Figure Legend
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 µm. b) The resulting 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 µm.
Fig. 2 Characterization of the in vitro vascularized microfluidic platforms. (a) Endothelial morphology and adhesion observed through PECAM-1 and DAPI (top row, scale bar: 100 µm), and F-actin and DAPI (middle row, scale bar: 200 µm) immunofluorescent staining, and SEM analysis of the endothelium (bottom, scale bar: 10 µm). 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<0.05, ** p< 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<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<0.05) compared to acellular TIME control and MDA-MB-231 platforms respectively.
Fig. 3 Collagen porosity measured with SEM. a) SEM images of tumor (scale bar: 2 µm), and TIME (scale bar: 20 µm) cells morphologies (top panels), and collagen matrix organization (bottom panels, scale bar: 1 µm). b) Collagen matrix porosity measurements calculated from SEM images of the ECM, *p<0.05, **p<0.01
Fig. 4  Vascular sprouting dynamically observed over a three week period in the MDA-IBC3/TIME 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 in vivo patient derived histological samples demonstrated by CD31 staining of vascular vessel (brown) surrounding IBC tumor emboli (blue). (f) 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).
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 1µm green fluorescent microspheres and presence of green microspheres away from the main vessel was indicative of lumen formation in the sprouts.
Fig. 6 Analysis of the vascular network. (a) A 45 µm 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 µm2 determined at 100, 200, 300 and 400 µm away from the vascular vessel. (e) Number of sprouts present at 100, 200, 300, and 400 µm 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 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 µm (scale bar indicates 100 µm) and correlate with the increase in larger area sprouts found at later time points in Fig. 6e.
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<0.05.