Materials and Methods

Cell Culture

Human breast carcinoma cell line MDA-MB-231(ATCC® HTB-26™) breast carcinoma, human breast inflammatory cancer cells MDA-IBC3 and SUM149, and telomerase-immortalized human microvascular endothelial (TIME) cells were used in this study. MDA-MB-231 and SUM149 are triple negative cell lines while MDA-IBC3 cells are negative for hormone receptors but overexpress human epidermal growth factor receptor 2 (HER2). GFP labeled MDA-MB-231 and mKate labeled TIME cells were a generous gift from Dr. Shay Soker at the Wake Forest Institute for Regenerative Medicine (Winston-Salem, NC). MDA-IBC3 and SUM149 IBC cells labeled with GFP were kindly provided by Dr. Wendy Woodward at MD Anderson Cancer Center (Houston, TX). MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle’s medium, nutrient mixture F-12 (DMEM/F12) (Sigma Aldrich) supplemented with 1% penicillin-streptomycin (Invitrogen), and 10 % fetal bovine serum (FBS). MDA-IBC3 and SUM149 cells were cultured in Ham’s F-12 media supplemented with 10% FBS, 1% antibiotic-antimycotic, 1 µg/ml hydrocortisone, and 5 µg/ml insulin. TIME cells were cultured in Endothelial Cell Growth Medium-2 BulletKitTM (EGM-2, Lonza). All cell cultures utilized in this study were maintained at 5% CO2 atmosphere and 37°C.

In Vitro 3D Tumor Platform Fabrication

The in vitro 3D tumor microfluidic platforms utilized in this study were composed of collagen type I matrix seeded with either GFP labeled MDA-MB-231, MDA-IBC3, or SUM 149 integrated with a hollow channel seeded with mKate labeled TIME cells housed in a polydimethylsiloxane (PDMS) scaffold. Collagen type I extracted from rat tails was prepared following our published protocols to produce stock collagen concentration of 14 mg/ml. Stock collagen was then neutralized with a solution consisting of 10x DMEM, 1N NaOH, and 1x DMEM to yield a final collagen concentration of 7 mg/ml giving comparable stiffness ofin vivo breast tumors (Buchanan et al., 2014b; Michna et al., 2018; Paszek et al., 2005; Szot et al., 2011, 2013). GFP labeled IBC and non-IBC cells were seeded at a density of 1x106cells/mL in the 7 mg/ml neutralized collagen solution and polymerized around a 22G needle at 37°C for 25 minutes. After polymerization, the needle was removed, and the resulting hollow void was filled with a solution of 2x105 TIME cells to form an endothelialized vessel lumen. Flow was introduced using a syringe pump system and a graded flow protocol was used to establish a confluent endothelium as we have previously published (Buchanan et al., 2014a; Buchanan et al., 2014b; Gadde et al., 2018; Michna et al., 2018). Briefly, EGM-2 media was perfused to expose the endothelium to wall shear stress (WSS) (τ) of 0.01 dyn/cm2 for 36 hours followed by a gradual increase in WSS to 0.1 dyn/cm2for the following 36 hours and a final increase to 1 dyn/cm2 for 6 hours. Additionally, EGM-2 media flow through the endothelial vessel provides nutrients and gas exchange allowing for long term culture of the TIME cells and the tumor cells in the collagen. Four conditions of the 3D in vitro vascularized tumor platforms were created: TIME cell only platform which served as control, and platforms consisting of co-culture of TIME cells with either MDA-MB-231, MDA-IBC3, or SUM149 cells. Endothelial morphology and adhesion, vessel permeability and coverage, matrix porosity, and expression of angiogenic cytokines were characterized in each platform with four replicates per platform type and were measured following completion of the graded flow protocol. Significance of the data was verified using one-way ANOVA and a 95% confidence criterion.

Characterization of Vascularized In Vitro IBC and non-IBC Platforms

Endothelial Morphology

Endothelial morphology and cell-cell junctions were analyzed by performing immunofluorescent staining for PECAM-1 and F-actin upon completion of the graded flow protocol. PECAM-1 (green) is expressed at endothelial intercellular junctions and functions in the maintenance of endothelial barrier functions (Privratsky et al., 2014). The staining protocol consisted of perfusing the platforms with 4% paraformaldehyde and 0.5% triton-X 100 for fixation and permeabilization of the cell membranes, respectively. Next, the platforms were incubated in 5% BSA followed by overnight incubation with antibodies for PECAM-1 (Abcam, ab215911) and Rhodamine Phalloidin (Thermo Scientific, R415).

