ABSTRACT Distant metastasis, which results in >90% of cancer-related deaths, is enabled by hematogenous dissemination of tumor cells via the circulation. This requires the completion of a

sequence of complex steps including transit, initial arrest, extravasation, survival and proliferation. Increased understanding of the cellular and molecular players enabling each of these

steps is key to uncovering new opportunities for therapeutic intervention during early metastatic dissemination. As a protocol extension, this article describes an adaptation to our existing

protocol describing a microfluidic platform that offers additional applications. This protocol describes an _in vitro_ model of the human microcirculation with the potential to recapitulate

vitro_ extravasation assays, robust and rapid scoring of extravascular cells, combined with high-resolution imaging, can be easily achieved because of the confinement of the vascular network

to one plane close to the surface of the device. This renders extravascular cells clearly distinct and allows tumor cells of interest to be identified quickly as compared with those in

thick tissues. The ability to generate large numbers of devices (∼36) per experiment further allows for highly parametric studies, which are required when testing multiple genetic or

pharmacological perturbations. This is coupled with the capability for live tracking of single-cell extravasation events, allowing both tumor and endothelial morphological dynamics to be

observed in high detail with a moderate number of data points. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS

OPTIONS Access through your institution Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription $29.99 / 30 days cancel any time Learn

more Subscribe to this journal Receive 12 print issues and online access $259.00 per year only $21.58 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to

full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our

FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS SQUEEZING THROUGH THE MICROCIRCULATION: SURVIVAL ADAPTATIONS OF CIRCULATING TUMOUR CELLS TO SEED METASTASIS Article

Open access 01 December 2020 IMPAIRING FLOW-MEDIATED ENDOTHELIAL REMODELING REDUCES EXTRAVASATION OF TUMOR CELLS Article Open access 23 June 2021 DISTINGUISHING HIGH-METASTASIS-POTENTIAL

CIRCULATING TUMOR CELLS THROUGH FLUIDIC SHEAR STRESS IN A BLOODSTREAM-LIKE MICROFLUIDIC CIRCULATORY SYSTEM Article 10 June 2024 REFERENCES * Nguyen, D.X., Bos, P.D. & Massagué, J.

Metastasis: from dissemination to organ-specific colonization. _Nat. Rev. Cancer_ 9, 274–284 (2009). Article  CAS  Google Scholar  * Shin, Y. et al. Microfluidic assay for simultaneous

culture of multiple cell types on surfaces or within hydrogels. _Nat. Protoc._ 7, 1247–1259 (2012). Article  CAS  Google Scholar  * Chen, M.B., Whisler, J.A., Jeon, J.S. & Kamm, R.D.

Mechanisms of tumor cell extravasation in an _in vitro_ microvascular network platform. _Integr. Biol._ 5, 1262–1271 (2013). Article  CAS  Google Scholar  * Whisler, J.A., Chen, M.B. &

Kamm, R.D. Control of perfusable microvascular network morphology using a multiculture microfluidic system. _Tissue Eng. Part C. Methods_ 20, 543–552 (2014). Article  CAS  Google Scholar  *

Ehsan, S.M. et al. A three-dimensional _in vitro_ model of tumor cell intravasation. _Integr. Biol._ 6, 603–610 (2015). Article  Google Scholar  * Ghajar, C.M. et al. The perivascular niche

regulates breast tumour dormancy. _Nat. Cell Biol._ 15, 807–817 (2013). Article  CAS  Google Scholar  * Hsu, Y.-H. et al. Full range physiological mass transport control in 3D tissue

cultures. _Lab Chip_ 13, 81–89 (2012). Article  Google Scholar  * Kim, S., Lee, H., Chung, M. & Jeon, N.L. Engineering of functional, perfusable 3D microvascular networks on a chip. _Lab

Chip_ 13, 1489–1500 (2013). Article  CAS  Google Scholar  * Kim, J. et al. Implantable microfluidic device for the formation of three-dimensional vasculature by human endothelial progenitor

cells. _Biotechnol. Bioprocess Eng._ 19, 379–385 (2014). Article  CAS  Google Scholar  * Labelle, M. & Hynes, R.O. The initial hours of metastasis: the importance of cooperative

host-tumor cell interactions during hematogenous dissemination. _Cancer Discov._ 2, 1091–1099 (2012). Article  CAS  Google Scholar  * Labelle, M., Begum, S. & Hynes, R.O. Direct

signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. _Cancer Cell_ 20, 576–590 (2011). Article  CAS  Google Scholar  *

