This study investigated how tumor secreted factors alter the responses of human endothelial cells (HUVECs) to matrix stiffness during capillary formation. The researchers used synthetic hydrogels of varying stiffness to culture HUVECs with or without tumor cells. They found that while HUVECs prefer a substrate of intermediate stiffness for optimal capillary formation in the absence of tumor factors, stimulation by tumor secreted factors disrupts the HUVECs' mechanosensitive behavior. Additionally, the anti-angiogenic drug vandetanib was less effective at reducing capillary formation when HUVECs were activated by tumor factors, particularly on stiffer gels. This suggests tumor activation alters the mechanosensitivity and drug responsiveness

1. Differential Effects of Tumor Secreted Factors on Mechanosensitivity,
Capillary Branching, and Drug Responsiveness in PEG Hydrogels
YANG WU, BINGXIN GUO, and GARGI GHOSH
Bioengineering Program, Department of Mechanical Engineering, University of Michigan, Dearborn, 4901 Evergreen Road,
Dearborn, MI 48128, USA
(Received 28 July 2014; accepted 14 January 2015)
Associate Editor Jennifer West oversaw the review of this article.
Abstract—Solid cancers induce the formation of new blood
vessels to promote growth and metastasis. Unlike the normal
vascular networks, the tumor induced vasculatures exhibit
abnormal shape and function. Past efforts have been focused
on characterizing the altered growth factor signaling path-
way in tumor capillary endothelial cells; however, the
mechanical microenvironment of tumor also plays a signif-
icant role in regulating the formation of vascular patterns.
Here, we used synthetic hydrogel based cell culture platforms
to probe how activation of human umbilical endothelial cells
(HUVECs) by tumor secreted factors alters the responses to
matrix modulus and in turn the capillary network formation
and drug sensitivity. Our study revealed that while in absence
of activation, HUVECs prefer a substrate of appropriate
stiffness for optimal capillary network formation; stimula-
tion by tumor cells disrupts the mechano-responsive behavior
of HUVECs. Additionally, the effect of vandetanib on
reducing the capillary network was also investigated. The
response of HUVECs to the anti-angiogenic agent was
substrate modulus dependent displaying increased sensitivity
on the compliant gels. Stimulation by tumor cells reduced the
responsiveness to vandetanib, particularly when plated on
stiffer gels.
Keywords—Angiogenesis, Hydrogel, Matrix compliance,
Tumor cell activation.
INTRODUCTION
Most solid tumors less than 1–2 mm in diameter can
survive by passive diffusion of oxygen and nutrition.20
To grow beyond 2 mm, the primary tumors induce
angiogenesis, i.e., form new vascular networks.12
This
process involves activation of the endothelial cells by
several tumor derived factors including vascular endo-
thelial growth factor (VEGF), basic fibroblast growth
factor (bFGF), platelet-derived growth factor (PDGF),
angiopoetins (ANG), and chemokines.6,15,25,34
These
newly formed highly complex vascular networks can
then supply oxygen and nutrients to enhance the growth
of tumor.33
Understanding the role of these growth
factors in stimulating angiogenesis facilitated the
development of cancer therapies targeting these mole-
cules or their receptors. However, these treatments have
only been moderately successful producing short-term
benefits.7
This underscores the importance of better
understanding the mechanisms regulating tumor vas-
cularization to achieve therapeutic success. A body of
evidence suggests that capillary morphogenesis and
vascular pattern formation is also governed by the
mechanical microenvironment of tumor.4,19,32
In con-
trast to the normal tissue extracellular matrix (ECM),
the tumor stroma is stiffer due to increased collagen
deposition, high interstitial pressure, and collagen
crosslinking.9,27,31
The progressively changing physical
environment of the tumor stroma alters the cell-ECM
interaction force equilibrium resulting in altered cell
shape, increased proliferation, and migration through
the clustering of force-sensing integrin receptors. How-
ever, it is not clear how activation via tumor secreted
factors modulates the responses of endothelial cells to
matrix rigidity and vascularization. Therefore,
understanding the bidirectional crosstalk between the
tumor cell-stimulated endothelial cells and ECM is
critical to evaluating the effectiveness of drugs designed
for inhibiting angiogenesis.
