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Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma Premal H Thaker1,10, Liz Y Han1,10, Aparna A Kamat1,10, Jesusa M Arevalo2, Rie Takahashi2, Chunhua Lu1, Nicholas B Jennings1, Guillermo Armaiz-Pena1, James A Bankson3, Murali Ravoori4, William M Merritt1, Yvonne G Lin1, Lingegowda S Mangala1, Tae Jin Kim1, Robert L Coleman1, Charles N Landen1, Yang Li1, Edward Felix5, Angela M Sanguino6, Robert A Newman5, Mary Lloyd7, David M Gershenson1, Vikas Kundra4,8, Gabriel Lopez-Berestein6, Susan K Lutgendorf 9, Steven W Cole2 & Anil K Sood1,7

Stress can alter immunological, neurochemical and

endocrinological functions, but its role in cancer progression

is not well understood. Here, we show that chronic behavioral

stress results in higher levels of tissue catecholamines, greater

tumor burden and more invasive growth of ovarian carcinoma

cells in an orthotopic mouse model. These effects are mediated

primarily through activation of the tumor cell cyclic AMP

(cAMP)–protein kinase A (PKA) signaling pathway by the b2 adrenergic receptor (encoded by ADRB2). Tumors in stressed

animals showed markedly increased vascularization and

enhanced expression of VEGF, MMP2 and MMP9, and we

found that angiogenic processes mediated the effects of stress

on tumor growth in vivo. These data identify b-adrenergic activation of the cAMP–PKA signaling pathway as a major

mechanism by which behavioral stress can enhance tumor

angiogenesis in vivo and thereby promote malignant cell

growth. These data also suggest that blocking ADRB-mediated

angiogenesis could have therapeutic implications for the

management of ovarian cancer.

Epidemiologic and experimental animal studies have shown that stress may alter tumor growth1–5. However, the biological mechanisms underlying such effects are not well understood, and their clinical significance for human disease remains controversial as a result. Data from animal models suggest that stress can modulate the growth of certain tumors via neuroendocrine regulation of the immune response to tumor cells1. However, the uncertain role of the immune system in controlling solid tumors led us to consider an alternative hypothesis: stress mediators from the sympathetic nervous system might directly modulate the growth and malignant behavior of tumor cells, inde- pendent of effects on the immune system. Based on our prior studies linking behavioral factors to circulating VEGF levels in vivo and

showing catecholamine regulation of tumor cell VEGF production in vitro6,7, we sought to determine whether sympathetic nervous system activity might mediate a causal effect of stress on the growth and metastasis of ovarian cancer in vivo. We also sought to define the role of angiogenic cytokines in mediating those effects.

These studies used an established orthotopic mouse model in which human ovarian carcinoma cells are inoculated into the peritoneal cavity of nude mice8. We then experimentally stressed animals with a new physical restraint system that we designed, in which periodic immobilization induces high levels of hypothalamic-pituitary-adrenal and sympathetic nervous system activity characteristic of chronic stress (Supplementary Fig. 1 online). We first examined the effects of stress on tumor growth and metastasis of HeyA8 ovarian cancer cells inoculated 7 d after the initiation of stress. In mice receiving 0, 2 or 6 h of immobilization daily for 21 d, the number of tumor nodules increased by 259% in the 2-h stress group (P ¼ 0.005, two-tailed Student’s t-test) and 356% in the 6-h stress group (P o 0.001; Fig. 1a). Mean tumor weight increased 242% in the 2-h stress group (P ¼ 0.01) and 275% in the 6-h group (P ¼ 0.005). Tumor growth was confined to the peritoneal cavity in all control mice, but it spread to the parenchyma of the liver or spleen in 50% of stressed mice (P ¼ 0.01). The number and weight of tumor nodules did not differ between the 2- and 6-h stress groups. Therefore, we used the 2-h restraint for all subsequent experiments. Additional experiments with the SKOV3ip1 ovarian cancer cell line and the MB-231 orthotopic breast cancer model (Fig. 1b) showed that stress effects on tumor growth were evident in a wide range of tumor cell lines (significant differences in mean tumor weight and nodule number, all P o 0.01; Fig. 1b). Subsequent studies using social isolation as an alternative stressor also showed a 187% increase in tumor weight (P o 0.01) and a 255% increase in nodule count (P o 0.001) compared with group- housed control animals (Fig. 1c). We also documented longitudinal