Kienast, Y. et al. Real-time imaging reveals the single steps of brain metastasis formation. _Nat. Med._ 16, 116–122 (2010). Article  CAS  Google Scholar  * Francia, G., Cruz-munoz, W., Man,

S., Xu, P. & Kerbel, R.S. Mouse models of advanced spontaneous metastasis for experimental therapeutics. _Nat. Rev. Cancer_ 11, 135–141 (2011). Article  CAS  Google Scholar  * Kitamura,

T. et al. CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages. _J. Exp. Med._ 212, 1043–1059 (2015). Article  CAS 

Google Scholar  * Qian, B. et al. A distinct macrophage population mediates metastatic breast cancer cell extravasation, establishment and growth. _PLoS One_ 4, e6562 (2009). Article  Google

Scholar  * Stoletov, K., Montel, V., Lester, R.D., Gonias, S.L. & Klemke, R. High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish. _Proc. Natl.

Acad. Sci. USA_ 104, 17406–17411 (2007). Article  CAS  Google Scholar  * Leong, H.S. et al. Invadopodia are required for cancer cell extravasation and are a therapeutic target for

metastasis. _Cell Rep._ 8, 1558–1570 (2014). Article  CAS  Google Scholar  * Koop, S. et al. Fate of melanoma cells entering the microcirculation: over 80% survive and extravasate. _Cancer

Res._ 55, 2520–2523 (1995). CAS  PubMed  Google Scholar  * Koop, S. et al. Independence of metastatic ability and extravasation: metastatic ras-transformed and control fibroblasts

extravasate equally well. _Proc. Natl. Acad. Sci. USA_ 93, 11080–11084 (1996). Article  CAS  Google Scholar  * Labelle, M., Begum, S. & Hynes, R.O. Platelets guide the formation of early

metastatic niches. _Proc. Natl. Acad. Sci. USA_ 111, E3053–E3061 (2014). Article  CAS  Google Scholar  * Roussos, E.T., Condeelis, J.S. & Patsialou, A. Chemotaxis in cancer. _Nat. Rev.

Cancer_ 11, 573–587 (2011). Article  CAS  Google Scholar  * Albini, A. & Benelli, R. The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation.

_Nat. Protoc._ 2, 504–511 (2007). Article  CAS  Google Scholar  * Mierke, C.T. Cancer cells regulate biomechanical properties of human microvascular endothelial cells. _J. Biol. Chem._ 286,

40025–40037 (2011). Article  CAS  Google Scholar  * Chrobak, K.M., Potter, D.R. & Tien, J. Formation of perfused, functional microvascular tubes _in vitro_. _Microvasc. Res._ 71, 185–196

(2006). Article  CAS  Google Scholar  * Zheng, Y. et al. _In vitro_ microvessels for the study of angiogenesis and thrombosis. _Proc. Natl. Acad. Sci. USA_ 109, 9342–9347 (2012). Article 

CAS  Google Scholar  * Kolesky, D.B., Homan, K.A., Skylar-Scott, M.A & Lewis, J.A. Three-dimensional bioprinting of thick vascularized tissues. _Proc. Natl. Acad. Sci. USA_ 113,

3179–3184 (2016). Article  CAS  Google Scholar  * Shin, M.K., Kim, S.K. & Jung, H. Integration of intra- and extravasation in one cell-based microfluidic chip for the study of cancer

metastasis. _Lab Chip_ 11, 3880–3887 (2011). Article  CAS  Google Scholar  * Song, J.W. et al. Microfluidic endothelium for studying the intravascular adhesion of metastatic breast cancer

cells. _PLoS One_ 4, e5756 (2009). Article  Google Scholar  * Jeon, J.S., Zervantonakis, I.K., Chung, S., Kamm, R.D. & Charest, J.L. _In vitro_ model of tumor cell extravasation. _PLoS

One_ 8, e56910 (2013). Article  CAS  Google Scholar  * Zervantonakis, I.K. et al. Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function. _Proc.

