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\begin{document}

\title{In Vitro Vascularized Tumor Platform for Modeling Tumor-Vasculature
Interactions of Inflammatory Breast Cancer}



\author[1]{Manasa Gadde}%
\author[1]{Caleb Phillips}%
\author[1]{Neda Ghousifam}%
\author[2]{Anna Sorace}%
\author[1]{Enoch Wong}%
\author[3]{Savitri Krishnamurthy}%
\author[1]{Anum Syed}%
\author[3]{Omar Rahal}%
\author[1]{Thomas Yankeelov}%
\author[3]{Wendy Woodward}%
\author[1]{Marissa Rylander}%
\affil[1]{University of Texas at Austin}%
\affil[2]{The University of Alabama at Birmingham School of Medicine}%
\affil[3]{University of Texas MD Anderson Cancer Center}%


\vspace{-1em}



  
  \date{March 14, 2025}


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\selectlanguage{english}
\begin{abstract}
Inflammatory breast cancer (IBC), a rare form of breast cancer
associated with increased angiogenesis and metastasis, is largely driven
by tumor-stromal interactions with the vasculature and the extracellular
matrix (ECM). However, there is currently a lack of understanding of the
role these interactions play in initiation and progression of the
disease. In this study, we developed the first three-dimensional, in
vitro, vascularized, IBC platform to quantify the spatial and temporal
dynamics of tumor-vasculature and tumor-ECM interactions specific to
IBC. Platforms consisting of collagen type 1 ECM with an endothelialized
blood vessel were cultured with IBC cells, MDA-IBC3 (HER2+) or SUM149
(triple negative), and for comparison to non-IBC cells, MDA-MB-231
(triple negative). An acellular collagen platform with an endothelial
blood vessel served as control. SUM149 and MDA-MB-231 platforms
exhibited a significantly (p\textless{}0.05) higher vessel permeability
and decreased endothelial coverage of the vessel lumen compared to the
control. Both IBC platforms, MDA-IBC3 and SUM149, expressed higher
levels of VEGF (p\textless{}0.05) and increased collagen ECM porosity
compared to non-IBC MDA-MB-231 (p\textless{}0.05) and control
(p\textless{}0.01) platforms. Additionally, unique to the MDA-IBC3
platform, we observed progressive sprouting of the endothelium over time
resulting in viable vessels with lumen. The newly sprouted vessels
encircled clusters of MDA-IBC3 cells replicating a feature of in vivo
IBC. The IBC in vitro vascularized platforms introduced in this study
model well-described in vivo and clinical IBC phenotypes and provide an
adaptable, high throughout tool for systematically and quantitatively
investigating tumor-stromal mechanisms and dynamics of tumor
progression.%
\end{abstract}%



\sloppy




\section*{Background}

{\label{background}}

Breast cancer accounts for 15\% of newly diagnosed cancer cases in
females (''CDC - Breast Cancer Statistics,'' 2017; ''Female Breast
Cancer - Cancer Stat Facts,''). Inflammatory breast cancer (IBC) a
highly metastatic and aggressive subtype of locally advanced breast
cancer and accounts for 10\% of all breast cancer related mortality
(Costa et al., 2017; Fouad et al., 2017; Fouad et al., 2014; Hance et
al., 2005; Lim et al., 2018). Compared to other metastatic breast
cancers, IBC is associated with a median survival of 4 years compared to
10 years in non-inflammatory breast cancer (non-IBC) cases (Hance et
al., 2005). Approximately 50\% of IBC cases lack a tumor mass and
present no radiographic evidence. Due to this, the diagnosis of IBC
occurs upon clinical manifestation of the disease including pain,
redness, and swelling of the breast. At this point, the tumor has
advanced to stage III or IV, most patients have lymph node metastases,
and 30\% of IBC patients exhibit distant metastases compared to 5\% for
non-IBC (Fernandez et al., 2013; Fouad et al., 2014; Giordano et al.,
2003). Additionally, contributing to its bleak prognosis, no molecular
or histological markers specific to IBC have been identified to
distinguish it from other non-IBC breast cancers.