Received 12 April; accepted 21 June; published online 23 July 2006; doi:10.1038/nm1447

1Department of Gynecologic Oncology, University of Texas (U.T.) M.D. Anderson Cancer Center, 1155 Herman Pressler, Unit 1362, Houston, Texas 77030, USA. 2Division of Hematology-Oncology, Department of Medicine, 11-934 Factor Building, University of California Los Angeles (UCLA) School of Medicine, Los Angeles, California 90095, USA. 3Department of Imaging Physics and 4Department of Experimental Diagnostic Imaging, U.T. M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. 5Department of Experimental Therapeutics, U.T. M.D. Anderson Cancer Center, 8000 El Rio Street, Houston, Texas 77054, USA. 6Department of Experimental Therapeutics, 7Department of Cancer Biology and 8Department of Diagnostic Radiology, U.T. M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. 9Department of Psychology, University of Iowa, E228 Seashore Hall, Iowa City, Iowa 52241, USA. 10These authors contributed equally to this work. Correspondence should be addressed to A.K.S. ([email protected]).

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development of stress-induced differences in tumor mass using a subcutaneous ovarian cancer model (Supplementary Fig. 2 online).

To assess the effects of stress on sympatho-adrenal-medullary activity, we measured the size of both adrenal glands9. In animals inoculated with HeyA8 cells, the left adrenal gland diameter averaged 1.85 ± 0.25 mm for the control group versus 2.88 ± 0.56 mm for the stressed group (P ¼ 0.003, with similar differences noted for right adrenal; data not shown). Based on our previous in vitro studies showing expression of b-adrenoreceptors on ovarian cancer cells7, we performed a series of experiments to evaluate their role in mediating stress effects on tumor growth. Beginning 4 d after tumor cell injection, we treated daily mice bearing HeyA8 ovarian cancer cells with either (i) phosphate-buffered saline (PBS), (ii) 10 mg/kg iso- proterenol (a nonspecific b-receptor agonist (hereafter ‘b agonist’)), (iii) 5 mg/kg terbutaline (a b2 agonist), (iv) 1 mg/kg xamoterol (a b1 agonist), or (v) isoproterenol plus 2 mg/kg of the nonspecific b antagonist propranolol. Isoproterenol increased the mean number of tumor nodules by 341% (P o 0.001) and the average tumor weight by 266% (P ¼ 0.001; Fig. 1d). We noted a similar increase in tumor

burden with the b2 agonist terbutaline but not the b1 agonist xamoterol. Propranolol blocked the isoproterenol-induced increase in tumor growth (Fig. 1d), showing that b-adrenergic signaling is essential for that effect. Propranolol also completely blocked the effects of immobilization stress on tumor growth, indicating a critical role for b-adrenergic signaling in behavioral stress effects on tumor growth (Fig. 1e). Among stressed animals, we observed invasive disease such as parenchymal metastasis to the liver, spleen, pancreas and diaphragm in 50% of the placebo-treated group, compared with 0% of the propranolol-treated group (P ¼ 0.01).

To confirm the relevance of b-adrenergic receptors (encoded by the ADRB genes) for stress-induced tumor growth, we screened 17 additional epithelial ovarian cancer cell lines and identified two (A2780 and RMG-II) that were negative for both ADRB1 and ADRB2, as shown by RT-PCR, western blot and lack of intracellular cAMP response to norepinephrine or isoproterenol (data not shown). In contrast to other ovarian cancer cells, stress did not significantly affect tumor weight, number of nodules or the pattern of spread in mice injected with ADRB1- and ADRB2-negative A2780 or RMG-II cells (Fig. 1f). To further define the specific b-adrenergic receptor subtype responsible for stress effects and assess the relative role of host (mouse) versus tumor (human) adrenergic receptors in this model, we used short interfering RNA (siRNA) specific for human ADRB1 or ADRB2 to selectively inhibit tumor cell adrenoreceptor expression (Fig. 1g). To facilitate in vivo distribution, we incorporated the siRNA into a neutral liposome, DOPC10. Both control and ADRB1 siRNA- DOPC failed to block stress-induced enhancement of tumor weight and nodules (stress effects continued to be significant at P o 0.01), but human ADRB2 siRNA-DOPC efficiently blocked stress-mediated enhancement of tumor cell growth (Fig. 1h). Together with data from b-adrenoreceptor–negative cell lines, these results identify ADRB2 receptors on the human tumor cell as the critical mediator of stress effects in this in vivo system.

