Live-cell imaging of invasion and intravasation in an artificial microvessel platform.

Fabricating a perfusable cylindrical ECM scaffold The ECM/vessel platform is comprised of a cylindrical collagen channel located within a polydimethylsiloxane (PDMS) housing that is perfused by a gravity flow system ( Fig. 1A ). An aluminum mold with 3 rectangular channels of dimension 1.2 mm × 1.5 mm × 5 cm (W × H × L) was used to form the housing by casting PDMS ( Fig. 1B ). After removal from the mold, holes are punched for connections to tubing and reservoirs, and the PDMS housing is plasma bonded to a glass slide. A custom nozzle, 1.6 mm in diameter and 1 cm in length, is inserted into each rectangular compartment to guide the insertion of the template rod and to direct flow into the channel during perfusion ( Fig. 1C ). The interior of the PDMS housing is silanized with (3-glycidyloxypropyl)trimethoxysilane (Sigma-Aldrich, St. Louis, MO) to improve adhesion of the subsequently introduced collagen gel. Prior to collagen introduction, devices and flow setups were sterilized by autoclaving. High concentration rat tail type I collagen (BD Biosciences, San Jose, CA) is used to form the ECM with the manufacture's recommended neutralizing protocol using 1N NaOH, 10x PBS, and distilled water. Collagen is the main structural protein within the body; types I, III, and IV are constitutively present in normal mammary glands and increasingly within the stroma of neoplastic mammary tissue and invasive carcinoma ( 9 ). Type I collagen was used to form a hydrogel scaffold that best represents the structural, biochemical, and transport properties of in vivo tumor tissue and permits both cellular adhesion and remodeling to facilitate endothelial vessel formation and tumor cell migration. Here we use a collagen density of 7 mg ml −1 resulting in a matrix stiffness of about 200 Pa ( 10 ). The ECM can be formed at lower gel concentrations although maintaining higher shear stresses becomes more difficult. A cancer cell suspension was introduced immediately after neutralizing the collagen solution to obtain a final concentration of 5 × 10 5 cells ml −1 . Neutralized collagen solutions were injected into the rectangular channels. Nitinol rods (McMaster-Carr, Princeton, NJ) of 150 μm in diameter were threaded through the nozzles and into the collagen solution to be used as cylindrical templates for collagen casting. During collagen neutralization and injection, all solutions and devices were kept on ice. Optically transparent collagen gels were formed by incubating the device at 37 °C during initial gelation for 15 min and allowed to complete gelation at room temperature for 1 h. Template rods were slowly removed, leaving behind a cylindrical channel ( Fig. 1C ). Endothelial cells were introduced into the channels at a concentration of 10 6 ml −1 . Channels typically yielded 50,000 cells cm −2 of coverage after seeding and would be confluent within 24 h. After forming a microvessel, the device is placed under steady laminar flow using an automatically recirculating gravity flow system. The device is kept under hydrostatic pressure of 10 cm of water with a height difference of about 5 cm between the upper and lower reservoirs ( Fig. 1D ). Vessels were maintained at a shear stress of 12 - 15 dyne cm −2 . During multiple day live-cell imaging experiments, both the device and the reservoirs are contained within separate chambers in the microscope incubator and maintained at 37 °C under constant humidity and 5% CO 2 ( Fig. 1E ). Most devices were overrun by proliferating tumor cells after 1 - 2 weeks of culture.

Cell lines and culture conditions Dual-labeled

MDA-MB-231 adenocarcinoma and HT-1080 fibrosarcoma cells (AntiCancer Inc., San Diego, CA) were cultured in RPMI (Corning Inc) supplemented with 10% fetal bovine serum (FBS) (Life Technologies), and 1% penicillin/streptomycin (Life Technologies) at physiological conditions (humidified 37 °C with 5% CO 2 ). Human umbilical vein endothelial cells (HUVEC) (PromoCell, Heidelberg, Germany) and adult human dermal microvascular endothelial cells (HMVEC) (Life Technologies) were cultured using EGM-2 (PromoCell) growth media with all supplementary ingredients: 2% fetal calf serum, hydrocortisone, heparin, ascorbic acid, hEGF, hVEGF, hbFGF, and R3 IGF-1. All cell lines were authenticated by their respective manufacturers and tested negative for mycoplasm.

Live-cell fluorescence imaging

Time-lapse, phase-contrast, and fluorescence images were captured using automated image acquisition software from Nikon with a TE-2000 U microscope ( Fig. 1E ) (Nikon Instruments Inc., Melville, NY). A 10× objective was used for all epifluorescence and phase-contrast imaging. Confocal z-stacks were obtained on a LSM 710 with a 20× water immersion objective (N.A. = 1.0) (Carl Zeiss, Oberkochen, Germany) from which 3D and cross-sectional projections of the stacks were obtained using LSM Image Browser (Carl Zeiss). Quantifying vessel permeability Permeability was measured through quantitative fluorescence detection of molecular transport from inside the vessel to the surrounding ECM ( 8 , 11 , 12 ). Bovine serum albumin (BSA) conjugated to Alexa Fluor 488 (Life Technologies, Carlsbad , CA) at 5 μg ml −1 was introduced into the vessels and imaged every 2 min. Image intensity profiles were obtained using ImageJ (NIH, Bethesa, MD) and permeability coefficients were calculated by extracting the initial step increase in fluorescence on introducing BSA into the vessel lumen and the slope corresponding to the rate of permeation from the vessel to the ECM ( Fig. 2F ). Image analysis of tumor/endothelial interactions Time-lapse videos of merged fluorescence and phase-contrast images were imported into ImageJ as stacks of TIFF files. The invasion front of a cluster of breast cancer cells (BCCs) was measured by manually tracing its perimeter using phase-contrast images, from which shape factor and tortuosity were extracted. The motility of single dual-labeled BCCs was quantified by selecting the approximate center of each GFP expressing nucleus and measuring its change in distance at 12 min intervals. For tracking individual endothelial cells, each cell was manually traced in phase-contrast images ( Supplementary Video S8 ), and its respective centroid position was calculated. To obtain the relative distance between a sprouting endothelial cell's protrusions and an adjacent tumor, the difference between the y-value of the tip's position and a reference point behind the tumor was taken and plotted vs. time ( Fig. 4A ); the tip of each endothelial protrusion was manually selected from phase-contrast images. The same operation was used to obtain the difference between the centroid positions of neighboring endothelial cells with an adjacent tumor. Particle image velocimetry (PIV) analysis PIV analysis of endothelial monolayers within the collagen channels was conducted in MATLAB using OpenPIV ( 13 ). Time-lapse phase-contrast images obtained at 10 min intervals were imported into OpenPIV as TIFF files with a resolution of 0.64 μm px −1 . Monolayers of endothelial cells in focus on the bottom or top of the collagen channels were selected as regions of interest. A 16x16 px area was used for both the interrogation window and spacing/overlap. Batch means of the velocity magnitudes of each vector in the 2D field were obtain from the unfiltered OpenPIV output.

Statistical Analysis

Values are represented as mean ± s.e.m. The principle statistical test used was a t-test (two-tailed with unequal variance). We considered a P-value less than 0.05 to be statistically significant.

Supplementary Material 1