While the small animal models are the gold stan-
dards for studying tumor vascularization, control over
various intrinsic variables including host cells, immune
response, endogenous growth factors, and hemody-
namics in these models are limited.23
Engineered tumor
Address correspondence to Gargi Ghosh, Bioengineering Pro-
gram, Department of Mechanical Engineering, University of Mich-
igan, Dearborn, 4901 Evergreen Road, Dearborn, MI 48128, USA.
Electronic mail: gargi@umich.edu
Annals of Biomedical Engineering (Ó 2015)
DOI: 10.1007/s10439-015-1254-2
Ó 2015 Biomedical Engineering Society

2. models capable of recapitulating the in vivo cellular
morphology and phenotypes are proving to be
invaluable for studying the dynamic and progressive
behavior of cancer under controlled conditions. The
majority of the existing models assess endothelial
morphogenesis in vitro when cultured on different
matrices including matrigel, collagen, or fibrin in pre-
sence of cancer cells or fibroblasts.1,8,21,24
However,
these systems suffer from the inability to decouple the
matrix mechanics from the porous architecture8
lead-
ing to restricted diffusional transport of putative
angiogenic factors which in turn can play an important
role in the regulation of capillary morphogenesis.13
This underscores the importance of decoupling differ-
ent intertwined ECM properties, e.g., the diffusional
characteristics from matrix compliance to parse the
specific contributions of these microenvironmental
cues in stimulating angiogenesis.
To investigate interplay between tumor cell activation
and mechano-responsive cellular behavior, we utilized
poly (ethylene glycol) diacrylate (PEGDA) and gelatin
methacrylate (GelMA) composite hydrogels as the cell
culture substrates. GelMA, obtained via conjugation of
methacrylate groups to gelatin, provides the cell binding
motifs, e.g., RGD as well as matrix metalloproteinase
(MMP) sensitive degradation groups.28
PEG is a bio-
inert polymer that resists non-specific adsorption of
proteins.18
This composite hydrogel system provides a
cell culture platform to evaluate the cellular responses to
the changes in the microenvironment. The stiffness of the
matrices was varied from 11 to 78 kPa to span the
mechanical properties reported for healthy and cancer-
ous breast tissue.36
Breast cancer cells, MDA-MB-231,
were encapsulated within these matrices. Human umbil-
ical vein endothelial cells (HUVECs) were then seeded on
the top of the cell-laden gels and capillary network for-
mation by HUVECs in the presence and absence of
cancer cells were evaluated. The inhibition of capillary
formation by vandetanib, a FDA approved anti-angio-
genic agent, was correlated with substrate stiffness. Our
results indicate that the activation of endothelial cells by
tumor cells alters the mechano-sensitivity, capillary net-
work formation, and drug sensitivity of endothelial cells.
MATERIALS AND METHODS
Materials
Poly (ethylene) glycol diacrylate 6000 (PEG6kDA),
Dulbecco’s phosphate buffer saline (DPBS), gelatin
from porcine skin, ethylene glycol, methacrylic
anhydride, dimethyl sulfoxide (DMSO) fluorescein
isothiocyanate—dextran (FITC-Dextran 70 kDa), and
photo-initiator (2-Hydroxy-4¢-(2-hydroxyethoxy)-2-
methylpropionphenone) were procured from Sigma
Aldrich (St. Louis, MO). Vandetanib was procured
from LC Laboratories (Woburn, MA), RPMI 1640
(Roswell Park Memorial Institute medium), Penicillin
Streptomycin L-Glutamine (Pen Strep) and Fetal bo-
vine serum (FBS) from GibcoÒ
(Grand Island, NY).
LIVE/DEADÒ
Cell Viability assay-kit was purchased
from Life Technologies (Grand Island, NY).
Synthesis of Methacrylated Gelatin
Gelatin methacrylate was synthesized as described
elsewhere.38
Methacrylation was achieved by adding
10% (w/v) of methacrylic anhydride to 10% (w/v)
gelatin solution at a rate 0.5 mL/min and reacting at
60 °C for 1 h. Following a 59 dilution with warm
DPBS (50 °C), the mixture was dialyzed using Slide-
A-lyzer against distilled water at 50 °C for 7 days. The
sample was then freeze dried and GelMA was gener-
ated as porous foam and stored at 280 °C until further
use.