Natl. Acad. Sci. USA_ 109, 13515–13520 (2012). Article  CAS  Google Scholar  * Chaw, K.C., Manimaran, M., Tay, E.H. & Swaminathan, S. Multi-step microfluidic device for studying cancer

metastasis. _Lab Chip_ 7, 1041–1047 (2007). Article  CAS  Google Scholar  * Zhang, Q., Liu, T. & Qin, J. A microfluidic-based device for study of transendothelial invasion of tumor

aggregates in realtime. _Lab Chip_ 12, 2837–2842 (2012). Article  CAS  Google Scholar  * Roberts, S.A., Waziri, A.E. & Agrawal, N. Development of a single-cell migration and

extravasation platform through selective surface modification. _Anal. Chem._ 88, 2770–2776 (2016). Article  CAS  Google Scholar  * Riahi, R. et al. A microfluidic model for organ-specific

extravasation of circulating tumor cells. _Biomicrofluidics_ 8, 024103 (2014). Article  CAS  Google Scholar  * Kim, Y. et al. Quantification of cancer cell extravasation _in vivo_. _Nat.

Protoc._ 11, 937–948 (2016). Article  CAS  Google Scholar  * Chen, M.B., Lamar, J.M., Li, R., Hynes, R.O. & Kamm, R.D. Elucidation of the roles of tumor integrin β1 in the extravasation

stage of the metastasis cascade. _Cancer Res._ 76, 2513–2524 (2016). Article  CAS  Google Scholar  * Stoletov, K. et al. Visualizing extravasation dynamics of metastatic tumor cells. _J.

Cell Sci._ 123, 2332–2341 (2010). Article  CAS  Google Scholar  * Jeon, J.S. et al. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. _Proc.

Natl. Acad. Sci. USA_ 112, 214–219 (2015). Article  CAS  Google Scholar  * Albelda, S.M. et al. Permeability characteristics of cultured endothelial cell monolayers. _J. Appl. Physiol._ 64,

308–322 (1988). Article  CAS  Google Scholar  * Quail, D.F. & Joyce, J.A. Microenvironmental regulation of tumor progression and metastasis. _Nat. Med._ 19, 1423–1437 (2013). Article 

CAS  Google Scholar  * Kitamura, T., Qian, B.-Z. & Pollard, J.W. Immune cell promotion of metastasis. _Nat. Rev. Immunol._ 15, 73–86 (2015). Article  CAS  Google Scholar  * Levario,

T.J., Zhan, M., Lim, B., Shvartsman, S.Y. & Lu, H. Microfluidic trap array for massively parallel imaging of _Drosophila_ embryos. _Nat. Protoc._ 8, 721–736 (2013). Article  Google

Scholar  * Spiegel, A. et al. Neutrophils suppress intraluminal NK-mediated tumor cell clearance and enhance extravasation of disseminated carcinoma cells. _Cancer Discov._ 6, 630–649

(2016). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank S. Chung for scientific discussions and A. Boussommier-Calleja for critical reading of the manuscript. We

thank B. Bista and R. Hynes of the Department of Biology, Massachusetts Institute of Technology and J. Massague of the Sloan-Kettering Institute for sharing cell lines. M.B.C. and R.D.K.

acknowledge support from the National Cancer Institute (CA202177). R.D.K. and J.A.W. thank the National Science Foundation for support (CBET-0939511). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA Michelle B Chen, Jordan A Whisler, Yoojin Shin & Roger D Kamm *

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA Julia Fröse * German Cancer Research Center (DKFZ), Heidelberg, Germany Julia Fröse * University of Heidelberg,

Heidelberg, Germany Julia Fröse * Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA Cathy Yu & Roger D Kamm Authors * Michelle B

Chen View author publications You can also search for this author inPubMed Google Scholar * Jordan A Whisler View author publications You can also search for this author inPubMed Google

Scholar * Julia Fröse View author publications You can also search for this author inPubMed Google Scholar * Cathy Yu View author publications You can also search for this author inPubMed 

Google Scholar * Yoojin Shin View author publications You can also search for this author inPubMed Google Scholar * Roger D Kamm View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS M.B.C., J.A.W. and R.D.K. conceived the project and designed the experiments; M.B.C. and C.Y. performed the experiments; J.F. designed fluorescent cell

lines; M.B.C. analyzed the data; R.D.K. supervised the project; Y.S. designed the figure schematics, M.B.C. and R.D.K. wrote the paper. CORRESPONDING AUTHOR Correspondence to Roger D Kamm.