Based on our previous in vitro data showing that catecholamines can promote VEGF production by ovarian cancer cells7, we evaluated the role of angiogenesis in stress effects on in vivo tumor growth. In the HeyA8 model, stress significantly enhanced mean vessel density (MVD) counts (P o 0.001), and that effect was blocked by the b antagonist propranolol (Fig. 2a). We observed similar changes in MVD counts in tumors from non-stressed animals treated with the b agonists isoproterenol or terbutaline, which had significantly higher

0 5 10 Number of nodules

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Figure 1 Effect of chronic stress on in vivo ovarian cancer growth.

(a) Quantification of tumor weights and tumor nodules in control mice and

in mice stressed for 2 h or 6 h daily that were injected intraperitoneally with

HeyA8 ovarian cancer cells. (b) Confirmation of the results in mice injected

with two different b-adrenoreceptor–positive ovarian cancer cell lines (HeyA8 and SKOV3ip1) intraperitoneally and MB-231 breast cancer cells injected

into the mammary fat pad. (c) Effects of social isolation stress on the HeyA8

ovarian cancer model. (d) The effects of immobilization are mimicked by the

chemical stressor isoproterenol (a nonspecific b agonist) and terbutaline (b2 agonist) but not by xamoterol (b1 agonist). Mice treated with both isoproterenol plus propranolol (b blocker) have a tumor burden similar to non-stressed mice. (e) Propranolol counteracts the effects of chronic stress,

confirming the importance of the b-adrenoreceptors. (f) Mice injected with b-adrenoreceptor–null ovarian cancer cell lines (A2780 and RMG-II) did not have accelerated tumor growth despite being chronically stressed. (g) Western blot of lysate from orthotopic HeyA8 tumors collected 48 h and

96 h after a single dose of siRNA (either control siRNA or siRNA targeted

to ADRB1 (b1) or ADRB2 (b2)) in DOPC liposomes. The targeted receptors were downregulated. (h) Effects of ADRB1- and/or ADRB2-targeted siRNA

on stress-induced in vivo HeyA8 tumor growth. Results represent the mean ±

s.e.m.; n ¼ 10 mice per group. *P r 0.01; **P r 0.001.

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MVD counts than did controls (P o 0.001); propranolol also blocked those effects (Fig. 2b). Stress-induced increases in angiogenesis were accompanied by significant elevation of VEGF protein (as shown by ELISA; P o 0.001) and mRNA (as shown by in situ hybridization; P o 0.001) within tumor tissue (Fig. 2a). We observed similar increases in vessel density and VEGF levels in the ADRB-positive MB-231 breast cancer model (Supplementary Fig. 3 online) and the HeyA8 model with social isolation stress (Supplementary Fig. 3 online), but not in the ADRB-null RMG-II tumors (Supplementary Fig. 4 online). Immunohistochemistry in the HeyA8 restraint model also demonstrated stress-induced increases in levels of bFGF, MMP-2 and MMP-9 proteins (Supplementary Fig. 5 online).

We carried out several additional studies to assess more compre- hensively the effects of stress on functional and anatomic character- istics of tumor vasculature. In an established Matrigel plug assay (Fig. 3a), exogenous VEGF (0.25 nM) enhanced angiogenesis as expected (P o 0.01, Fig. 3b), and conditioned medium from norepinephrine-treated tumor cells also significantly enhanced angio- genesis (P o 0.01) (Fig. 3a,b). Both catecholamines had modest effects on in vitro migration and tube formation of mouse mesenteric