Fabrication and Characterization of Hydrogel Matrices
The scaffolds were fabricated by adding 150 lL of
the pre-polymer solution in 48 well plates and then
exposing the plates to UV (CL-1000 UV Crosslinker
(UVP), 365 nm) for 5 min. The pre-polymer solution
consisted of PEG6kDA, 5% (w/v) GelMA, 1% (w/v)
photoinitiator and 20% (v/v) RPMI media. The con-
centration of PEGDA was varied from 5 to 15%.
Diffusion
To estimate the release kinetics of proteins from the
hydrogels, the scaffolds were fabricated by incorpo-
rating FITC-dextran 70 kDa within the precursor
solution. The concentration of dextran within each
sample was maintained at 50 lg/mL. After photo-
polymerization, the hydrogels were washed with PBS.
To facilitate the release of dextran from these matrices,
the scaffolds were incubated in 500 lL PBS at 37 °C.
At different time points (1, 3, 5, 7, 9, 18, 24 h post-
incubation in PBS), 200 lL of PBS was collected and
replaced with fresh PBS. All the collected samples were
analyzed using PerkinElmer LS55 fluorescence spec-
trometer to assess the release of FITC.
For hydrogel samples with thickness much smaller
than the diameter, the diffusion of dextran from the gel
phase to the surrounding can be considered as one
dimensional. To determine the mechanism of the
transport of these macromolecules, the dextran release
WU et al.

3. data were fitted into Korsmeyer–Peppas transport
model given by the Eq. (1):
F ¼
Mt
Mo
¼ k1tn
ð1Þ
where the (F) defined as Mt/Mo is the fractional release
of the molecule, Mt is the amount of dextran released
at any given time, Mo is the total mass of dextran
encapsulated within the hydrogel matrices, k1 is the
kinetic constant (s2n
), t is the release time (s), and n is
the diffusional exponent, which informs the mecha-
nism of diffusional release. For n £ 0.5, the transport
of the molecules can be defined by Fick’s law. The data
fitting was performed on the first 60% cumulative re-
lease, i.e., Mt/Mo = 0.6. The effective diffusivity of the
macromolecules is related to the cumulative release
according to the Eq. (2):
Mt=Mo ¼ 4 Ã
Dt
pL2
n
ð2Þ
Effective diffusivity (D, cm2
/s) can be then related to
Korsmeyer–Peppas constants
D ¼ pL2
k1=4ð Þ1=n
ð3Þ
Cell Culture
HUVECs were purchased from American Type
Culture Collection (ATCC, Manassas, VA) and ex-
panded in vascular basal medium with endothelial
growth supplement (Ascorbic, FBS, rh EGF, heparin
sulfate, L-glutamine, hydrocortisone, bovine brain ex-
tract) and 1% (v/v) penicillin streptomycin (PS).
MDA-MB-231 (ATCC) were expanded in RPMI
(Roswell Park Memorial Institute) 1640 medium con-
taining 1% (v/v) PS and 10% (v/v) FBS. The confluent
cells were trypsinized to detach them from the flask
surfaces, counted using cell automated counter (Bio-
Rad TC10TM
), and centrifuged to obtain cell pellets.
To fabricate cancer cell impregnated scaffolds, cancer
cells were suspended in 30 lL of RPMI and mixed with
120 lL pre-polymer solutions. The cancer cell seeding
density was 3.2 9 104
cells/scaffold. Cells up to pas-
sages 6 were used in this study.
Cell Morphology
RPMI conditioned media was collected from the
MDA-MB-231 cell line when the flask was over 70%
of confluent. The conditioned media was filtered and
stored at 4 °C for future use. 5 9 103
HUVECs were
seeded on scaffolds of different compliances. To
investigate the spreading of HUVECs, the cells were
seeded on scaffolds of varying stiffness and incubated
with either HR (50% HUVECs media and 50% RPMI
media) or HCR (50% HUVECs media and 50%
RPMI conditioned media). The images were captured
18 h after seeding using Zeiss Axio Observer A1 with
integrated CCD camera. The morphology of the cells
was assessed based on aspect ratio which indicates the
ratio between cell width and cell length.
Viability of Encapsulated Cancer Cells
To investigate the viability of the cancer cells within
PEG matrices, cells were encapsulated within the
scaffolds at a density of 3.2 9 104
cells/scaffold and
incubated for 24 h. Viability of the cancer cells was
assessed by using LIVE/DEADÒ
Viability/Cytotoxic-
ity assay kit as per the manufacturer’s instructions.
Characterization of Capillary Formation
To investigate the impact of activation by tumor
cells on capillary formation, 5 9 104
HUVECs were
seeded on the scaffolds with and without cancer cells
(Fig. 1). 3.2 9 104
cancer cells were encapsulated
within each scaffold. To monitor and quantify capil-
lary branching, images were captured 18 h post-seed-
ing. At least 5 images were captured for each scaffold.
Experiments were performed in three replicates. Cap-
illary formation was quantified by manually counting
the number of networks branching out from a branch
point/node and number of nodes per image. To eval-
uate the ability of vandetanib to inhibit the capillary
morphogenesis, HUVECs were seeded on the cell-la-
den as well as blank scaffolds and vandetanib at var-
ious concentrations was introduced 10 h post-seeding.
The concentration of the inhibitor was varied from 0 to
2 lM. The images of the branches were captured 8 h
post-treatment with the anti-angiogenic agent.
Statistical Analysis
For all experiments, data reported as mean ± SEM
of three independent experiments. Statistical analyses
were carried out with one way ANOVA. Differences
between two sets of data were considered significant at
p value 0.05.
RESULTS
Integrated Effects of Substrate Rigidity and Tumor
Secretory Factors on Cell–Matrix Interactions
To assess the effects of tumor secreted stimulatory
factors on endothelial cell responses to increasing
matrix stiffness, the hydrogel compliances were
Differential Effects of Tumor Secreted Factors

4. manipulated by varying the concentration of PEGDA
from 5 to 15% while maintaining uniform presentation
of cell adhesion molecules (i.e., constant gelatin con-
centration) (Fig. 2a). Alterations in the potential
crosslinking groups per unit volume increased the
hydrogel modulus from 11.4 ± 0.72 kPa for 5% PEG-
DA, to 35.8 ± 2.52 for 10% PEGDA, and 78 ± 5.03
for 15% PEGDA.38
Scanning electron microscopy
(SEM) analysis revealed that the increase in modulus
had minimal effect on the porosity of the gels.38
Increase
in crosslinking density which alters the free volume
available for transport attenuates the diffusion of
macromolecules through the hydrogels. So, to investi-
gate whether increasing the PEGDA concentration in
the gels affect the macromolecular release kinetics,
hydrogel disks were fabricated encapsulating FITC
conjugated (70 kDA) dextran. Figure 2b demonstrates
the cumulative release (% released) of dextran into the
solution for each crosslinking density over a span of
24 h. No significant difference was observed in the
dextran release profile from these hydrogel disks, indi-
cating that the transport of dextran molecules was
independent of the crosslinking density over the range
of PEGDA studied. Further analysis using Korsmeyer–
Peppas model confirmed the crosslinking density inde-
pendent nature of the dextran transport through
PEGDA hydrogels as no significant difference was
observed in the fitting parameters (p value 0.05) for
different matrices (Table 1). The average values of dif-
fusional exponent (n) for the three different crosslinking
density were found to be less than 0.5 indicating that the
transport mechanism is Fickian diffusion. Effective
diffusivity was calculated from Eq. (3). As demon-
strated in Fig. 2c, no correlation was observed between
effective diffusion coefficient and crosslinking density.
The values of diffusivity ranged from 0.5 9 1027
±
0.007 cm2
/s for 5% PEGDA gel to 0.6 9 1027
±
0.006 cm2
/s for 10% and 0.5 9 1027
± 0.006 cm2
/s for
15% PEGDA.
To evaluate the effect of tumor secretory factors on
endothelial cell–matrix interactions, HUVECs were
seeded on the top of the hydrogels with different
compliances in the presence or absence of cancer cell
(MDA-MB-231) conditioned media samples. Fig-
ure 3a illustrates the morphology of HUVECs incu-
bated with and without conditioned media samples.
Quantitative analysis revealed that, in the absence of
conditioned media, the increase in matrix rigidity from
11 to 36 kPa altered the aspect ratio of the cells (cell
width/cell length) from 0.59 ± 0.03 to 0.56 ± 0.03,
respectively; indicating no significant difference (p va-
lue 0.05) (Fig. 3b). However, when the matrix stiff-
ness was further increased to 78 kPa, an aspect ratio of
0.65 ± 0.03 was obtained, suggesting that the cells
plated on the stiffer matrices had less elongated mor-
phology (p value 0.05) and displayed more isotropic
spreading. In the presence of cancer cell conditioned
media, the aspect ratio varied from 0.46 ± 0.02
(11 kPa) to 0.49 ± 0.03 (36 kPa) to 0.53 ± 0.01
(78 kPa). Thus, tumor secreted factors stimulated the
cells to adopt more elongated spindle shaped
morphology.
FIGURE 1. Schematic representation of the approach. MDA-MB-231 cells were encapsulated within the hydrogel matrices.
HUVECs were then seeded on the top of the matrices. Integrated effects of matrix compliances and activation by tumor cells on
capillary network formation were then monitored.
WU et al.

5. FIGURE 2. (a) Schematic representing the synthesis of composite hydrogel matrices. (b) Comparison of the release of FITC-
dextran 70 kDa from the hydrogel matrices over 24 h. (c) Correlation between diffusion coefficient and substrate compliances.
Error bars are SEM (n 5 3). *Represents p value 0.05.
TABLE 1. Compression modulus, diffusional exponent, and kinetic constants of the hydrogels with different PEGDA concen-
trations.
PEGDA concentration wt(%)
Compression
modulus (kPa)
Diffusional
exponent n
Kinetic
constant k1 (s2n
)
5 11 ± 0.7 0.33 ± 0.01 0.14 ± 0.01
10 36 ± 2.5 0.33 ± 0.01 0.17 ± 0.01
15 78 ± 5.03 0.36 ± 0.02 0.16 ± 0.01
Differential Effects of Tumor Secreted Factors

6. Cancer Cell Viability Within Three Dimensional
Hydrogel Matrices
To investigate the viability of cancer cells within the
PEG hydrogels, MDA-MB-231 cells were encapsu-
lated within the matrices of varying stiffness. The
seeding density was 3.2 9 104
cells/scaffold. Figure 4a
illustrates a typical live dead image of the encapsulated
cancer cells. For all the conditions, MDA-MB-231
exhibited a viability level greater than 40% (Fig. 4b).
This was anticipated since increased compliances had
no effect on transport of macromolecules as mani-
fested from the release of dextran molecules.
Capillary Morphogenesis of HUVECs on Cancer Cell
Laden PEG Gels
To understand how HUVECs interpret, assimilate,
and integrate the biochemical and mechanical signals
from microenvironment to direct capillary network
formation, MDA-MB-231 cells were encapsulated
within PEG gels of varying stiffness. HUVECs were then
FIGURE 3. (a) HUVECs were seeded on scaffolds of varying stiffness without (i–iii) and with (iv–vi) conditioned media samples.
The substrate stiffness was varied from 11 kPa (i, iv), to 36 kPa (ii, v), and 78 kPa (iii, vi). Integrated effect of substrate stiffness and
tumor secreted factors on the morphology of HUVECs. (b) Quantification of the aspect ratio of HUVECs. HR corresponds to (50%
HUVECs media and 50% RPMI media) and HCR to (50% HUVECs media and 50% RPMI conditioned media). Error bars are SEM
(n 5 3). *Represents p value 0.05.
WU et al.

7. seeded on the top of the cell laden gels. To assess the
contribution of matrix mechanics, HUVECs were also
seeded on the top of the gels without encapsulated
cancer cells. Figure 5a illustrates the capillary network
formation in the presence and absence of MDA-MB-231
cells. To quantify the capillary branching, number of
nodes per image and number of sprouts branching out
from individual nodes were calculated (Figs. 5b, 5c). As
demonstrated in Fig. 5b, in the absence of tumor stim-
ulation maximum number of nodes (6.8 ± 0.5 nodes per
image) were observed on hydrogel of intermediate
stiffness as compared to compliant (5.3 ± 0.40 nodes
per image) and stiff (4.0 ± 0.3 nodes per image) gels.
