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 bloodvessels to promote growth and metastasis. Unlike the normalvascular networks, the tumor induced vasculatures exhibitabnormal shape and function. Past efforts have been focusedon characterizing the altered growth factor signaling path-way in tumor capillary endothelial cells; however, themechanical microenvironment of tumor also plays a signif-icant role in regulating the formation of vascular patterns.Here, we used synthetic hydrogel based cell culture platformsto probe how activation of human umbilical endothelial cells(HUVECs) by tumor secreted factors alters the responses tomatrix modulus and in turn the capillary network formationand drug sensitivity. Our study revealed that while in absenceof activation, HUVECs prefer a substrate of appropriatestiffness for optimal capillary network formation; stimula-tion by tumor cells disrupts the mechano-responsive behaviorof HUVECs. Additionally, the effect of vandetanib onreducing the capillary network was also investigated. Theresponse of HUVECs to the anti-angiogenic agent wassubstrate modulus dependent displaying increased sensitivityon the compliant gels. Stimulation by tumor cells reduced theresponsiveness to vandetanib, particularly when plated onstiffer gels.

Keywords—Angiogenesis, Hydrogel, Matrix compliance,

Tumor cell activation.

INTRODUCTION

Most solid tumors less than 1–2 mm in diameter cansurvive by passive diffusion of oxygen and nutrition.20

To grow beyond 2 mm, the primary tumors induceangiogenesis, i.e., form new vascular networks.12 Thisprocess involves activation of the endothelial cells by

several tumor derived factors including vascular endo-thelial growth factor (VEGF), basic fibroblast growthfactor (bFGF), platelet-derived growth factor (PDGF),angiopoetins (ANG), and chemokines.6,15,25,34 Thesenewly formed highly complex vascular networks canthen supply oxygen and nutrients to enhance the growthof tumor.33 Understanding the role of these growthfactors in stimulating angiogenesis facilitated thedevelopment of cancer therapies targeting these mole-cules or their receptors. However, these treatments haveonly been moderately successful producing short-termbenefits.7 This underscores the importance of betterunderstanding the mechanisms regulating tumor vas-cularization to achieve therapeutic success. A body ofevidence suggests that capillary morphogenesis andvascular pattern formation is also governed by themechanical microenvironment of tumor.4,19,32 In con-trast to the normal tissue extracellular matrix (ECM),the tumor stroma is stiffer due to increased collagendeposition, high interstitial pressure, and collagencrosslinking.9,27,31 The progressively changing physicalenvironment of the tumor stroma alters the cell-ECMinteraction force equilibrium resulting in altered cellshape, increased proliferation, and migration throughthe clustering of force-sensing integrin receptors. How-ever, it is not clear how activation via tumor secretedfactors modulates the responses of endothelial cells tomatrix rigidity and vascularization. Therefore,understanding the bidirectional crosstalk between thetumor cell-stimulated endothelial cells and ECM iscritical to evaluating the effectiveness of drugs designedfor inhibiting angiogenesis.

While the small animal models are the gold stan-dards for studying tumor vascularization, control overvarious intrinsic variables including host cells, immuneresponse, 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 (� 2015)

DOI: 10.1007/s10439-015-1254-2

� 2015 Biomedical Engineering Society

models capable of recapitulating the in vivo cellularmorphology and phenotypes are proving to beinvaluable for studying the dynamic and progressivebehavior of cancer under controlled conditions. Themajority of the existing models assess endothelialmorphogenesis in vitro when cultured on differentmatrices 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 thematrix mechanics from the porous architecture8 lead-ing to restricted diffusional transport of putativeangiogenic factors which in turn can play an importantrole in the regulation of capillary morphogenesis.13

This underscores the importance of decoupling differ-ent intertwined ECM properties, e.g., the diffusionalcharacteristics from matrix compliance to parse thespecific contributions of these microenvironmentalcues in stimulating angiogenesis.

