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On-chip human microvasculature assay for visualization and quantification of tumor cell extravasation dynamics

Nature Protocols volume 12, pages 865–880 (2017)Cite this article

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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 discrete steps of early metastatic seeding, including arrest, transendothelial migration and early micrometastases formation. The microdevice features self-organized human microvascular networks formed over 4–5 d, after which the tumor can be perfused and extravasation events are easily tracked over 72 h via standard confocal microscopy. Contrary to most in vivo and in 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.

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Figure 1: Schematics of device fabrication and cell–fibrin gel seeding protocol.
Figure 2: Microvessel bed formation and tumor cell perfusion procedure.
Figure 3: Scoring and quantification of extravasation efficiency.
Figure 4: Tracking and quantification of extravasation kinetics.
Figure 5: Resolving temporal–spatial organization of endothelial and tumor cell proteins at a single-cell level.
Figure 6: Beyond the endothelium: the basement membrane.
Figure 7: Addition of noncancer host cells.
Figure 8: Tracking tumor cell proliferation post extravasation.

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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).

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Authors and Affiliations

  1. Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Michelle B Chen, Jordan A Whisler, Yoojin Shin & Roger D Kamm

  2. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Julia Fröse

  3. German Cancer Research Center (DKFZ), Heidelberg, Germany

    Julia Fröse

  4. University of Heidelberg, Heidelberg, Germany

    Julia Fröse

  5. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Cathy Yu & Roger D Kamm

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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.

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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)

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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

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