ETHICS DECLARATIONS COMPETING INTERESTS R.K. is a cofounder of and has a substantial financial interest in AIM Biotech, a company that has commercialized microfluid assays of design similar

to the one described in the present protocol. All the reported studies, however, were performed with devices designed and fabricated at the Kamm laboratory at MIT. INTEGRATED SUPPLEMENTARY

INFORMATION SUPPLEMENTARY FIGURE 1 SHEAR STRESS DETERMINATION IN MICROVASCULAR NETWORKS. A pressure drop of ~4 mmH2O is applied across the vascular network using a suspension of 2 micron red

polystyrene spheres in EGM. Velocity of beads (those near the centerline only) are calculated using the “streak-length method”1, and diameters of vessels are estimated via corresponding

phase contrast images using Image J. Viscosity of media is assumed to be ~0.0008 Pa s. (A) Example fluorescent images of flowing beads taken at 50 fps with 20 ms exposure (note images shown

are post-processed to be oversaturated to clearly show streaks. Actual quantification should be done with raw (properly exposed) images to ensure streak length calculations are correct). (B)

Distribution of shear stresses found in 1 device over 30 vessel segments. Mean velocity is taken as half of the centerline velocity. Pipe flow is assumed as an approximation. (C) Table of

individual values of diameter, centerline bead velocity and corresponding shear stress estimated for individual vessel segments in a single device. (D) Table of average shear values of 20

vessels per device, for a total of 10 separate devices. (E) Table of the average shear over 10 devices per experiment (20 vessels per device), for a total of 5 experiments. 1. Al-Khazraji,

B. K., Novielli, N. M., Goldman, D., Medeiros, P. J. & Jackson, D. N. A Simple 'Streak Length Method' for Quantifying and Characterizing Red Blood Cell Velocity Profiles and

Blood Flow in Rat Skeletal Muscle Arterioles. _Microcirculation_ 19, 327–335 (2012). SUPPLEMENTARY FIGURE 2 LUMENS ARE SURROUNDED ON ALL SIDES BY HYDROGEL. Confocal reconstruction of various

lumens formed in micro devices (white=reflectance; red=HUVEC; green=MDA-MB-213 LifeAct GFP). While most lumens lie in roughly in one plane, the surface of lumens are at least >30 microns

away from the bottom glass and top PDMS layers. SUPPLEMENTARY FIGURE 3 DETERMINING PERFUSABILITY OF MICROVASCULAR NETWORKS. A perfusable device satisfies 2 criteria: (1) 50% of interpost

regions on one side allow for tumor cell entry and (2) more than 25% of tumor cells in the network are distributed beyond the centerline of the gel region. (A) Histogram of the number of

devices (49 devices over 3 experiments) with different numbers of perfusable interpost regions. Perfusable interpost regions are counted for each device via bright field microscopy during

tumor cell perfusion. Out of the 49 devices, 43 showed more than 10 (50%) perfusable interpost regions. (B) 40 out of 43 of these devices showed a distribution of tumor cells across the

vascular network of more than 25% past the centerline of the gel. In these set of experiments, the perfusability is thus ~82% of total devices. (C) Phase contrast images (20X) of typical

perfusable openings. (D) 10X phase contrast images of a good device with many openings (device 1) and a poor device with few openings (device 2). SUPPLEMENTARY INFORMATION SUPPLEMENTARY

FIGURES AND TABLE Supplementary Figures 1–3, Supplementary Methods and Supplementary Table 1. (PDF 727 kb) SUPPLEMENTARY DATA Photo-mask with microdevice design. (ZIP 106 kb) TIME-LAPSE

VIDEO OF EXTRAVASATING TUMOR CELL. Time-lapse video of a transmigrating MDA-MB-231 cell (LifeAct GFP, green) from a microvessel (HUVEC, red). Frames are 40 min apart. (MOV 237 kb) FLOW OF

TUMOR CELLS WITHIN MICROVASCULAR NETWORKS. Real-time video via phase-contrast at 10× magnification, depicting the flow of MDA-MB-231 cells and human platelets within microvascular networks

upon introduction of a 4-mm H2O hydrostatic pressure drop. Tumor cells are seen to decelerate, arrest and at times dislodge from the capillaries under flow conditions. (MOV 3909 kb) RIGHTS

AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Chen, M., Whisler, J., Fröse, J. _et al._ On-chip human microvasculature assay for visualization and

quantification of tumor cell extravasation dynamics. _Nat Protoc_ 12, 865–880 (2017). https://doi.org/10.1038/nprot.2017.018 Download citation * Received: 17 August 2016 * Accepted: 20

October 2016 * Published: 30 March 2017 * Issue Date: May 2017 * DOI: https://doi.org/10.1038/nprot.2017.018 SHARE THIS ARTICLE Anyone you share the following link with will be able to read

this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative