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.