endothelial cells11, but these effects were much smaller than those observed when endothelial cells were exposed to conditioned medium from norepinephrine-treated tumor cells (P o 0.001; Supplementary Fig. 6 online). Consistent with a key mechanistic role of ADRB regulation of VEGF, the stimulatory effects of norepinephrine-treated tumor cell conditioned medium was efficiently blocked by PTK787 (inhibits VEGFR2 and other tyrosine kinases) or by pretreatment of tumor cells with propranolol before norepinephrine treatment for collection of conditioned medium (Supplementary Fig. 6). These results paralleled confocal microscopy studies showing that tumors from stressed animals contained more tortuous and numerous blood vessels than controls (Fig. 3c). This was accompanied by a 24% decrease (P o 0.01) in the proportion of blood vessels with pericyte coverage in tumors from stressed animals (Fig. 3d), suggesting a more immature vasculature. In addition, in vivo magnetic resonance ima- ging and kinetic analysis showed stress-induced increases in tumor size (Fig. 3e) and an increase in the volume transfer constant between plasma and the extravascular extracellular space (Ktrans, a reflection of flow and permeability; P o 0.05; Fig. 3f). Tumors from control mice showed a higher return rate constant between the extravascular

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Figure 2 Angiogenesis is increased in chronically stressed animals. (a) HeyA8 tumor samples from control and stressed animals treated with placebo or

propranolol were stained for VEGF and CD31 (for quantification of mean vessel density (MVD) counts) by immunohistochemistry. We quantified tumor

VEGF levels using an ELISA kit. We also stained tumor samples for VEGF by in situ hybridization (ISH) and calculated optical density (OD) measurements.

(b) HeyA8 tumor samples from mice treated with a placebo, isoproterenol (nonspecific b agonist), terbutaline (b2 agonist), xamoterol (b1 agonist), or isoproterenol plus propranolol were stained for CD31 and VEGF by immunohistochemistry and ISH. We quantified tumor VEGF protein levels by ELISA. MVD was higher in the isoproterenol and terbutaline groups than in controls or in the combination therapy group. All photographs were taken at 200�. The bars in the graphs correspond sequentially to the labeled columns of images at left. Error bars represent s.e.m. *P o 0.001.

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extracellular space and blood plasma (kep, the return rate of contrast to plasma; Fig. 3g). In comparison, the volume of the extravascular extracellular space per unit volume of tissue (ve) was greater in the stressed animals (Fig. 3h).

Given that behavioral stress induced significant anatomical and functional alterations in tumor vasculature, we sought to determine whether VEGF activity had a key role in mediating stress-induced acceleration in tumor growth. Stress effects on the number and weight of HeyA8 tumor nodules were completely blocked by co-administra- tion of either the VEGF-R2 inhibitor PTK787 (50 mg/kg orally, daily) or the monoclonal VEGF-specific antibody bevacizumab (5 mg/kg intraperitoneally twice per week) (Fig. 4a,b and Supplementary Fig. 7 online).

To more clearly define the mechanism by which b-adrenergic signaling regulates VEGF production, we examined transcriptional

changes in activity of the VEGF gene after catecholamine stimulation. In SKOV3ip1 cells, VEGF mRNA expression increased by a peak 842% 1.5 h after exposure to norepinephrine (Fig. 4c), and promoter activity increased by 1,240% (Fig. 4d). This effect was blocked by propranolol, was mimicked by isoproterenol and was not observed in ADRB-negative A2780 ovarian cancer cells, implicating b-adrenoreceptors as a key mediator (Fig. 4c and data not shown). Subsequent studies showed that b-adrenergic stimulation modulates VEGF gene expression via the cAMP–protein kinase A (PKA) signal- ing pathway; the adenylyl cyclase activator forskolin (1 mM) and the PKA activator dibutyryl cAMP (db-cAMP; 1 mM) also stimulated

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Figure 3 Effects of chronic stress on angiogenesis. (a) Representative images of Matrigel containing either serum-free SKOV3ip1 conditioned medium (control), VEGF (0.25 nM), or serum-free conditioned medium from norepinephrine-

treated SKOV3ip1 cells (SKOV3ip1 NE CM). Newly formed blood vessels in the Matrigel are indicated by arrowheads.