IBC has been shown to be highly angiogenic and metastatic, but a deeper
understanding of the diseases dynamics has remained elusive and would
enable identification of new diagnostic and therapeutic markers. Current
pre-clinical experimental models used to study IBC consist primarily of
xenograft animal models, two dimensional (2D) monolayers, and three
dimensional (3D) \emph{in vitro} models (Charafe-Jauffret et al., 2010;
Klopp et al., 2010; Lehman et al., 2013; Silvera et al., 2009a; Silvera
et al., 2009b; van Golen et al., 2002a; van Golen et al., 2002b; van
Golen et al., 2000b; van Uden et al., 2015). 2D models do not
recapitulate the complex and dynamic nature of the tumor
microenvironment which hosts multi-cellular and cell-matrix interactions
and evolving biomechanical and chemical features (Jang et al., 2003; Kim
et al., 2012; Tr\selectlanguage{ngerman}édan et al., 2007). Compared to monolayers, patient
derived xenograft (PDX) models are more favorable among researchers as
preclinical models for IBC as they provide physiologically relevant
tumor microenvironment conditions (Alpaugh et al., 2002; Alpaugh et al.,
1999; Lim et al., 2018; Robertson et al., 2012; Shirakawa et al., 2003;
Wurth et al., 2015). The Woodward lab has recreated skin invasion and
diffuse spread characteristic of IBC in a mouse model with the addition
of mesenchymal stem cells (Lacerda et al., 2015). Other examples of
xenograft systems for modelling IBC consist of Mary-X and WIBC-9 models.
Mary-X established from an IBC patient, recapitulated the human IBC
phenotype of extensive lymphovascular invasion of the tumor cell emboli
(Alpaugh et al., 1999), while the WIBC-9 model recreated an invasive
ductal carcinoma with a hypervascular structure of solid nests and
lymphatic permeation (Shirakawa et al., 2003). While PDX models provide
a more comprehensive model, determining the influence of specific
signaling pathways and microenvironmental stimuli on IBC progression is
challenging and frequently cost prohibitive due to the large animal
numbers needed. Additionally, dynamic tracking and quantification of
tumor presentation and development at a high spatial and temporal
resolution is limited in xenograft models. While less common than PDX
models, three dimensional (3D) \emph{in vitro} models provide a
compromise between 2D and xenograft models as they recapitulate key
spatial and physiological facets of the complex tumor microenvironment
while maintaining temporal sampling comparable to 2D models. Common
3D\emph{in vitro} IBC models are avascular and consist of culturing IBC
monolayers or tumor spheroids on an ECM layer consisting of Matrigel,
Culturex, or collagen. (Allen et al., 2016; Arora et al., 2017;
Hoffmeyer et al., 2005; Lacerda et al., 2015; Lacerda et al., 2014;
Lehman et al., 2013; Mohamed et al., 2008; Mohamed et al., 2014; Morales
et al., 2009; Nokes et al., 2013). These experiments are typically
evaluated under static conditions, thereby lacking physiological flow
which has been shown to influence tumor response to treatment. (Lacerda
et al., 2015; Lacerda et al., 2014; Lehman et al., 2013; Mohamed et al.,
2008). Our lab previously established a 3D vascularized microfluidic
breast cancer platform incorporated with MDA-MB-231 cells and a
vascularized endothelial vessel that addressed the limitation described
earlier with existing \emph{in vitro} tumor models. Using this
vascularized platform, we determined the relationship between wall shear
stress and signaling between cancer and endothelial cells on the
vasculature (Buchanan et al., 2014a; Gadde et al., 2018; Michna et al.,
2018) but similar to other non-IBC \emph{in vitro} tumor models (Bersini
et al., 2014; Buchanan et al., 2012; Buchanan et al., 2014a; Buchanan et
al., 2014b; Duinen et al., 2017; Gadde et al., 2018; Huang et al., 2019;
Jeon et al., 2015; Kim et al., 2015; Kim et al., 2016; Ko et al., 2019;
Koh et al., 2008; Ma et al., 2018; Malandrino et al., 2018; Meer et al.,
2013; Michna et al., 2018; Nguyen et al., 2013; Osaki et al., 2018;
Ozcelikkale et al., 2017; Pagano et al., 2014; Pouliot et al., 2013;
Pradhan et al., 2018; Rhodes et al., 2007; Shang et al., 2019; Sleeboom
et al., 2018; Sontheimer-Phelps et al., 2019; Szot et al., 2011, 2013;
Tsai et al., 2017; Vickerman et al., 2008) the platform does not account
for the complex tumor dynamics inherent to IBC.

In this study, we described the development and characterization of a
versatile, first of its kind, 3D \emph{in vitro} vascularized IBC
platform as a tool for gaining a deeper understanding of the uniqueness
of the IBC phenotype. Specifically, we focused on tumor-vasculature
interactions due to the highly angiogenic and metastatic nature of IBC,
as well as the significant role that these interactions have in
impacting disease phenotype (Castells et al., 2012; Mendoza et al.,
2008; Reid et al., 2017; Schaaf et al., 2018; Senthebane et al., 2017;
Ungefroren et al., 2011; Whiteside, 2008). Conditions representative
of\emph{in vivo} tumor vasculature interface including physiological
flow and associated shear stress were utilized for development of a
continuous, aligned, and functional endothelium in the 3D \emph{in
vitro} vascularized IBC platform (Buchanan et al., 2014a). The
vascularized IBC platforms consisted of one of two aggressive IBC cells,
MDA-IBC3 (HER2+) and SUM149 (triple negative), and the non-IBC platform
consisted of MDA-MB-231 cells (triple negative) cultured within the
collagen ECM incorporated with an endothelialized vessel in the center.
We quantified the differential effects of the IBC platforms and compared
the response, specifically, vascular permeability, endothelial coverage
of the vessel lumen, ECM porosity, and cytokine secretion, compared to
non-IBC platforms to demonstrate the utility of the platform in the
investigation of the spatial and functional interactions not readily
quantified in existing \emph{in vivo} IBC models. Additionally, we
characterized the spatiotemporal angiogenesis of the MDA-IBC3 platforms
and recreated behaviors characteristic of \emph{in vivo} IBC phenotypes
including increased angiogenesis, emboli formation, and vascular nesting
of tumor emboli. The platforms introduced in this study provide a tool
to elucidate unique disease dynamics of IBC and determine the
tumor-vasculature interactions driving IBC development and progression.

\section*{Materials and Methods}

{\label{materials-and-methods}}

\subsection*{Cell Culture}

{\label{cell-culture}}

Human breast carcinoma cell line MDA-MB-231(ATCC® HTB-26\selectlanguage{english}) 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 \selectlanguage{greek}µ\selectlanguage{english}g/ml hydrocortisone, and 5 \selectlanguage{greek}µ\selectlanguage{english}g/ml insulin. TIME cells were cultured in
Endothelial Cell Growth Medium-2 BulletKit\textsuperscript{TM} (EGM-2,
Lonza). All cell cultures utilized in this study were maintained at 5\%
CO\textsubscript{2} atmosphere and 37\selectlanguage{ngerman}°C.

\subsection*{\texorpdfstring{\emph{In Vitro} 3D Tumor Platform
Fabrication}{In Vitro 3D Tumor Platform Fabrication}}

{\label{in-vitro-3d-tumor-platform-fabrication}}

The \emph{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
of\emph{in 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
1x10\textsuperscript{6}cells/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 2x10\textsuperscript{5} 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) (\selectlanguage{greek}τ\selectlanguage{english}) of 0.01 dyn/cm\textsuperscript{2} for 36 hours
followed by a gradual increase in WSS to 0.1
dyn/cm\textsuperscript{2}for the following 36 hours and a final increase
to 1 dyn/cm\textsuperscript{2} 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 \emph{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.

\subsection*{\texorpdfstring{Characterization of Vascularized \emph{In
Vitro} IBC and non-IBC
Platforms}{Characterization of Vascularized In Vitro IBC and non-IBC Platforms}}

{\label{characterization-of-vascularized-in-vitro-ibc-and-non-ibc-platforms}}

\subsubsection*{Endothelial Morphology}

{\label{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).

\subsubsection*{Matrix Porosity and Endothelial
Adhesion}

{\label{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 CO\textsubscript{2}. Platforms were
coated with a thin layer of platinum-palladium and SEM imaging was
performed with Zeiss Supra40 SEM-Electron Microscope.

\subsubsection*{Endothelium Coverage}

{\label{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.

