Results

In Vitro IBC Platform Development

The graded flow preconditioning protocol with a graded increase in WSS from 0.01 dyn/cm2 to 1 dyn/cm2resulted in a confluent endothelium as shown in Fig. 1a, which shows the evolution of the vascular endothelium in the TIME only in vitrovascularized 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 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 in vitrovascularized 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.

Characterization of In Vitro Tumor Platforms

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/TIMEin 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 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.

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<0.05) and MDA-MB-231/TIME (p<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.

Endothelial Permeability

Measured effective permeability for TIME, MDA-IBC3/TIME, SUM149/TIME, and MDA-MB-231/TIME platforms were 0.016 ± 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 in vitro vascularized platforms were statistically significant (p <0.05) with 1.6 and 1.3 fold higher permeability than TIME and MDA-IBC3/TIME in vitro vascularized platforms respectively. SUM149/TIME in vitrovascularized platform also differed significantly from the TIME platforms (p< 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).

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 < 0.05) in MDA-IBC3/TIME 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<0.05). bFGF was expressed highest in the MDA-MB-231/TIME platform, 1.2 times, compared to both the IBC platforms (p<0.05).

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 in vivo (Debeb et al., 2016). 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<0.01), 1.6 (p<0.01), and 1.3 (p<0.05) times higher in matrix porosity compared to MDA-MB-231/TIME, TIME only, and MDA-IBC3/TIME in vitro vascularized platforms, respectively. MDA-IBC3 in vitro platforms also showed an increase in ECM porosity of 1.1 (p<0.05) and 1.2 (p<0.01) times compared to the MDA-MB-231/TIME and TIME only platforms.

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

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, 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 in vitro platform (Fig. 4f) is reminiscent of IBC tumor behavior 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<0.001 (Fig. 4b).

Lumen Formation

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 µm 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.

Quantification of Sprout Properties and Vascular Network

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 µm section at the center of the vessel was used (Fig. 6a). The computational recreation of the vascular network from the 45 µm 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 400µm 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 µm2 which correspond to a cross sectional diameter of approximately 11 µm, 16 µm, 20 µm, 23 µm, 25 µm, and 36 µm were counted. On Day 4, the longest vessel was measured 300 µm away from the edge of the vessel. At later time points of Day 8 and Day 12, sprouts were present 400 µm away from the vessel. With time, larger vessels of areas 1000 µm2 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 in vitro MDA-IBC3/TIME platform.

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<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<0.05) and then decreasing back to levels comparable to Day 0 on Days 14 and 21.