Similarly, maximum capillary branching (3.6 ± 0.3
branches per node) was observed when HUVECs were
seeded on the hydrogel matrices of intermediate stiffness
(36 kPa) as compared to the compliant (11 kPa) and
stiff (78 kPa) gels (p value 0.05). To investigate whe-
ther hydrogel matrices of intermediate stiffness promote
maximal branching, HUVECs were seeded on 28 kPa
scaffolds. No significant difference in capillary branch-
ing per nodes (2.7 ± 0.2 vs. 2.4 ± 0.1) or nodes per
image (5.3 ± 0.40 vs. 5.1 ± 0.30) was observed between
11 and 28 kPa. These observations suggested that opti-
mal matrix stiffness enhanced capillary morphogenesis
of endothelial cells.
Next, HUVECs were seeded on gels impregnated
with MDA-MB-231 cells. Since, no significant differ-
ence was observed in endothelial tubulogenesis on 11
and 28 kPa scaffolds, MDA-MB-231 cells were
encapsulated within 11, 36, and 78 kPa matrices. In the
presence of cancer cells, number of branching points
increased on 11 kPa gels (6.9 ± 0.2 nodes per image).
However, no significant difference was observed in the
case of intermediate and stiff matrices (p value 0.5).
On the other hand, endothelial cell assembly increased
to 3.8 ± 0.2 capillary branches per node for compliant
gels, 4.1 ± 0.1 for gels with intermediate stiffness, and
3.1 ± 0.2 for stiffer gels. The network formation on
compliant and intermediate gels in presence of MDA-
MB-231 cells was significantly higher (p value 0.05)
from control; however, no significant difference was
observed when HUVECs were plated on cell laden stiff
gels (p value 0.05).
Integrated Effects of Substrate Stiffness and Tumor Cell
Activation on Drug Sensitivity of Endothelial Cells
To evaluate how matrix rigidity regulates the sen-
sitivity of endothelial cells to drug treatment, HUVECs
were plated on PEG matrices (without cancer cells).
10 h post-plating HUVECs were incubated with
FIGURE 4. Viability of MDA-MB-231 cells encapsulated within the hydrogel matrices of varying stiffness. (a) Typical images of live
and dead cells encapsulated within (i) 11 kPa, (ii) 36 kPa and (iii) 78 kPa scaffold. (b) Quantification of live cells within the three
dimensional PEG matrices. Error bars are SEM (n 5 3). *Represents p value 0.05.
Differential Effects of Tumor Secreted Factors

8. vandetanib for 8 h. Vandetanib is a FDA approved
anti-angiogenic agent used for treating solid cancers
including thyroid cancer. It works as a kinase inhibitor
of several receptor tyrosine kinases including the vas-
cular endothelial growth factor receptor (VEGFR), the
epidermal growth factor receptor (EGFR), and the
FIGURE 5. (a) Phase contrast images of capillary branching on control (without cancer cells) (i–iii) and cell laden hydrogel
scaffolds (iv–vi). The substrate stiffness was varied from 11 kPa (i, iv), to 36 kPa (ii, v), and 78 kPa (iii, vi). Arrow indicates the
branching and *indicates the branching points or nodes. Quantification of capillary network formation: (b) nodes per image and (c)
sprouts number per nodes. Error bars are SEM (n 5 3). *Represents p value 0.05.
WU et al.

9. RET-tyrosine kinase.2
By inhibiting the kinase activity
of VEGFR, this multi-targeted kinase inhibitor can
limit the growth of new blood vessels and thereby re-
strict the growth of tumor. The concentration of the
anti-angiogenic agent was varied from 0 to 2 lM.