\subsubsection*{Endothelial Permeability}

{\label{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
\selectlanguage{greek}μ\selectlanguage{english}g/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 \selectlanguage{ngerman}± standard deviation.

\subsubsection*{Enzyme-linked Immunosorbent
Assay}

{\label{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).

\subsection*{\texorpdfstring{Characterization of Angiogenic Sprouting in
the \emph{In Vitro} Vascularized MDA-IBC3
Platforms}{Characterization of Angiogenic Sprouting in the In Vitro Vascularized MDA-IBC3 Platforms}}

{\label{characterization-of-angiogenic-sprouting-in-the-in-vitro-vascularized-mda-ibc3-platforms}}

\subsubsection*{Endothelial Sprouting}

{\label{endothelial-sprouting}}

MDA-IBC3/TIME \emph{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 \emph{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 \textless{}0.001.

\subsubsection*{Quantification of Sprout Properties and Vascular
Network}

{\label{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 (\textasciitilde{}45 \selectlanguage{greek}µ\selectlanguage{english}m 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.

\subsubsection*{Lumen formation}

{\label{lumen-formation}}

To confirm the formation of lumen in the newly formed sprouts, platforms
were injected with a 20 \selectlanguage{greek}µ\selectlanguage{english}l solution of 1 \selectlanguage{greek}µ\selectlanguage{english}m 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.

\subsubsection*{Cytokine Analyses}

{\label{cytokine-analyses}}

Cytokine analyses for CD31, ANG1, ANG2, TGF-\selectlanguage{greek}α, \selectlanguage{english}bFGF, PDGF-bb, EGF,
VEGF-A, VEGFR3, VEGF-C, TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-8, IL-6, IL-6 R\selectlanguage{greek}α, \selectlanguage{english}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.

\section*{Results}

{\label{results}}

\subsection*{\texorpdfstring{\emph{In Vitro} IBC Platform
Development}{In Vitro IBC Platform Development}}

{\label{in-vitro-ibc-platform-development}}

The graded flow preconditioning protocol with a graded increase in WSS
from 0.01 dyn/cm\textsuperscript{2} to 1
dyn/cm\textsuperscript{2}resulted in a confluent endothelium as shown in
Fig. 1a, which shows the evolution of the vascular endothelium in the
TIME only \emph{in vitro}vascularized platform. The platforms initiated
with a vascular vessel seeded with rounded clusters of TIME cells (0
hour time point) which began to spread out and elongate (24 and 48 hour
time points), followed by proliferation and alignment of the cells in
the direction of flow to ultimately form the confluent endothelium
observed at the 78 hour time point. The resulting endothelium served as
the baseline for evaluation of the influence of different cancer cells,
IBC and non-IBC, on the surrounding vessel with respect to endothelial
morphology, barrier function, and secretion of protumor cytokines (Fig.
1b). In addition to the TIME only \emph{in vitro} vascularized platform,
platforms with co-culture of TIME cells with MDA-IBC3, SUM149, and
non-IBC MDA-MB-231 breast cancer cells were developed (Fig. 1B).
Co-culture of TIME cells with MDA-MB-231 and SUM149 cells resulted in a
sparsely covered endothelium evidenced by the presence of large voids in
red signal from the endothelium representing areas of the vessel lumen
with no endothelial coverage. Both MDA-IBC3/TIME and TIME only \emph{in
vitro}vascularized platforms presented a confluent and intact
endothelium. The difference in the tumor cells in the platform groups is
related to their fluorescent expressions. Emission of the GFP signal
from the MDA-IBC3 is much brighter and stronger compared to the other
two cells lines. Initial cell seeding shown in Supplementary Fig. A
revealed a similar tumor population in the different groups.

\subsection*{\texorpdfstring{Characterization of \emph{In Vitro} Tumor
Platforms}{Characterization of In Vitro Tumor Platforms}}

{\label{characterization-of-in-vitro-tumor-platforms}}

\subsubsection*{Endothelial morphology and cell-cell
junctions}

{\label{endothelial-morphology-and-cell-cell-junctions}}

Endothelial morphology and cell-cell junctions as measured by PECAM-1
and F-actin staining, and SEM are illustrated in Fig. 2a. A compromised
endothelium with holes and gaps was observed in the SUM149/TIME and
MDA-MB-231/TIME. Staining patterns of PECAM-1 (green, top row) and
F-actin (red, middle row) revealed a bright fluorescent signal present
continuously across the endothelium in the TIME and
MDA-IBC3/TIME\emph{in vitro} vascularized platforms. However,
expressions of PECAM-1 and F-actin in SUM149/TIME and MDA-MB-231/TIME
were discontinuous with regions of endothelium lacking any signal
(pointed out by white arrows) indicating formation of intercellular gaps
between neighboring endothelial cells which are typical of a leaky
endothelium. Additionally, F-actin staining of MDA-IBC3/TIME platform
displayed early signs of angiogenic sprouting with TIME cells starting
to bud from the borders of the endothelial vessel (boxed areas) towards
MDA-IBC3 cells replicating another important phenomenon characteristic
of \emph{in vivo} IBC tumors. High resolution SEM images (bottom row)
displayed a tight endothelium with endothelial cell edges overlapping
between neighboring cells in the TIME only and the MDA-IBC3/TIME
platforms, whereas SUM149/TIME and MDA-MB-231/TIME platforms showed
voids between adjacent endothelial cells as denoted by the white arrows.

\subsubsection*{Endothelial Lumen
Coverage:}

{\label{endothelial-lumen-coverage}}

Quantitative comparison of endothelial coverage of the lumen, Fig. 2b,
exhibited a significant decrease in the endothelium coverage in the
SUM149/TIME (p\textless{}0.05) and MDA-MB-231/TIME (p\textless{}0.01)
platforms, compared to the MDA-IBC3/TIME and control platform as
illustrated in Fig. 3. SUM149/TIME had a 1.3 fold and 1.4 fold decrease,
and MDA-MB-231/TIME had a 1.5 and 1.6 fold decrease in endothelial
coverage compared to control TIME and MDA-IBC3/TIME respectively. There
was no significant difference between the TIME and the MDA-IBC3/TIME
platforms.