To investigate interplay between tumor cell activationand mechano-responsive cellular behavior, we utilizedpoly (ethylene glycol) diacrylate (PEGDA) and gelatinmethacrylate (GelMA) composite hydrogels as the cellculture substrates. GelMA, obtained via conjugation ofmethacrylate groups to gelatin, provides the cell bindingmotifs, e.g., RGD as well as matrix metalloproteinase(MMP) sensitive degradation groups.28 PEG is a bio-inert polymer that resists non-specific adsorption ofproteins.18 This composite hydrogel system provides acell culture platform to evaluate the cellular responses tothe changes in themicroenvironment. The stiffness of thematrices was varied from 11 to 78 kPa to span themechanical 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 onthe top of the cell-laden gels and capillary network for-mation by HUVECs in the presence and absence ofcancer cells were evaluated. The inhibition of capillaryformation by vandetanib, a FDA approved anti-angio-genic agent, was correlated with substrate stiffness. Ourresults indicate that the activation of endothelial cells bytumor 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), gelatinfrom porcine skin, ethylene glycol, methacrylicanhydride, dimethyl sulfoxide (DMSO) fluoresceinisothiocyanate—dextran (FITC-Dextran 70 kDa), and

photo-initiator (2-Hydroxy-4¢-(2-hydroxyethoxy)-2-methylpropionphenone) were procured from SigmaAldrich (St. Louis, MO). Vandetanib was procuredfrom LC Laboratories (Woburn, MA), RPMI 1640(Roswell Park Memorial Institute medium), PenicillinStreptomycin L-Glutamine (Pen Strep) and Fetal bo-vine serum (FBS) from Gibco� (Grand Island, NY).LIVE/DEAD� Cell Viability assay-kit was purchasedfrom Life Technologies (Grand Island, NY).

Synthesis of Methacrylated Gelatin

Gelatin methacrylate was synthesized as describedelsewhere.38 Methacrylation was achieved by adding10% (w/v) of methacrylic anhydride to 10% (w/v)gelatin solution at a rate 0.5 mL/min and reacting at60 �C for 1 h. Following a 59 dilution with warmDPBS (50 �C), the mixture was dialyzed using Slide-A-lyzer against distilled water at 50 �C for 7 days. Thesample was then freeze dried and GelMA was gener-ated as porous foam and stored at 280 �C until furtheruse.

Fabrication and Characterization of Hydrogel Matrices

The scaffolds were fabricated by adding 150 lL ofthe pre-polymer solution in 48 well plates and thenexposing the plates to UV (CL-1000 UV Crosslinker(UVP), 365 nm) for 5 min. The pre-polymer solutionconsisted 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 thehydrogels, the scaffolds were fabricated by incorpo-rating FITC-dextran 70 kDa within the precursorsolution. The concentration of dextran within eachsample 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 andreplaced with fresh PBS. All the collected samples wereanalyzed using PerkinElmer LS55 fluorescence spec-trometer to assess the release of FITC.

For hydrogel samples with thickness much smallerthan the diameter, the diffusion of dextran from the gelphase to the surrounding can be considered as onedimensional. To determine the mechanism of thetransport of these macromolecules, the dextran release

WU et al.

data were fitted into Korsmeyer–Peppas transportmodel given by the Eq. (1):

F ¼ Mt

Mo¼ k1t

n ð1Þ

where the (F) defined as Mt/Mo is the fractional releaseof the molecule, Mt is the amount of dextran releasedat any given time, Mo is the total mass of dextranencapsulated within the hydrogel matrices, k1 is thekinetic constant (s2n), t is the release time (s), and n isthe diffusional exponent, which informs the mecha-nism of diffusional release. For n £ 0.5, the transportof the molecules can be defined by Fick’s law. The datafitting was performed on the first 60% cumulative re-lease, i.e., Mt/Mo = 0.6. The effective diffusivity of themacromolecules is related to the cumulative releaseaccording to the Eq. (2):

Mt=Mo ¼ 4 � Dt

pL2

� �n

ð2Þ

Effective diffusivity (D, cm2/s) can be then related toKorsmeyer–Peppas constants

