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.