\subsubsection*{Endothelial Permeability}

{\label{endothelial-permeability-1}}

Measured effective permeability for TIME, MDA-IBC3/TIME, SUM149/TIME,
and MDA-MB-231/TIME platforms were 0.016 \selectlanguage{ngerman}± 0.002, 0.019 ± 0.002, 0.023 ±
0.002, and 0.025 ± 0.002 respectively, as portrayed in Fig. 2c. Vascular
permeability of the MDA-MB-231/TIME \emph{in vitro} vascularized
platforms were statistically significant (p \textless{}0.05) with 1.6
and 1.3 fold higher permeability than TIME and MDA-IBC3/TIME \emph{in
vitro} vascularized platforms respectively. SUM149/TIME \emph{in
vitro}vascularized platform also differed significantly from the TIME
platforms (p\textless{} 0.05) with a 1.4 fold increase in permeability.
The increased permeability in the MDA-MB-231/TIME and SUM149/TIME
platforms confirm the presence of a compromised endothelium and
reaffirms the observations from immunofluorescent stained images (Fig.
2a).

\subsubsection*{Expression of VEGF and
bFGF}

{\label{expression-of-vegf-and-bfgf}}

ELISA measurements for VEGF and bFGF are illustrated in Fig. 2d with the
TIME platform serving as the control. VEGF expression was higher in both
the IBC groups (MDA-IBC3, SUM149) compared to non-IBC (MDA-MB-231) and
TIME platforms while bFGF was higher in the non-IBC group. VEGF
expression was significantly higher (p \textless{} 0.05) in
MDA-IBC3/TIME \emph{in vitro} vascularized platforms compared to the
TIME (1.6 times higher) and MDA-MB-231/TIME (2 times higher) platforms.
SUM149/TIME had a higher VEGF expression, 1.5 times, compared to
MDA-MB-231/TIME (p\textless{}0.05). bFGF was expressed highest in the
MDA-MB-231/TIME platform, 1.2 times, compared to both the IBC platforms
(p\textless{}0.05).

\subsubsection*{Matrix Porosity}

{\label{matrix-porosity}}

Tumor cell morphology and matrix porosity measurements are illustrated
in Fig. 3. IBC cells, MDA-IBC3 and SUM149, displayed an epithelial like
rounded phenotype while the MDA-MB-231 presented a mesenchymal like
phenotype replicating behavior found \emph{in vivo} (Debeb et al.,
2016)\emph{.} Porosity measurements in Fig. 3 revealed significantly
more porous collagen ECM in the IBC platforms compared MDA-MB-231/TIME
and TIME platforms. SUM149/TIME platforms were 1.5 (p\textless{}0.01),
1.6 (p\textless{}0.01), and 1.3 (p\textless{}0.05) times higher in
matrix porosity compared to MDA-MB-231/TIME, TIME only, and
MDA-IBC3/TIME \emph{in vitro} vascularized platforms, respectively.
MDA-IBC3 \emph{in vitro} platforms also showed an increase in ECM
porosity of 1.1 (p\textless{}0.05) and 1.2 (p\textless{}0.01) times
compared to the MDA-MB-231/TIME and TIME only platforms.

\subsection*{\texorpdfstring{Reproduction of Relevant IBC Tumor Biology
and Phenotypic Comparisons to Published \emph{In
Vivo}Models}{Reproduction of Relevant IBC Tumor Biology and Phenotypic Comparisons to Published In VivoModels}}

{\label{reproduction-of-relevant-ibc-tumor-biology-and-phenotypic-comparisons-to-published-in-vivo-models}}

\subsubsection*{Longitudinal Characterization of Vascular
Sprouting}

{\label{longitudinal-characterization-of-vascular-sprouting}}

Following characterization of the IBC and non-IBC platforms, angiogenic
sprouting observed in the MDA-IBC3/TIME was followed for a three week
period as illustrated in Fig. 4. This phenomenon was only observed in
the presence of MDA-IBC3 cells and not in the presence of SUM149 or
MDA-MB-231. Additionally, \emph{in vitro} vascularized platforms
composed of BT474, a HER2+ non-IBC cell type, also failed at recreating
the extensive angiogenesis present in MDA-IBC3/TIME platforms (data not
shown). Fig. 4 reveals the ability of the MDA-IBC3/TIME platforms to
promote angiogenic sprouting of the vascular endothelium, formation of
MDA-IBC3 tumor emboli, and the capability of the platform for
spatiotemporal tracking of the sprouting behavior. Day 0, which
represents the endothelium formed after the graded flow protocol, the
endothelium exhibited very few sprouts. On Day 4, more sprouts were
present with TIME cells extending out from the vessel wall into the
collagen. By Days 12 and 16, numerous sprouts formed along the length of
the vessel wall with multiple branches and patent lumen (Fig. 4d)
invading deeper into the collagen ECM. The sprouts extended towards
clusters of MDA-IBC3 and started to encircle these clusters leading to
formation and proliferation of MDA-IBC3 emboli as pointed out by the
white arrows in the later time points of Day 12 and 16 (Fig. 4a) and in
the higher magnification images in Fig. 4c. Vascular encircling of
MDA-IBC3 clusters in the \emph{in vitro} platform (Fig. 4f) is
reminiscent of IBC tumor behavior \emph{in vivo} in both IBC patients
(Fig. 4e) and PDX models of IBC (Colpaert et al., 2003; Mahooti et al.,
2010). K-S analysis of vessel sprouting using the center plane of the
vessel confirmed a consistent and significant increase in sprout lengths
compared to Day 0, p\textless{}0.001 (Fig. 4b).

\subsubsection*{Lumen Formation}

{\label{lumen-formation-1}}

Lumen presence in the new angiogenic sprouts were confirmed if green
fluorescent microspheres were observed. In TIME only platforms and
acellular platforms without an endothelialized vessel (data not shown),
perfusion of 1 \selectlanguage{greek}µ\selectlanguage{english}m green fluorescent microsphere through the vessel
resulted in their aggregation at the vessel walls without entering the
surrounding collagen ECM. Fig. 5a and b, confocal images of vessel
sprouts taken on Day 14 and 21 reveal the presence of fluorescent
microsphere in the vessel sprouts and not in the surrounding collagen
matrix indicating the formation of a lumen that allowed for the beads to
be transported from the main vessel. By Day 21, we observed an increase
in the number of sprouts positive for the presence of the green
fluorescent microspheres.