(b) We excised the Matrigel plugs and used them for quantification of angiogenesis by measuring the hemoglobin (Hgb)

content in the Matrigel matrix. *P o 0.01 versus control. (c) We visualized tumor vasculature from HeyA8 tumors in control and stressed animals by confocal fluorescence microscopy after perfusion with FITC-lectin. (d) We examined

vessel maturation by determining the extent of pericyte coverage in tumors from control and chronic stress-exposed

animals using triple immunofluorescence (CD31 (red) for endothelial cells, desmin (green) for pericytes and Hoechst

(blue) for nuclei). *P o 0.01. (e) Magnetic resonance imaging (MRI) was used to evaluate the functional characteristics of the tumor vasculature. Representative in vivo axial images of intraperitoneal HeyA8 ovarian tumors (arrowheads) from control and stressed animals were acquired in a 4.7-T scanner

using a T2-weighted fast spin echo sequence (FSE) and respiratory gating (day 18 post-inoculation with HeyA8 cells). Vascular parameters derived from

dynamic contrast–enhanced MRI included (f) Ktrans, the volume transfer constant between blood plasma and extravascular extracellular space, (g) kep, the

rate constant between extravascular extracellular space and blood plasma and (h) ve, the volume of extravascular extracellular (interstitial) space per unit

volume of tissue. We made measurements from the enhancing periphery of each tumor (n ¼ 8 per group; *P o 0.05). Error bars represent s.e.m.

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Figure 4 Effect of VEGF on stress-induced tumor growth. (a) HeyA8-injected

mice were randomly assigned to one of the following four groups (4 d after tumor cell injection; n ¼ 10 per group): control, oral placebo with stress, 50 mg/kg oral PTK787 (VEGF-R2 inhibitor) daily with no stress, or PTK787

with stress. (b) HeyA8-injected mice were randomly assigned to one of the

following four groups (4 d after tumor cell injection; n ¼ 10 per group): control, oral placebo with stress, bevacizumab (VEGF-specific antibody;

5 mg/kg intraperitoneally, twice per week) with no stress, or bevacizumab

with stress. Treatment with PTK787 or bevacizumab blocked the stress-

induced increase in tumor weight and number of nodules compared with

treatment with the oral placebo (PBS) plus stress. (c) VEGF mRNA

increased significantly when SKOV3ip1 cells were stimulated with

norepinephrine (1 mM), isoproterenol (1 mM), forskolin (activator of cAMP) (1 mM), or dibutyryl cAMP (db-cAMP) (1 mM). KT5720 (1 mM) is a selective inhibitor of the cAMP-dependent protein kinase A and blocked VEGF

induction by norepinephrine and forskolin. (d) In SKOV3ip1 cells, the VEGF

promoter activity was increased by 1,280% after norepinephrine treatment

compared with vehicle control. *P r 0.01; **P r 0.001.

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VEGF mRNA (Fig. 4c), and the PKA inhibitor KT5720 could efficiently block VEGF mRNA induction by norepinephrine and forskolin (Fig. 4c).

Together, these results demonstrate a complete pathway by which behavioral stress can directly regulate the growth of ovarian carcino- mas in vivo. Stress-induced release of catecholamines can activate ADRB2 on ovarian carcinoma cells to trigger increased expression of the VEGF gene, which results in enhanced tumor vascularization and more aggressive growth and spread of malignant cells. In addition to providing the first experimental demonstration that behavioral stres- sors can enhance the pathogenesis of ovarian carcinoma in vivo, these data also suggest new approaches for adjuvant therapy in metastatic disease (for example, b-blockade).

The present study also expands our understanding of the general pathways by which stress can regulate cancer pathogenesis. Previous research emphasized the role of the immune system in mediating stress effects on tumor growth and metastasis1,12,13, but the present results show that the neuroendocrine stress response can also directly affect the growth and activity of malignant tissue through hormone receptors expressed by tumor cells. Key to the demonstration of this direct effect on tumor cell biology is the use of a mouse host–human tumor hybrid model that provides opportunities for species-specific molecular manipulations that focally affect the tumor, such as the use of siRNA specific to human ADRB1 and ADRB2 and the comparison of human tumors positive for ADRB expression with those lacking ADRB expression. In addition to these tumor tissue-specific manipulations, we carried out several additional studies to ensure that generalized changes in host physio- logy (such as effects of the host cardiovascular or lymphatic systems) or direct effects on tumor cell proliferation are not responsible for the observed effects of stress on tumor growth (Supplementary Fig. 8 online).