Figure 6 demonstrates the impact of the inhibitor on
capillary network formation. As observed, incubation
with vandetanib disrupted the capillary formation on
all the three hydrogel matrices (Figs. 6a, 6b). To
compare the efficacy of the anti-angiogenic agent in
reducing sprout formation, IC50 of vandetanib was
calculated for each condition (Table 2). As can be seen,
IC50 values increased from 0.14 to 0.21 lM when the
rigidity was varied from 11 to 78 kPa. These studies
suggested that the matrix stiffness may play a signifi-
cant role in regulating the response of endothelial cells
towards angiogenic inhibitors.
To investigate the impact of activation, HUVECs
were seeded on cancer cell laden gels and incubated
with vandetanib. As demonstrated in Fig. 7, incuba-
tion with the drug reduced the numbers of capillary
branches per node from 3.8 ± 0.2 (no inhibitor) to 0
(2 lM), 4.1 ± 0.1 (no inhibitor) to 0.6 ± 0.2 (2 lM),
and 3.1 ± 0.2 (no inhibitor) to 1 ± 0.2 (2 lM) for
compliant, intermediate, and stiff gels, respectively.
Furthermore, as observed from Figs. 7a and 7b, most
dramatic effects of vandetanib was observed when
HUVECs were seeded on 11 kPa hydrogels. A pro-
found increase in IC50 value from 0.24 to 0.57 lM was
observed when the matrix rigidity was changed from
11 to 78 kPa. This observation suggests that tumor cell
activation accentuates the substrate dependent drug
sensitivity of HUVECs.
DISCUSSION
Past attempts at elucidating the mechanisms
underlying tumor vessel formation focused solely on
the differences in the canonical biochemical signaling
pathways. It is well known that the compliance of the
stroma within breast carcinomas is 5–20 times more
FIGURE 6. Effect of vandetanib on capillary branching when HUVECs were seeded on hydrogel matrices without cancer cells on
(a) sprouts per nodes and (b) nodes per image, Error bars are SEM (n 5 3). *Represents p value 0.05.
TABLE 2. IC50 of Vandetanib (lM).
Compression
modulus (kPa)
Without cancer
cells
With cancer
cells
11 0.14 0.24
36 0.16 0.36
78 0.21 0.57
Differential Effects of Tumor Secreted Factors

10. rigid than the normal breast tissue.36
In this study,
PEG based hydrogel systems were used as cell culture
platforms to assess how stimulation by cancer cells
affect the mechanosensitivity, capillary formation, and
drug sensitivity of endothelial cells.
Towards this, HUVECs were seeded on hydrogel
matrices with stiffness varying from 11 to 78 kPa in the
presence and absence of tumor conditioned media.
This matrix rigidity covers a broad range of stiffness
reported for different human tissue including breast
tissue, stromal tissue, as well as cancer tissue,10,11,35
thereby highlighting the physiological relevance of
these matrices. Our in vitro studies revealed that acti-
vation by tumor cells induced the endothelial cells to
display more elongated spindle shaped morphology as
compared to the isotropic spreading of cells in absence
of tumor stimulation. The fact that the endothelial cells
respond differently to the same mechanical environ-
ment upon stimulation by tumor cells suggests that the
endothelial cells may acquire an altered phenotype
when confronted by the tumor microenvironment. Our
observations are in agreement with earlier report which
suggested that the aberrant behavior of tumor capil-
lary endothelial cells results from the dysregulation of
mechanosensing mechanism of the cells.14
The abnor-
mal mechanosensitivity can be attributed to the higher
Rho mediated tension which disrupts the ability of
tumor capillary endothelial cells to sense and respond
to the physical cues emanating from tumor microen-
vironment.14
Though the roles of cell adhesion and matrix com-
pliances during vascularization are well defined, how
endothelial cells integrate the biophysical cues in the
context of tumor cell activation is not very clear. Any
variations in the cell–matrix force equilibrium change
the cell behavior as manifested in altered cell shape,
cell proliferation, and motility.3,5
This behavior when
repeated over space and time can lead to the formation
of tubular networks as observed in normal vasculari-
zation.17
Since, activation via tumor secreted factors
altered the responsiveness of endothelial cells to
mechanical stimuli arising from variation in substrate
compliances, we examined how the presence of tumor
cells affects capillary network formation by HUVECs.