D ¼ pL2 k1=4ð Þ1=n ð3Þ

Cell Culture

HUVECs were purchased from American TypeCulture Collection (ATCC, Manassas, VA) and ex-panded in vascular basal medium with endothelialgrowth supplement (Ascorbic, FBS, rh EGF, heparinsulfate, 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 confluentcells were trypsinized to detach them from the flasksurfaces, counted using cell automated counter (Bio-Rad TC10TM), and centrifuged to obtain cell pellets.To fabricate cancer cell impregnated scaffolds, cancercells were suspended in 30 lL of RPMI and mixed with120 lL pre-polymer solutions. The cancer cell seedingdensity 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 theMDA-MB-231 cell line when the flask was over 70%of confluent. The conditioned media was filtered andstored at 4 �C for future use. 5 9 103 HUVECs wereseeded on scaffolds of different compliances. Toinvestigate the spreading of HUVECs, the cells wereseeded on scaffolds of varying stiffness and incubated

with either HR (50% HUVECs media and 50% RPMImedia) or HCR (50% HUVECs media and 50%RPMI conditioned media). The images were captured18 h after seeding using Zeiss Axio Observer A1 withintegrated CCD camera. The morphology of the cellswas assessed based on aspect ratio which indicates theratio between cell width and cell length.

Viability of Encapsulated Cancer Cells

To investigate the viability of the cancer cells withinPEG matrices, cells were encapsulated within thescaffolds at a density of 3.2 9 104 cells/scaffold andincubated for 24 h. Viability of the cancer cells wasassessed by using LIVE/DEAD� Viability/Cytotoxic-ity assay kit as per the manufacturer’s instructions.

Characterization of Capillary Formation

To investigate the impact of activation by tumorcells on capillary formation, 5 9 104 HUVECs wereseeded on the scaffolds with and without cancer cells(Fig. 1). 3.2 9 104 cancer cells were encapsulatedwithin 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 countingthe number of networks branching out from a branchpoint/node and number of nodes per image. To eval-uate the ability of vandetanib to inhibit the capillarymorphogenesis, 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 to2 lM. The images of the branches were captured 8 hpost-treatment with the anti-angiogenic agent.

Statistical Analysis

For all experiments, data reported as mean ± SEMof three independent experiments. Statistical analyseswere carried out with one way ANOVA. Differencesbetween two sets of data were considered significant atp value <0.05.

RESULTS

Integrated Effects of Substrate Rigidity and TumorSecretory Factors on Cell–Matrix Interactions

To assess the effects of tumor secreted stimulatoryfactors on endothelial cell responses to increasingmatrix stiffness, the hydrogel compliances were

Differential Effects of Tumor Secreted Factors

manipulated by varying the concentration of PEGDAfrom 5 to 15% while maintaining uniform presentationof cell adhesion molecules (i.e., constant gelatin con-centration) (Fig. 2a). Alterations in the potentialcrosslinking groups per unit volume increased thehydrogel modulus from 11.4 ± 0.72 kPa for 5% PEG-DA, to 35.8 ± 2.52 for 10% PEGDA, and 78 ± 5.03for 15% PEGDA.38 Scanning electron microscopy(SEM) analysis revealed that the increase in modulushadminimal effect on the porosity of the gels.38 Increasein crosslinking density which alters the free volumeavailable for transport attenuates the diffusion ofmacromolecules through the hydrogels. So, to investi-gate whether increasing the PEGDA concentration inthe gels affect the macromolecular release kinetics,hydrogel disks were fabricated encapsulating FITCconjugated (70 kDA) dextran. Figure 2b demonstratesthe cumulative release (% released) of dextran into thesolution for each crosslinking density over a span of24 h. No significant difference was observed in thedextran release profile from these hydrogel disks, indi-cating that the transport of dextran molecules wasindependent of the crosslinking density over the rangeof PEGDA studied. Further analysis using Korsmeyer–Peppas model confirmed the crosslinking density inde-pendent nature of the dextran transport throughPEGDA hydrogels as no significant difference wasobserved in the fitting parameters (p value >0.05) fordifferent matrices (Table 1). The average values of dif-fusional exponent (n) for the three different crosslinkingdensity were found to be less than 0.5 indicating that the

transport mechanism is Fickian diffusion. Effectivediffusivity was calculated from Eq. (3). As demon-strated in Fig. 2c, no correlation was observed betweeneffective 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 for15% PEGDA.

