, Campus Lübeck, 23535 Lübeck, Germany 11Department of Diagnostic and Interventional Neuroradiology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 12Center for Diagnostics, Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 13CNRS UMR7504, Institut de Physique et Chimie des Matériaux de Strasbourg (IPCMS), Strasbourg, F-67000, France. 14LabEx NIE, Université de Strasbourg, Strasbourg, F-67000, France. 1 bioRxiv preprint doi: https://doi.org/10.1101/183046; this version posted August 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Materials and correspondance (*): Jacky G. GOETZ, [email protected] INSERM UMR_S1109, The Microenvironmental Niche in Tumorigenesis and Targeted Therapy, 67200 Strasbourg, France; LabEx Medalis, Fédération de Médecine Translationnelle de Strasbourg (FMTS), 67000 Strasbourg, France Web: www.goetzlab.com, twitter: @GoetzJacky 2 bioRxiv preprint doi: https://doi.org/10.1101/183046; this version posted August 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT Metastatic seeding is driven by cell-intrinsic and environmental cues, yet the contribution of biomechanics is poorly known. We aim to elucidate the impact of blood flow on the arrest and the extravasation of circulating tumor cells (CTCs) in vivo. Using the zebrafish embryo, we show that arrest of CTCs occurs mostly in vessels with favorable flow profiles where flow forces control the adhesion efficacy of CTCs to the endothelium. We biophysically identified the threshold values of flow and adhesion forces allowing successful arrest of CTCs. In addition, flow forces fine-tune tumor cell extravasation by impairing the remodeling properties of the endothelium. Importantly, we also observe endothelial remodeling at arrest sites of CTCs in mouse brain capillaries. Finally, we observed that human supratentorial brain metastases preferably develop in areas with low perfusion. Altogether, these results demonstrate that hemodynamic profiles at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth. KEYWORDS Blood flow, extravasation, metastasis, endothelial remodeling, circulating tumor cells, cell adhesion, biomechanics, zebrafish 3 bioRxiv preprint doi: https://doi.org/10.1101/183046; this version posted August 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. INTRODUCTION Metastatic progression is a complex process resulting in the formation of lethal secondary tumors at distance of its origin1. Metastatic cancer cells disseminate very efficiently throughout the body upon intravasation in the blood circulation. Recent work on breast cancer suggests that about 80% of metastases originate from early disseminated cancer cells2,3. Once in the blood stream, circulating tumor cells (CTCs) may find a location favoring arrest and stable adhesion before extravasating, and avoiding the hostile shear forces4,5. After extravasation, metastatic cells either remain dormant6 or grow successfully into life-threatening secondary tumors7. Although multiple mechanisms have been postulated for successful extravasation and outgrowth of metastatic cells7–10, there are only little informations on the role played by mechanical cues encountered in the blood, the main route for hematogenous metastatic dissemination. Biomechanical forces are known to have a major impact on tumor progression, leading to metastatic growth. For example, tumor cells sense and respond to stiffening of the surrounding stroma by increasing their invasive potential11–13. High extravascular stress caused by tumor growth14,15 and interstitial fluid pressure16 leads to vascular compression that impairs perfusion and eventually promotes tumor progression, immunosuppression, and treatment resistance. Locally, invading tumor cells need to overcome physical tissue constraints by cellular and nuclear deformability17,18, possibly inducing nuclear envelope rupture and DNA damage19 leading eventually to inheritable genomic instability20. Overall, while the impact of biomechanics on tumor growth and invasion are mechanistically relatively well understood, the in vivo mechanisms driving arrest, survival and successful extravasation of CTCs, preceding metastatic growth, remain to be elucidated. Indeed, very little is known about how CTCs arrest and adhere to the endothelium of small capillaries and leave the blood stream by crossing the vascular wall. While the “seed and soil” concept states that metastasis will occur at sites where the local microenvironment is favorable21, the “mechanical” concept argues that arrest and metastasis of CTC occur at sites of optimal flow patterns22. CTCs in the blood circulation are subjected to vascular routing23, collisions and associations with blood cells24, hemodynamic shear forces25 and physical constraints imposed by the vessel architecture7,9. Only CTCs capable of overcoming or exploiting the deleterious effects of shear forces will eventually arrest, adhere to and exit the vasculature to form a 4 bioRxiv preprint doi: https://doi.org/10.1101/183046; this version posted August 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. secondary tumor26. Interestingly, upon arrest in lung microvessels, shear flow disrupts CTCs into microparticles that prime and recruit myeloid cells thereby stimulating successful metastasis from surviving CTC9. Nevertheless, a direct contribution of mechanical cues to the arrest and successful extravasation of CTCs has only been poorly studied so far26. Therefore, new in vivo models, where modeling, visualization and biophysical quantification of the extravasation parameters are easily performed, are of utmost importance for assessing whether biomechanics regulate metastatic extravasation. Here, we aim to address the direct impact of the blood flow on the arrest and extravasation of CTCs in vivo. We developed an original experimental approach to measure and modulate blood flow with intravascular injection of CTCs within zebrafish (ZF) embryos. We observed that blood flow controls the sequential steps of arrest, adhesion and extravasation of CTCs in vivo. In parallel, using microfluidics and optical tweezers, we identified the critical adhesion force (80 pN) that CTCs require to initiate adhesion to the endothelium which rapidly stabilizes under shear flow. This value matches the threshold dragging force measured in vivo at extravasation sites. Finally, we used our recently-developed intravital correlative light and electronic microscope (CLEM)27–29, to identify endothelial remodeling as one of the major extravasation mechanisms in vivo, and that endothelial remodeling scales with flow velocities. Overall our studies demonstrate that blood flow forces at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth. 5 bioRxiv preprint doi: https://doi.org/10.1101/183046; this version posted August 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. RESULTS Arrest and adhesion of CTC is favored by permissive flow velocities In order to test the impact of blood flow on the arrest, adhesion and extravasation of CTCs, we experimentally modeled metastatic seeding in endothelium-labeled ZF embryo (Tg(Fli1a:EGFP)) at 2 days post-fertilization (dpf) by injecting fluorescently-