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) 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; Trédan 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) 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 3Din 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 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 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 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 ofin 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 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 in vivo IBC models. Additionally, we
characterized the spatiotemporal angiogenesis of the MDA-IBC3 platforms
and recreated behaviors characteristic of 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.