To evaluate the effect of tumor secretory factors onendothelial cell–matrix interactions, HUVECs wereseeded on the top of the hydrogels with differentcompliances 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 ofconditioned media, the increase in matrix rigidity from11 to 36 kPa altered the aspect ratio of the cells (cellwidth/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 of0.65 ± 0.03 was obtained, suggesting that the cellsplated on the stiffer matrices had less elongated mor-phology (p value <0.05) and displayed more isotropicspreading. In the presence of cancer cell conditionedmedia, 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 thecells to adopt more elongated spindle shapedmorphology.

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 oncapillary network formation were then monitored.

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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

Cancer Cell Viability Within Three DimensionalHydrogel Matrices

To investigate the viability of cancer cells within thePEG hydrogels, MDA-MB-231 cells were encapsu-lated within the matrices of varying stiffness. Theseeding density was 3.2 9 104 cells/scaffold. Figure 4aillustrates a typical live dead image of the encapsulatedcancer cells. For all the conditions, MDA-MB-231exhibited 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 CellLaden PEG Gels

To understand how HUVECs interpret, assimilate,and integrate the biochemical and mechanical signalsfrom microenvironment to direct capillary networkformation, MDA-MB-231 cells were encapsulatedwithin PEGgels of varying stiffness.HUVECswere 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 andtumor 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.

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seeded on the top of the cell laden gels. To assess thecontribution of matrix mechanics, HUVECs were alsoseeded on the top of the gels without encapsulatedcancer cells. Figure 5a illustrates the capillary networkformation in the presence andabsence ofMDA-MB-231cells. To quantify the capillary branching, number ofnodes per image and number of sprouts branching outfrom individual nodes were calculated (Figs. 5b, 5c). Asdemonstrated in Fig. 5b, in the absence of tumor stim-ulationmaximumnumber of nodes (6.8 ± 0.5 nodes perimage) were observed on hydrogel of intermediatestiffness as compared to compliant (5.3 ± 0.40 nodesper image) and stiff (4.0 ± 0.3 nodes per image) gels.Similarly, maximum capillary branching (3.6 ± 0.3branches per node) was observed when HUVECs wereseeded on the hydrogelmatrices of intermediate stiffness(36 kPa) as compared to the compliant (11 kPa) andstiff (78 kPa) gels (p value <0.05). To investigate whe-ther hydrogel matrices of intermediate stiffness promotemaximal branching, HUVECs were seeded on 28 kPascaffolds. No significant difference in capillary branch-ing per nodes (2.7 ± 0.2 vs. 2.4 ± 0.1) or nodes perimage (5.3 ± 0.40 vs. 5.1 ± 0.30) was observed between11 and 28 kPa. These observations suggested that opti-mal matrix stiffness enhanced capillary morphogenesisof endothelial cells.

Next, HUVECs were seeded on gels impregnatedwith MDA-MB-231 cells. Since, no significant differ-ence was observed in endothelial tubulogenesis on 11and 28 kPa scaffolds, MDA-MB-231 cells wereencapsulated within 11, 36, and 78 kPa matrices. In thepresence of cancer cells, number of branching pointsincreased on 11 kPa gels (6.9 ± 0.2 nodes per image).However, no significant difference was observed in thecase of intermediate and stiff matrices (p value <0.5).On the other hand, endothelial cell assembly increasedto 3.8 ± 0.2 capillary branches per node for compliantgels, 4.1 ± 0.1 for gels with intermediate stiffness, and3.1 ± 0.2 for stiffer gels. The network formation oncompliant and intermediate gels in presence of MDA-MB-231 cells was significantly higher (p value <0.05)from control; however, no significant difference wasobserved when HUVECs were plated on cell laden stiffgels (p value >0.05).

Integrated Effects of Substrate Stiffness and Tumor CellActivation on Drug Sensitivity of Endothelial Cells

To evaluate how matrix rigidity regulates the sen-sitivity of endothelial cells to drug treatment, HUVECswere 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 liveand dead cells encapsulated within (i) 11 kPa, (ii) 36 kPa and (iii) 78 kPa scaffold. (b) Quantification of live cells within the threedimensional PEG matrices. Error bars are SEM (n 5 3). *Represents p value <0.05.

Differential Effects of Tumor Secreted Factors

vandetanib for 8 h. Vandetanib is a FDA approvedanti-angiogenic agent used for treating solid cancersincluding thyroid cancer. It works as a kinase inhibitor

of several receptor tyrosine kinases including the vas-cular endothelial growth factor receptor (VEGFR), theepidermal 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 hydrogelscaffolds (iv–vi). The substrate stiffness was varied from 11 kPa (i, iv), to 36 kPa (ii, v), and 78 kPa (iii, vi). Arrow indicates thebranching 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.

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RET-tyrosine kinase.2 By inhibiting the kinase activityof VEGFR, this multi-targeted kinase inhibitor canlimit the growth of new blood vessels and thereby re-strict the growth of tumor. The concentration of theanti-angiogenic agent was varied from 0 to 2 lM.Figure 6 demonstrates the impact of the inhibitor oncapillary network formation. As observed, incubationwith vandetanib disrupted the capillary formation onall the three hydrogel matrices (Figs. 6a, 6b). Tocompare the efficacy of the anti-angiogenic agent inreducing sprout formation, IC50 of vandetanib wascalculated for each condition (Table 2). As can be seen,IC50 values increased from 0.14 to 0.21 lM when therigidity was varied from 11 to 78 kPa. These studiessuggested that the matrix stiffness may play a signifi-cant role in regulating the response of endothelial cellstowards angiogenic inhibitors.

To investigate the impact of activation, HUVECswere seeded on cancer cell laden gels and incubatedwith vandetanib. As demonstrated in Fig. 7, incuba-tion with the drug reduced the numbers of capillarybranches 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) forcompliant, intermediate, and stiff gels, respectively.

Furthermore, as observed from Figs. 7a and 7b, mostdramatic effects of vandetanib was observed whenHUVECs were seeded on 11 kPa hydrogels. A pro-found increase in IC50 value from 0.24 to 0.57 lM wasobserved when the matrix rigidity was changed from11 to 78 kPa. This observation suggests that tumor cellactivation accentuates the substrate dependent drugsensitivity of HUVECs.

DISCUSSION

Past attempts at elucidating the mechanismsunderlying tumor vessel formation focused solely onthe differences in the canonical biochemical signalingpathways. It is well known that the compliance of thestroma 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

rigid than the normal breast tissue.36 In this study,PEG based hydrogel systems were used as cell cultureplatforms to assess how stimulation by cancer cellsaffect the mechanosensitivity, capillary formation, anddrug sensitivity of endothelial cells.

Towards this, HUVECs were seeded on hydrogelmatrices with stiffness varying from 11 to 78 kPa in thepresence and absence of tumor conditioned media.This matrix rigidity covers a broad range of stiffnessreported for different human tissue including breasttissue, stromal tissue, as well as cancer tissue,10,11,35

thereby highlighting the physiological relevance ofthese matrices. Our in vitro studies revealed that acti-vation by tumor cells induced the endothelial cells todisplay more elongated spindle shaped morphology ascompared to the isotropic spreading of cells in absenceof tumor stimulation. The fact that the endothelial cellsrespond differently to the same mechanical environ-ment upon stimulation by tumor cells suggests that theendothelial cells may acquire an altered phenotypewhen confronted by the tumor microenvironment. Ourobservations are in agreement with earlier report whichsuggested that the aberrant behavior of tumor capil-lary endothelial cells results from the dysregulation ofmechanosensing mechanism of the cells.14 The abnor-mal mechanosensitivity can be attributed to the higherRho mediated tension which disrupts the ability of

tumor capillary endothelial cells to sense and respondto the physical cues emanating from tumor microen-vironment.14