Pharmacologic and genetic manipulations identify b-adrenergic signaling as a central mediator of stress effects in the model of stress used here, but these findings do not rule out the possibility that other neuroendocrine signaling pathways might modulate tumor cell bio- logy under other circumstances (for example, stress effects involving the hypothalamic-pituitary-adrenal/glucocorticoid axis14 or inhibitory effects of dopamine on VEGF activity15 and tumor growth16). To the extent that behavioral processes modulate the activity of multiple hormones via the central nervous system17–19 and those hormones influence tumor cell biology6,20, interventions targeting neuro- endocrine function at the level of the central nervous system could represent new strategies for protecting individuals with cancer from the detrimental effects of stress biology on the progression of malignant disease.

METHODS Cell lines and culture conditions. We grew the HeyA8, SKOV3ip1, A2780,

RMG-II (from N. Ueno and H. Itamochi, M.D. Anderson Cancer Center) and

MB-231 (from J. Price, M.D. Anderson Cancer Center) cancer cell lines as

previously described8.

Chronic stress model. We obtained 10- to 12-week-old female athymic nude

mice from the US National Cancer Institute. All experiments were approved by

the Institutional Animal Care and Use Committee of the M.D. Anderson

Cancer Center. We used a restraint-stress procedure based on previous

studies21, using a new physical restraint system that we designed (Supplemen-

tary Fig. 1). For the social isolation model, we randomly assigned mice to be

group housed (n ¼ 5 per cage) or individually housed (n ¼ 1 per cage). There was a wall and minimum distance of 24 inches between cages for the isolated

mice. For both models, we injected the ovarian cancer cells intraperitoneally

into mice 7 d after starting stress in all groups. We necropsied animals 21 d after

tumor cell injection. For beta-blockade, we inserted the Alzet osmotic mini-

pumps (DURECT) containing PBS or s-propranolol hydrochloride (Sigma;

2 mg/kg/d)22,23 on the nape of neck 7 d before initiation of restraint stress.

Short interfering RNA (siRNA) preparation. We purchased siRNAs targeted

against ADRB1 (target sequence 5¢-CCGATAGCAGGTGAACTCGAA-3¢) or ADRB2 (target sequence: 5¢-CAGAGTGGATATCACGTGGAA-3¢) from Qiagen and incorporated them into a neutral liposome (1,2-dioleoyl-sn-glycero-3-

phosphatidlycholine (DOPC)), as previously described10,24.

Inhibition of VEGF-R signaling. We blocked VEGF-R signaling using PTK787

(Novartis) daily25. We also used the monoclonal VEGF-specific antibody

bevacizumab (Genentech) in separate experiments.

Real-time RT-PCR. We extracted total RNA (Trizol reagent; Invitrogen) and

performed reverse transcription using an oligo(dT) primer and Moloney

murine leukemia virus (M-MLV) reverse transcriptase (Life Technologies).

After PCR amplification, we obtained quantitative values (we normalized each

sample on the basis of its I8S content). We obtained the VEGF primers from

Applied Biosystems (number HS00173626-m1).

Promoter analysis. We transiently transfected ovarian cancer cells

(Invitrogen Lipofectamine 2000) with the VEGF promoter-reporter constructs

(obtained from L. Ellis, M.D. Anderson Cancer Center) and determined

luciferase activity in triplicate as previously described26.

Immunohistochemistry. We stained paraffin sections for CD31 (1:800 dilu-

tion, Pharmingen), MMP-2 (1:500 dilution, Chemicon), MMP-9 (1:200 dilu-

tion, Calbiochem), VEGF (1:500 dilution, Santa Cruz Biotechnology) and

bFGF (1:1,000 dilution, Sigma) at 4 1C (refs. 8,24). We exposed control samples

to secondary antibody alone, and they did not show any nonspecific staining.

Details for immunostaining fixed frozen sections for CD31 and desmin have

been reported previously8. To quantify MVD, we examined ten random

0.159 mm2 fields at 100� magnification for each tumor and counted the microvessels within those fields8. We evaluated the degree of pericyte coverage

in five fields per tumor (200� magnification) as either absent or present (defined as covering 450% of the vessel). We then calculated, for each tumor, the average percentage of covered vessels relative to uncovered vessels.