\subsubsection*{Quantification of Sprout Properties and Vascular
Network}

{\label{quantification-of-sprout-properties-and-vascular-network-1}}

Total length of the vascular network, number of sprouts, and analysis of
sprout area along the length of the sprouts are shown in Fig. 6. For
determining the number of newly formed sprouts and the total network
length, a 45 \selectlanguage{greek}µ\selectlanguage{english}m section at the center of the vessel was used (Fig. 6a).
The computational recreation of the vascular network from the 45 \selectlanguage{greek}µ\selectlanguage{english}m
region of interest and the corresponding measurements of number of
sprouts and total vascular network are shown in Fig. 6b and c
respectively. As expected, the total vascular network and number of
sprouts at each subsequent time point increased indicating continuous
angiogenic sprouting (Fig. 6b and c). While the growth trends in
vascular network and number of newly formed sprouts at each time point
are similar between the platforms, the number of sprouts and length of
network varies between the different platforms. The analysis for the
sprout areas along the sprout lengths showed an increase in the sprout
area at later time points. Each sprout was analyzed 100, 200, 300, and
400\selectlanguage{greek}µ\selectlanguage{english}m from the edge of the vessel as depicted in the schematic in Fig.
6d. At each distance from the vessel, the number of sprouts of varying
area: 100, 200, 300, 400, 500, and 1000 \selectlanguage{greek}µ\selectlanguage{english}m\textsuperscript{2} which
correspond to a cross sectional diameter of approximately 11 \selectlanguage{greek}µ\selectlanguage{english}m, 16 \selectlanguage{greek}µ\selectlanguage{english}m,
20 \selectlanguage{greek}µ\selectlanguage{english}m, 23 \selectlanguage{greek}µ\selectlanguage{english}m, 25 \selectlanguage{greek}µ\selectlanguage{english}m, and 36 \selectlanguage{greek}µ\selectlanguage{english}m were counted. On Day 4, the longest
vessel was measured 300 \selectlanguage{greek}µ\selectlanguage{english}m away from the edge of the vessel. At later
time points of Day 8 and Day 12, sprouts were present 400 \selectlanguage{greek}µ\selectlanguage{english}m away from
the vessel. With time, larger vessels of areas 1000
\selectlanguage{greek}µ\selectlanguage{english}m\textsuperscript{2} which correlate to larger sprouts with lumen were
detected and in accordance with observations of lumen formation in image
Fig. 6f taken on Day 12 in the \emph{in vitro} MDA-IBC3/TIME platform.

\subsubsection*{Cytokine Analyses of Vascular
Sprouting}

{\label{cytokine-analyses-of-vascular-sprouting}}

Cytokine analysis of the perfusion media at the outlet was measured at
multiple time points illustrated in Fig. 7 and performed to understand
the driving factors behind the sustained angiogenic sprouting and
kinetics of their expression. VEGF-A, ANG-2, PDGF-bb, IL-8, IL-6, and
MMP2 expressions were significantly higher (p\textless{}0.05) on Day 21
compared to the earlier timepoints. VEGF-A expression was higher at the
later timepoints (Day 7, 14, and 21) compared to Day 0 and IL-8
expression increased significantly on Day 14 and 21 compared to Day 0.
bFGF and EGF both showed a similar trend with expression peaking on Day
7 (p\textless{}0.05) and then decreasing back to levels comparable to
Day 0 on Days 14 and 21.

\section*{Discussion}

{\label{discussion}}

In this study, we developed the first 3D \emph{in vitro} vascularized
IBC platform to model the unique interactions of IBC cells (SUM149 and
MDA-IBC3) with the vasculature and ECM in a dynamic and spatial manner
and compared the observations to non-IBC (MDA-MB-231) cells cultured in
the platform. Tumor specific \emph{in vivo} responses including
increased vascular permeability, ECM remodeling, and vessel sprouting as
a result of the tumor-vasculature and tumor-ECM interactions were
reproduced and we showed a differential response of the three different
cells lines (IBC vs non-IBC and HER2+ vs triple negative) in modulating
these behaviors. After identifying the differences between the tumor
cells, we investigated the vascular sprouting nature of HER2+ MDA-IBC3
with the platform providing the first opportunity to spatially observe
and quantify this behavior \emph{in vitro} and were able to recreate and
validate previously published \emph{in vivo} phenotypes including
endothelial sprouting, and vascular encircling of tumor emboli.

\subsection*{\texorpdfstring{Characterization of \emph{In Vitro} Tumor
Platforms}{Characterization of In Vitro Tumor Platforms}}

{\label{characterization-of-in-vitro-tumor-platforms-1}}

For comparison between IBC and non-IBC as well as HER2+ and triple
negative IBC cells, we investigated the influence of cell type on
vascular permeability of the endothelium, endothelial coverage of the
vessel lumen, expression of angiogenic factors VEGF and bFGF as well as
remodeling of the collagen ECM. Tumor vasculature is characterized by
the presence of leaky blood vessels which has been implicated in
inefficient delivery of chemotherapies as well as playing a crucial role
in tumor intravasation (Azzi et al., 2013; Claesson-Welsh, 2015;
Hashizume et al., 2000; Jain et al., 2014; Shenoy et al., 2016; Uldry et
al., 2017). In this study, we demonstrated that the presence of triple
negative, both IBC (SUM149) and non-IBC (MDA-MB-231) cells compromised
the vasculature with formation of large pores and gaps in the
endothelium, increased vascular permeability, and decreased endothelial
coverage of the vessel lumen. Vascular permeability, a measurement of
the integrity of the endothelium, was significantly higher in the
platforms with the triple negative cancer cells (SUM149 and MDA-MB-231)
regardless of IBC or non-IBC status and is in accordance with results
from multiple groups where introduction of highly invasive tumor cells
increased permeability of the endothelium (Jeon et al., 2013; Kim et
al., 2017; Kim et al., 2013; Lee et al., 2014a; Lee et al., 2014b; Tang
et al., 2017; Terrell-Hall et al., 2017; Tsai et al., 2017;
Zervantonakis et al., 2012). In addition to vascular permeability, these
same platforms exhibited a significant decrease in endothelial
population correlating with previous studies that showed direct contact
between triple negative MDA-MB-231 and endothelial cells disrupted
endothelial monolayers and resulted in anoikis of endothelial cells
allowing for dextran to cross into the collagen unhindered correlating
with results seen in other experimental studies (Brenner et al., 1995;
Haidari et al., 2012; Haidari et al., 2013; Kebers et al., 1998; Mierke,
2011; Peyri et al., 2009; Zervantonakis et al., 2012; Zhang et al.,
2012). In contrast to the triple negative cells, HER2+ MDA-IBC3, did not
significantly alter the endothelium barrier function and maintained a
confluent endothelium. Bright patches of red fluorescent signal in the
F-actin stained images, as well as the increased coverage of the
endothelium in the MDA-IBC3/TIME \emph{in vitro} vascularized platform,
suggest the presence of a larger endothelial population consistent with
previous studies that demonstrated a strong association between IBC and
increased proliferation levels of endothelial cells (Colpaert et al.,
2003; Costa et al., 2017; Shirakawa et al., 2002; van Uden et al., 2015;
Vermeulen et al., 2010).

