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.