In vivo angiogenesis assay with Matrigel. We exposed SKOV3ip1 cells

to serum-free medium with 1 mM of norepinephrine or vehicle for 24 h. We collected supernatants and centrifuged them to remove cells. We infused

the conditioned medium with phenol-red free Matrigel (2:3 proportion,

total 500 ml; BD Biosciences)27. We performed subcutaneous injections in mice for the three treatment groups (n ¼ 3), and VEGF (0.25 nM) served as positive control. We killed the mice after 8 d and extracted the

Matrigel plug for hemoglobin content (QuantiChrom hemoglobin assay;

BioAssay Systems).

In situ hybridization. We performed in situ hybridization for VEGF and

analyzed results as described previously (Supplementary Methods online)28.

Tumor vessel imaging. We anesthetized tumor-bearing mice and injected

them intravenously with 100 ml of fluorescein Lycopersicon esculentum lectin (Vector Laboratories). Ten minutes later, we perfused mice through the

ascending aorta with 4% paraformaldehyde for 2 min. Tumors were extracted,

fixed, embedded in OCT, and sectioned for visualization using confocal

fluorescence microscopy29.

Magnetic resonance imaging. We performed all magnetic resonance imaging

experiments using a 4.7-T Biospec small animal imaging system (Bruker

Biospin USA). For anatomic tumor imaging, we used axial T2-weighted fast

spin echo sequence (TE/TR 60 ms/1,050 ms; in-plane resolution 120 mm; slice thickness 1 mm; skip 0.25 mm; 3 nex) to delineate the tumor. For functional

imaging, we acquired a series of axial, dynamic-contrast-enhanced images

during and after contrast (diluted Magnevist) injection through a tail-vein

catheter. We used an interleaved fast spoiled gradient echo sequence (TE/TR

1.4 ms/40 ms; in-plane resolution 312 mm � 234 mm; 1 mm slice thickness;

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update rate 5.5 s per slice package) to monitor contrast accumulation in blood

vessels and tumor. We analyzed regions of interest encompassing the enhancing

periphery of the tumor. We measured functional tissue parameters by fitting

contrast levels to a two-compartment pharmacokinetic model30.

Assessment of tumor VEGF levels. We quantified concentrations of VEGF in

tumor tissue homogenates using an ELISA kit (R&D Systems).

Statistical analysis. We compared continuous variables with either Student’s

t-test or analysis of variance (ANOVA) and compared categorical variables with

w2. We used nonparametric tests (Mann-Whitney test), if appropriate, to compare differences. We considered P o 0.05 to be significant.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTS The authors thank I.J. Fidler, J. Price, C. Bucana, G. Gallick and A. Johnson for their helpful input and discussions regarding this work. We thank D. Reynolds for assistance with immunohistochemistry. We also thank J. Johnson and E. Schrock for assistance with manuscript preparation. This work was supported by US National Institutes of Health (NIH) grants (CA-11079301 and CA- 10929801), the Donna Marie Cimitile-Fotheringham Award for Ovarian Cancer Research, the U.T. M.D. Anderson Ovarian Cancer SPORE (2P50 CA083639- 06A1), and a Program Project Development Grant from the Ovarian Cancer Research Fund, Inc. to A.K.S., NIH grant CA-104825 to S.K.L., and NIH grant A152737 to S.W.C.

AUTHOR CONTRIBUTIONS P.H.T., L.Y.H. and A.A.K. designed and performed experiments and wrote portions of the manuscript. J.M.A. and R.T. designed and performed VEGF transcription and promoter assays and edited the manuscript. C.L., N.B.J., G.A.-P., W.M.M., Y.G.L., M.L., L.S.M., T.J.K., C.N.L. and Y.L. designed and performed experiments and edited the manuscript. E.F. and R.A.N. designed and performed catecholamine analyses and edited the manuscript. J.A.B., M.R. and V.K. designed and performed the imaging experiments and edited the manuscript. A.M.S. and G.L.-B. performed siRNA incorporations and edited the manuscript. R.L.C. and D.M.G. performed statistical analyses and edited the manuscript. S.K.L., S.W.C. and A.K.S. designed the overall study, analyzed data and edited the manuscript.

COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturemedicine/

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