While vascular permeability and endothelial coverage of the lumen were
not deterministic factors between IBC and non-IBC cells, expression of
the proangiogenic factor VEGF and ECM remodeling were significantly
higher in the IBC platforms regardless of receptor status. Both the
HER2+ MDA-IBC3 and triple negative SUM149 platforms expressed increased
amounts of VEGF and were more active in remodeling the collagen ECM as
evidenced by the increased ECM porosity. van Golen et al. determined
increased levels of VEGF mRNA in IBC tumors vs non-IBC tumors (van Golen
et al., 2000a) corresponding with the increased levels of VEGF
expression in both the IBC MDA-IBC3 and SUM149 \emph{in
vitro}vascularized platforms. Along with being highly angiogenic, IBC
tumors are also highly invasive. Analysis of SEM images of the acellular
collagen matrix (data not shown) revealed a pore size of
\textasciitilde{}1 \selectlanguage{greek}µ\selectlanguage{english}m, much smaller than cell width. Pore sizes smaller
than a cell's width induce cellular degradation of the ECM through
secretion of matrix metalloproteinases (MMPs) to allow for motility of
cancer cells (Guzman et al., 2014; Holle et al., 2016; Lautscham et al.,
2015; Sabeh et al., 2009; Seo et al., 2014; Wolf et al., 2011).
Al-Raawi\emph{et al} found an overexpression of MMPs by IBC carcinoma
tissues (Al-Raawi et al., 2011) which are involved in degradation of
collagen I and widening of pore size to allow for cell migration and
invasion (Lang et al., 2015; Sabeh et al., 2004; Sabeh et al., 2009;
Wolf et al., 2011; Wolf et al., 2013; Wolf et al., 2007). Rizwan
\emph{et al} demonstrated an increased migratory and invasive behavior
in IBC cells as well as increased levels of MMP9 compared to MDA-MB-231
cells (Rizwan et al., 2015). Higher proteolytic activity of IBC breast
tumors compared to non-IBC tumors coincides with the increased matrix
porosity in the SUM149 and MDA-IBC3 \emph{in vitro} vascularized
platforms.

\subsection*{\texorpdfstring{Reproduction of Relevant IBC Tumor Biology
and Phenotypic Comparisons to Published \emph{In
Vivo}Models}{Reproduction of Relevant IBC Tumor Biology and Phenotypic Comparisons to Published In VivoModels}}

{\label{reproduction-of-relevant-ibc-tumor-biology-and-phenotypic-comparisons-to-published-in-vivo-models-1}}

To the best of our knowledge, this is the first demonstration of the
dynamics of vascular sprouting in a 3D \emph{in vitro} platform
sustained through interactions between tumor and endothelial cells
without the influence of any exogenous supplements or additional stromal
cells. IBC is characterized as highly angiogenic with a significantly
higher population of tumor infiltrating and proliferating endothelial
cells compared to non-IBC cells (Colpaert et al., 2003; Shirakawa et
al., 2002) which is evidenced with the sustained angiogenesis occurring
and directed towards tumor cells in our MDA-IBC3 \emph{in
vitro}platforms. We performed further studies to confirm whether the
vascular angiogenesis seen in the HER2+ MDA-IBC3 was due to HER2+ status
or HER2+ and IBC status. MDA-IBC3 platforms were compared to HER2+
non-IBC BT474 vascularized platforms, and observed vascular sprouting
only in the MDA-IBC3 platforms (data not shown), revealing the
angiogenic behavior attributed to the cells being both HER2+ and IBC.
Along with vessel sprouting, we saw the formation and growth of MDA-IBC3
emboli enveloped by newly formed vascular vessels which is
characteristic of \emph{in vivo} IBC tumors. Histology samples of IBC
tumors and 3D spheroid IBC assays reveal tightly packed clusters of IBC
cells similar to MDA-IBC3 emboli developed in the \emph{in vitro}
platforms (Arora et al., 2017; Kleer et al., 2001). In an invasion
independent metastasis mechanism proposed by Sugino et al., tumor
clusters accessed blood vessels by being surrounded by the vessels
rather than intravasation, similar to behavior seen in the MDA-IBC3/TIME
vascularized \emph{in vitro} breast tumor platforms (Fig. 4C) (Sugino et
al., 2002). Published work by Mahooti et al. describe a phenotype of
encircling vasculogenesis in the Mary-X IBC mouse model (Mahooti et al.,
2010), behavior reproduced by the endothelial sprouts in our \emph{in
vitro} platform encircling MDA-IBC3 cells in the matrix demonstrating
this \emph{in vivo} phenotype (Fig. 4). Analysis of cytokine expression
in the MDA-IBC3 platforms revealed a significant increase in the
proangiogenic factors by Day 21 compared to Day 0 associated with
significant amount of angiogenesis occurring at the later time point.
The highest expression of most of the measured factors, occurred on Day
21 but bFGF and EGF both displayed similar trends in expression levels
with the highest expression on Day 7. Additionally, we determined VEGF,
an important angiogenic factor (Carmeliet, 2005; Hoeben et al., 2004),
to be a key contributor of angiogenesis in our system as continued
increase in expression of VEGF paralleled the increase in angiogenic
response in the MDA-IBC3 platform. We also confirmed for lumen
development in the newly formed sprouts as an indicator of viable
vessels and saw the formation of larger and longer vessels over time
reminiscent of angiogenic processes. Along with lumen formation, we
determined the presence of a larger population of vessels with patent
lumen extending further out into the collagen. The trends observed in
both the sprout area, number of sprouts and length of the total vascular
network showed an increase with each subsequent point yet there were no
significant differences between time points. Upon looking at the trends
of the individual platforms, we observed one platform presented a much
larger vascular network compared to the other two leading to large
variation between the platforms.

