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.