Discussion

In this study, we developed the first 3D 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 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 in vitro and were able to recreate and validate previously published in vivo phenotypes including endothelial sprouting, and vascular encircling of tumor emboli.

Characterization of In Vitro Tumor Platforms

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 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 in vitrovascularized 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 ~1 µm, 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-Raawiet 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 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 in vitro vascularized platforms.

Reproduction of Relevant IBC Tumor Biology and Phenotypic Comparisons to Published In VivoModels

To the best of our knowledge, this is the first demonstration of the dynamics of vascular sprouting in a 3D 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 in vitroplatforms. 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 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 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 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 in vitro platform encircling MDA-IBC3 cells in the matrix demonstrating this 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 thein vitro IBC vascularized platforms to reproduce in vivoIBC phenotypes and serve as an investigative tool for studying IBC. There are some limitations to our study and the platforms presented. While the 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 in vivomicrovasculature. 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).