The focus of our study was to introduce and show the ability of
the\emph{in vitro} IBC vascularized platforms to reproduce \emph{in
vivo}IBC phenotypes and serve as an investigative tool for studying IBC.
There are some limitations to our study and the platforms presented.
While the \emph{in vitro} platforms developed in this study do not
encompass the entire complexity of the tumor microenvironment and
utilize immortalized endothelial cells, they recapitulate key IBC
characteristics in their current form not available with existing
platforms and provide an initial insight into the behavior of aggressive
breast tumors and enabling us to recapitulate key phenotypic behaviors
specific to IBC. Future work utilizing this platform can be expanded to
incorporate stromal and immune cells known to influence tumor behavior
as well as the use of non-immortalized endothelial cells. Macrophages
have been shown to be a key player in driving IBC phenotype and
therefore will be an important factor to include in future studies
(Allen et al., 2016; Mohamed et al., 2014; Wolfe et al., 2016). Other
cells for incorporation in the platform include mesenchymal stem cells,
adipocytes and fibroblasts all of which have shown to also contribute to
IBC phenotype. Additionally, we acknowledge that the size of the
endothelial vessels is larger than the size of \emph{in
vivo}microvasculature. Platforms can be adapted to present a more
comparable vessel with the use of smaller gauge needles for formation of
the cylindrical vessels as published in our previous work (Michna et
al., 2018).

\section*{Conclusion}

{\label{conclusion}}

The 3D \emph{in vitro} vascularized IBC platforms presented in this work
enabled us to dynamically characterize and model the IBC tumor-vascular
interactions, as well as determine the spatiotemporal response of these
interactions on vascular permeability and matrix porosity not possible
with existing \emph{in vitro} or \emph{in vivo} models. The platforms
provide a robust and cost-effective means to systematically and
quantitatively investigate IBC in a controlled and replicable manner
compared to the current standard of using PDX models. Using the \emph{in
vitro} platform, we determined IBC cells were more active in remodeling
of the collagen ECM as well as secretion of proangiogenic and
tumorigenic factor VEGF compared to non-IBC MDA-MB-231, revealing
potential targets for IBC therapeutics. For the first time, we induced
angiogenic sprouting of the vascular endothelium and vascular
surrounding of tumor emboli (unique behaviors of IBC tumors) purely
through tumor-endothelial cell interactions and characterized sprouting
in a spatial and temporal fashion in an \emph{in vitro} setting.
Furthermore, our system captures blood vessel leakiness and increased
matrix porosity, representative behavior of \emph{in vivo} invasive
tumors. With the \emph{in vitro} vascularized IBC tumor platforms,
behavioral variations that are representative of \emph{in vivo} tumors
can be identified and distinguished as a result of the different breast
cancer cells. This platform allows for spatiotemporal imaging and
identification of biological proteins and responses which may play a
direct role in tumorigenesis and vascularization \emph{in vivo} and
represent a useful tool for studying various aggressive breast cancers
whose phenotype is driven by tumor-stromal-vascular interactions. These
platforms can be further expanded to investigate increasingly complex
cell type interactions, thereby providing a tool to further decipher the
mechanisms behind development of these tumors.

\section*{Acknowledgements}

{\label{acknowledgements}}

TEY is a CPRIT Scholar in Cancer Research.

\section*{Funding}

{\label{funding}}

We thank the National Cancer Institute for funding through R01CA186193
and U01CA174706, National Institute of Health for funding through
1R21CA158454-01A1 and R21EB019646, Cancer Prevention Research Institute
of Texas Grant RR160005 and the American Cancer Society for funding
through RSG-18-006-01-CCE.

\section*{Author Disclosure Statement}

{\label{author-disclosure-statement}}

No competing financial interests exist.

\section*{List of abbreviations:}

{\label{list-of-abbreviations}}

ECM: Extracellular Matrix

IBC: Inflammatory Breast Cancer

2D: 2 Dimensional

3D: 3 Dimensional

TIME: Telomerase Immortalized Microvascular Endothelial

GFP: Green Fluorescent Protein

RFP: Red Fluorescent Protein

WSS: Wall Shear Stress

VEGF: Vascular Endothelial Growth Factor

HER2: Human Epidermal Growth Factor Receptor 2

PECAM-1: Platelet Endothelial Cell Adhesion Molecule-1



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\textbf{Figure Legend}

\textbf{Fig. 1} Development of a confluent endothelium in the vessel
using the graded flow protocol in various co-culture conditions. a)
Progression of endothelium alignment and confluence ~throughout the flow
protocol for the TIME only platform. The 0 hour time point, imaged after
channel formation, initiated with TIME cells in a rounded morphology.
The subsequent 48 hour of flow promoted TIME cell spreading and
proliferation followed by alignment of the TIME cells in the direction
of flow. The resulting confluent endothelium at 78 hours serves to
function as a barrier for transendothelial flow; scale bar: 200 \selectlanguage{greek}µ\selectlanguage{english}m. b)
The resulting \emph{in vitro} vascularized breast tumor platforms
consisting of monoculture of TIME cell seeded lumen (red) or co-culture
of GFP labeled (green) MDA-IBC3, SUM149, and MDA-MB-231 tumor cells
around a TIME cell seeded lumen (red); scale bar: 500 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Fig. 2} Characterization of the \emph{in vitro} vascularized
microfluidic platforms. (a) Endothelial morphology and adhesion observed
through PECAM-1 and DAPI (top row, scale bar: 100 \selectlanguage{greek}µ\selectlanguage{english}m), and F-actin and
DAPI (middle row, scale bar: 200 \selectlanguage{greek}µ\selectlanguage{english}m) immunofluorescent staining, and SEM
analysis of the endothelium (bottom, scale bar: 10 \selectlanguage{greek}µ\selectlanguage{english}m). PECAM-1 (green)
staining revealed difference in endothelial cell-cell junctions between
neighboring TIME cells, F-actin (red) staining and SEM images revealed
morphological difference in the endothelium. White arrows denote gaps
between the neighboring cells and the boxed areas in the F-actin images
show early signs of angiogenic sprouting. (b) Quantification of
endothelium coverage of the vessel lumen from F-actin stained images
revealed a decrease in coverage in the MDA-MB-231 and SUM149 platforms;
*p\textless{}0.05, ** p\textless{} 0.01. (c) Measured effective
permeability of 70 kDA green fluorescent dextran perfusion through the
platforms showed a significant decrease in vessel permeability in the
MDA-MB-231 and SUM149 platforms; *p\textless{}0.05. (d) VEGF and bFGF
expression measured by ELISA showed significantly higher VEGF expression
in the IBC platforms while bFGF was higher in the non-IBC and acellular
control platforms; #, ^denote significance (p\textless{}0.05) compared
to acellular TIME control and MDA-MB-231 platforms respectively.