Matrix Porosity and Endothelial Adhesion

Scanning electron microscopy (SEM) was performed to determine collagen matrix porosity and observe endothelial adhesion to the collagen matrix. After exposure to the graded flow protocol, the platforms were fixed in an aldehyde mixture overnight at room temperature followed by fixation with osmium on ice for 4 hours. Post fixation, the platforms were dehydrated in an ascending series of ethanol solutions (50-70-95-100%) and then critical point dried by CO2. Platforms were coated with a thin layer of platinum-palladium and SEM imaging was performed with Zeiss Supra40 SEM-Electron Microscope.

Endothelium Coverage

Vessel volume occupied by TIME cells was quantified using 3D F-actin stained images of the endothelium with LASX image processing software. Briefly, the software the computes the area of the platform expressing fluorescence signal from F-actin staining and the total area of each platform. Reported values for the co-culture platforms were normalized to the control.

Endothelial Permeability

Endothelial vessel permeability as a function of paracrine signaling between tumor and vasculature was determined by perfusing the channels with 70 kDa Oregon green dextran according to our published protocols (Buchanan et al., 2014a; Grainger et al., 2011). After completion of the graded flow protocol for establishing a confluent endothelium, green fluorescent dextran suspended in serum free endothelial growth media (10 μg/ml) was perfused through the platforms with images taken every five minutes. The average fluorescent intensity was measured from the images and used to determine the diffusion permeability coefficient as previously published (Buchanan et al., 2014a). Three samples (n=3) were used for each platform condition with the resulting permeability factor expressed as a mean value ± standard deviation.

Enzyme-linked Immunosorbent Assay

Expression of VEGF, a growth factor known to promote angiogenesis that is excreted from endothelial and tumor cells, was measured using enzyme-linked immunosorbent assays (ELISA) upon completion of the graded flow protocol. 1 ml samples of perfusion media were collected from the flow outlet and ELISA was performed as per manufacturer’s protocol (R&D Systems, DVE00).

Characterization of Angiogenic Sprouting in the In Vitro Vascularized MDA-IBC3 Platforms

Endothelial Sprouting

MDA-IBC3/TIME in vitro vascularized platforms were cultured for an additional three weeks following the graded flow protocol in order to characterize endothelial sprouting spatially and temporally. 3D images of the MDA-IBC3/TIME in vitro platforms were acquired using Leica TCS SP8 confocal microscope to observe sprout formation and growth. Cross sectional images from the center plane of each channel were used to analyze sprout growth and quantified using ImageJ. Fluorescent intensity histograms for each image were generated using ImageJ’s plot profile function. Differences between fluorescence intensity histograms at each time point were quantified using the two-sample Kolmogorov–Smirnov (K-S) statistic, a distance measure between each sample pair’s empirical distribution functions. The K-S statistic was calculated between the baseline fluorescence intensity distribution at Day 0 and subsequent imaging time points and significance was determined using p <0.001.

Quantification of Sprout Properties and Vascular Network

Confocal microscopy images acquired on Day 0, 4, 8, 12 and 16 from 3 different platform replicates were analyzed for length of the total vascular network and number of sprouts. Briefly, 11 slices from z-stack of the vessel (~45 µm height) at the center or the widest part of the vessel were analyzed using a Matlab algorithm adapted from work done by Kollmannsberger et al. and Crosby et al. (Crosby et al., 2019; Kerschnitzki et al., 2013; Kollmannsberger et al., 2017) to quantify total vascular network and number of sprouts. Analysis of cross sectional areas of the vessel sprouts was performed using methodology developed in house and detailed in supplementary section S1 on Days 0, 4, 8, 12.

Lumen formation

To confirm the formation of lumen in the newly formed sprouts, platforms were injected with a 20 µl solution of 1 µm Fluoro-Max dyed green aqueous fluorescent microspheres (Thermo Scientific, G0100) on Day 14 and Day 21. The vessels were then imaged using the Leica TCS SP8 confocal microscope and sprouts with beads present were deemed to have formed lumen.

Cytokine Analyses

Cytokine analyses for CD31, ANG1, ANG2, TGF-α, bFGF, PDGF-bb, EGF, VEGF-A, VEGFR3, VEGF-C, TNF-α, IL-8, IL-6, IL-6 Rα, MMP9, MMP2, MMP13 were performed using a custom human magnetic luminex assay (R&D Systems). Analyses were performed on platform perfusion effluent on Days 0, 7, 14 and 21 according to manufacturer’s instructions. Significance of the data was verified using one-way ANOVA and a 95% confidence criterion.