Though the roles of cell adhesion and matrix com-pliances during vascularization are well defined, howendothelial cells integrate the biophysical cues in thecontext of tumor cell activation is not very clear. Anyvariations in the cell–matrix force equilibrium changethe cell behavior as manifested in altered cell shape,cell proliferation, and motility.3,5 This behavior whenrepeated over space and time can lead to the formationof tubular networks as observed in normal vasculari-zation.17 Since, activation via tumor secreted factorsaltered the responsiveness of endothelial cells tomechanical stimuli arising from variation in substratecompliances, we examined how the presence of tumorcells affects capillary network formation by HUVECs.For the purpose, HUVECs were plated on the top ofcancer cell laden hydrogel matrices. The viability ofcancer cells within the scaffolds was found to begreater than 40%. Even though other studies havereported viability >80% of cells encapsulated within3D hydrogels, the discrepancy can be attributed to thecell type as well as cell seeding density. Increasing theseeding density from 1.6 9 104 to 6.4 9 104 cells/scaffold, reduced the viability of MDA-MB-231 cellsfrom 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 pernodes and (b) nodes per image. Error bars are SEM (n 5 3). *Represents p value <0.05.

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initiator and UV exposure time can also affect theviability of encapsulated cells. However, matrix rigid-ity did not have any effect on the viability of theencapsulated cancer cells. We observed that stimula-tion by tumor cells promoted the reorganization ofendothelial cells into capillary networks. The increasednumber of network formation can be attributed to theelongated morphology exhibited by the endothelialcells in response to tumor cell activation which in turnreflects the propensity of the endothelial cells toorganize into vascular networks. While assessing thequantitative relation between tumor stimulation, ma-trix mechanics, and sprout formation, our study re-vealed that in the absence of activation by tumor cellsmaximum sprouting was observed on hydrogel matri-ces of intermediate stiffness. Our results corroborateswith earlier studies which demonstrated that appro-priate matrix compliance is required for optimalsprouting.26,39 However, interestingly when stimulatedby the tumor secreted factors, preference for optimalmatrix compliance was abrogated. This result reiter-ates the previous findings that activation by tumor cellsdisrupts the ability of endothelial cells to sense andrespond to the biomechanical cues.

Since, VEGF is a ubiquitous tumor angiogenicfactor, we assessed how activation by tumor cellsaffects the sensitivity of endothelial cells to vandetaniband whether or not the drug sensitivity is dependent onsubstrate stiffness. We observed that the responsive-ness of endothelial cells to anti-angiogenic agentshowed a dependence on matrix compliance both inthe presence and absence of stimulation by tumor cells.When HUVECs were challenged with drug treatment,increased level of inhibition of capillary formation wasobserved on the compliant gels as compared to thestiffer matrices. It is likely that in addition to growthfactors, capillary morphogenesis is regulated by themechanical force balance between the cells and matrixand thus modulated by the cytoskeletal regulatorymolecules. It is well documented that matrix stiffnessregulates the focal adhesion formation. Formation ofadhesions due to clustering of integrins to ECMligands in turn leads to the recruitment of growthfactor 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 reportedto mediate cell contractility by organizing actin fila-ments into stress fibers16,30 and regulate endothelialcell organization during angiogenesis. It has beensuggested that the aberrant behavior of tumor capil-lary endothelial cells correlates with a constitutivelyhigh level of baseline activity of the small GTPaseRho.14 The interaction between the pathwaysinvolved in cytoskeletal reorganization and subsequent

signaling cascade may play a critical role in endothelialcell responsiveness to the anti-angiogenic agent. Per-haps the interplay between these signaling pathwaysand the interaction of tumor cell activated endothelialcells with the matrices of varying stiffness dictates theefficacy of the drug.

CONCLUSIONS

We investigated the use of PEG based hydrogelsystems as cell culture platforms to understand theimpact of activation by tumor cells on mechanosensi-tivity, ability to form capillary networks, and anti-angiogenic drug sensitivity of endothelial cells. Ourstudy revealed that stimulation by tumor secretedfactors reduces the ability of the HUVECs to sense andrespond to the variation of substrate stiffness which inturn alters the pattern of vascular network formationby the endothelial cells. In addition, activation bytumor 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 thefinancial support.

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