\textbf{Fig. 3} Collagen porosity measured with SEM. a) SEM images of
tumor (scale bar: 2 \selectlanguage{greek}µ\selectlanguage{english}m), and TIME (scale bar: 20 \selectlanguage{greek}µ\selectlanguage{english}m) cells morphologies
(top panels), and collagen matrix organization (bottom panels, scale
bar: 1 \selectlanguage{greek}µ\selectlanguage{english}m). b) Collagen matrix porosity measurements calculated from SEM
images of the ECM, *p\textless{}0.05, **p\textless{}0.01

\textbf{Fig. 4} ~Vascular sprouting dynamically observed over a three
week period in the MDA-IBC3/TIME \emph{in vitro} vascularized tumor
platforms. (a) Longitudinal cross section images of the vessel show
vessel sprouting, branching, as well formation of tumor emboli pointed
out by white arrows (top panels), and front view of the vessels (bottom
panels). (b) K-S analysis of vessel sprouting revealed a significant
increase in sprouting at later time points compared to Day 0. (c)
F-actin (red) staining of GFP labeled MDA-IBC3 cells (green) showed
formation and growth of tumor emboli. (d) Lumen formation followed over
time in one of the vessel sprouts. (e) Vascular nesting phenomenon of
IBC tumors in \emph{in vivo} patient derived histological samples
demonstrated by CD31 staining of vascular vessel (brown) surrounding IBC
tumor emboli (blue). (f) \emph{In vitro} recreation of vascular nesting
of IBC tumors as shown by the encircling of MDA-IBC3 tumor cells (green)
by mKate labeled sprouts (red).

\textbf{Fig. 5} Confirmation of lumen formation in the vessel sprouts on
Day 14 (a) and Day 21 (b). Platforms were injected with a solution of
1\selectlanguage{greek}µ\selectlanguage{english}m green fluorescent microspheres and presence of green microspheres
away from the main vessel was indicative of lumen formation in the
sprouts.

\textbf{Fig. 6} Analysis of the vascular network. (a) A 45 \selectlanguage{greek}µ\selectlanguage{english}m region of
interest at the center of the vascular vessel was used to quantify the
number of sprouts and the network length. (b) Representative images of
the vascular network in the region of interest derived by applying the
algorithm developed by Kollmannsberger et al. and Crosby et al. (Crosby
et al., 2019; Kerschnitzki et al., 2013; Kollmannsberger et al., 2017)
used for determining (c) fold change comparisons of total network length
and number of sprouts normalized to the Day 0 values. Results show a
steady increase in both number of sprouts and total vascular network
length at each subsequent time point. (d) A schematic cross-section of
the vessel with vascular sprouting used for calculation of number of
sprouts with cross sectional areas ranging from 100 to 1000
\selectlanguage{greek}µ\selectlanguage{english}m\textsuperscript{2} determined at 100, 200, 300 and 400 \selectlanguage{greek}µ\selectlanguage{english}m away from
the vascular vessel. (e) Number of sprouts present at 100, 200, 300, and
400 \selectlanguage{greek}µ\selectlanguage{english}m away from the vessel as well as the cross sectional areas of
those sprouts were determined. ~Over time, the new sprouts increase in
both cross sectional area, an indication of lumen formation, and length.
(f) Measurement of the diameters of the \emph{in vitro} sprouts with
lumen capable of particle perfusion (vessels with green signal from
microspheres overlaying red signal from endothelial sprouts) taken on
Day 12. Sprout area range from 15-42 \selectlanguage{greek}µ\selectlanguage{english}m (scale bar indicates 100 \selectlanguage{greek}µ\selectlanguage{english}m) and
correlate with the increase in larger area sprouts found at later time
points in Fig. 6e.

\textbf{Fig. 7} Cytokine analysis of angiogenic associated factors
measured over a three week period. ANG2, VEGF-A, PDGF-bb, IL-8, IL-6,
and MMP2 showed a significant increase in expression on Day 21 compared
to earlier timepoints while bFGF and EGF both peaked on Day 7,
*p\textless{}0.05.

\textbf{Supplementary Information}

S1. Sprouting Quantification Methodology

Analysis for Vessel Quantification: We have developed an in-house
algorithm for quantifying angiogenesis in the platform. We map the
experimental vessel that is a cylinder with a near elliptical cross
section onto a true cylinder. This is reasonable since a
\textasciitilde{} 0.95b, where a is the semi-major axis and b is the
semi-minor axis of the elliptical cross section. We then map the
elliptical cross section onto a circular cross section and assume this
preserves area since a is nearly equal to b. Since the true cylindrical
vessel has radius R and we are interested in quantifying vascular
sprouting at distances of 50, 100, 150, etc. from the vessel, we create
cylindrical shells that are 50, 100, 150, etc. microns from the
experimental vessel. We construct this surface using intensity values of
fluorescence and unroll the cylindrical shells into rectangular
surfaces. We use an intensity threshold on these surfaces and then
quantify vessel sprouting by taking area thresholds of varying size that
correspond to the cross-sectional area of the vessel going through these
constructed surfaces at varying distances from the parent vessel.

\textbf{Supplementary Fig. A} : Calcein staining (red) of live GFP tumor
cells (green) in the platforms without TIME cells 12 hours after initial
seeding to visualize cell numbers; scale bar 300\selectlanguage{greek}µ\selectlanguage{english}m. All cells were
seeded at an initial density of 1 million cells/ml. Difference in
inherent GFP expression are shown as the IBC cells lines MDA-IBC3 and
SUM149 express a strong GFP signal while the GFP expression of
MDA-MB-231 cells is much weaker.\selectlanguage{english}
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