For the purpose, HUVECs were plated on the top of
cancer cell laden hydrogel matrices. The viability of
cancer cells within the scaffolds was found to be
greater than 40%. Even though other studies have
reported viability 80% of cells encapsulated within
3D hydrogels, the discrepancy can be attributed to the
cell type as well as cell seeding density. Increasing the
seeding density from 1.6 9 104
to 6.4 9 104
cells/
scaffold, reduced the viability of MDA-MB-231 cells
from 72 to 18%. In addition, concentration of photo-
FIGURE 7. Effect of vandetanib on capillary branching when HUVECs were seeded on cell laden hydrogel on (a) sprouts per
nodes and (b) nodes per image. Error bars are SEM (n 5 3). *Represents p value 0.05.
WU et al.

11. initiator and UV exposure time can also affect the
viability of encapsulated cells. However, matrix rigid-
ity did not have any effect on the viability of the
encapsulated cancer cells. We observed that stimula-
tion by tumor cells promoted the reorganization of
endothelial cells into capillary networks. The increased
number of network formation can be attributed to the
elongated morphology exhibited by the endothelial
cells in response to tumor cell activation which in turn
reflects the propensity of the endothelial cells to
organize into vascular networks. While assessing the
quantitative relation between tumor stimulation, ma-
trix mechanics, and sprout formation, our study re-
vealed that in the absence of activation by tumor cells
maximum sprouting was observed on hydrogel matri-
ces of intermediate stiffness. Our results corroborates
with earlier studies which demonstrated that appro-
priate matrix compliance is required for optimal
sprouting.26,39
However, interestingly when stimulated
by the tumor secreted factors, preference for optimal
matrix compliance was abrogated. This result reiter-
ates the previous findings that activation by tumor cells
disrupts the ability of endothelial cells to sense and
respond to the biomechanical cues.
Since, VEGF is a ubiquitous tumor angiogenic
factor, we assessed how activation by tumor cells
affects the sensitivity of endothelial cells to vandetanib
and whether or not the drug sensitivity is dependent on
substrate stiffness. We observed that the responsive-
ness of endothelial cells to anti-angiogenic agent
showed a dependence on matrix compliance both in
the presence and absence of stimulation by tumor cells.
When HUVECs were challenged with drug treatment,
increased level of inhibition of capillary formation was
observed on the compliant gels as compared to the
stiffer matrices. It is likely that in addition to growth
factors, capillary morphogenesis is regulated by the
mechanical force balance between the cells and matrix
and thus modulated by the cytoskeletal regulatory
molecules. It is well documented that matrix stiffness
regulates the focal adhesion formation. Formation of
adhesions due to clustering of integrins to ECM
ligands in turn leads to the recruitment of growth
factor receptors within the focal adhesions.22,37
Growth factor signals synergize with integrins to acti-
vate Rho-ERK pathway thereby modulating angio-
genesis.29
In addition, GTPase Rho has been reported
to mediate cell contractility by organizing actin fila-
ments into stress fibers16,30
and regulate endothelial
cell organization during angiogenesis. It has been
suggested that the aberrant behavior of tumor capil-
lary endothelial cells correlates with a constitutively
high level of baseline activity of the small GTPase
Rho.14
The interaction between the pathways
involved in cytoskeletal reorganization and subsequent
signaling cascade may play a critical role in endothelial
cell responsiveness to the anti-angiogenic agent. Per-
haps the interplay between these signaling pathways
and the interaction of tumor cell activated endothelial
cells with the matrices of varying stiffness dictates the
efficacy of the drug.
CONCLUSIONS
We investigated the use of PEG based hydrogel
systems as cell culture platforms to understand the
impact of activation by tumor cells on mechanosensi-
tivity, ability to form capillary networks, and anti-
angiogenic drug sensitivity of endothelial cells. Our
study revealed that stimulation by tumor secreted
factors reduces the ability of the HUVECs to sense and
respond to the variation of substrate stiffness which in
turn alters the pattern of vascular network formation
by the endothelial cells. In addition, activation by
tumor cells significantly reduces the ability of vande-
tanib to inhibit the capillary network formation,
especially at higher substrate rigidity.
ACKNOWLEDGMENTS
We would like to thank University of Michigan-
Dearborn and University of Michigan-Ann Arbor:
Office of the Vice President for Research for the
financial support.
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