Nature Protocols: doi: /nprot Supplementary Figure 1. Shear stress determination in microvascular networks.

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1 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 ~ 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, (2012).

2 Supplementary Figure 2 Lumens are surrounded on all sides by hydrogel. 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.

3 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).

4 Supplementary Methods Creation of fluorescently labeled stable HUVECs LifeAct-labeled HUVEC lines were made by lentiviral transduction using the plv-lifeact-mruby2 and plv-lifeact-egfp plasmids (LifeAct plasmids were generous gifts from Tsukasa Shibue 1 ). Similarly, GFP- (plenti CMV GFP Puro (658-5) was a gift from Eric Campeau (Addgene plasmid # 17448) 2 ) and Azurite- (plv-azurite was a gift from Pantelis Tsoulfas; unpublished (Addgene plasmid #36086)) labeled HUVEC lines were produced. For lentiviral production, HEK 293T cells were transduced with the plasmid of interest at % confluency. In brief, 1.5 µg of the plasmid of interest, 1 µg of pcmv-vsv-g (pcmv-vsv-g was a gift from Bob Weinberg (Addgene plasmid # 8454) 3 ) and 1 µg of pcmv-dr8.2 (pcmv-dr8.2 dvpr was a gift from Bob Weinberg (Addgene plasmid # 8455) 3 ) were mixed with X-tremeGENE 9 DNA transfection reagent (Roche) in Opti-MEM (ThermoFisher Scientific) and incubated for 20 min. Afterwards the transfection mix was added to the HEK 293T cells and incubated for 24 h. Subsequently, the media was changed and virus was harvested 48 h and 72 h post-transfection. For that purpose, the virus-containing supernatant of the cells was filtered through a 0.45 µm filter and stored at -80 C until use. On the day prior to viral transduction, HUVECs were seeded at low confluency in 75cm 2 cell culture flasks. HUVECs were transduced overnight using 500 µl of the before produced virus and Polybrene transfection reagent (1:1500 dilution; Millipore). The transduced cells were washed with PBS, supplied with fresh media and plasmid-containing cells were sorted by FACS. 1. Shibue, T., Brooks, M.W., Inan, M.F., Reinhardt, F. & Weinberg, R.A. The outgrowth of micrometastases is enabled by the formation of filopodium-like protrusions. Cancer Discov 2, (2012). 2. Campeau, E. et al. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS One 4, e6529 (2009). 3. Stewart, S.A. et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA (New York, N.Y.) 9, (2003).

5 Supplementary Information SI Table 1: Optimal settings on microscopes to visualize micro devices Purpose Visualizing flow of tumor cells through networks Scoring of extravascular or intravascular cells Time-lapse for tumor cell protrusion kinetics Microscope type Bright field ) Objective N.A. Time step Pinhole Minimum z- Resolution size step size 10X 0.25 N/A N/A N/A N/A 20X 0.75 N/A 80 2 micron 800 X 800 At least 20X >3 min intervals in between images to prevent photo toxicity and bleaching of dyes 80 2 micron 800 X 800 Visualizing cellular protein localization, matrix protein deposition (at fixed time point) At least 30X <20 min between time points to capture protrusion kinetics 1.05 N/A 80 < 1 micron (dependent on fluorescent protein of interest). Use microscope s optimal step size when possible. 800 X 800