tgf-b blockade controls ascites by preventing ... · shan liao 1, jieqiong liu1,2, peichun lin ,...

Cancer Therapy: Preclinical

TGF-b Blockade Controls Ascites by Preventing Abnormalization ofLymphatic Vessels in Orthotopic Human Ovarian Carcinoma Models

Shan Liao1, Jieqiong Liu1,2, Peichun Lin1, Tony Shi3, Rakesh K. Jain1, and Lei Xu1

AbstractPurpose: Ovarian cancer patients with malignant ascites have poor prognosis. The accumulation of

ascites is caused by an imbalance between fluid extravasation from the blood vessels and reabsorption by

lymphatic vessels. Whereas, the role of TGF-b in tumor progression has been well studied, the role of TGF-bin lymphatic vessel function is far from understood. Here, we sought to dissect the role of TGF-b blockade

in the formation of ascites.

Experimental Design: We used soluble TGF-b Receptor II (sTbRII) to block TGF-b signaling in two

orthotopic human ovarian carcinoma models: SKOV3ip1 and Hey-A8. We measured tumor proliferation,

apoptosis, lymphangiogenesis, and angiogenesis by immunohistochemical staining, and examined dia-

phragm lymphatic vessel network by intraperitoneal injection of a fluorescent dye. Diaphragm lymphatic

vessel function was assessed by tracking fluorescent beads in the diaphragm and measuring their drainage

rate.

Results: TGF-b blockade impaired tumor growth in both models, accompanied by a decreased tumor

cell proliferation and angiogenesis. More strikingly, TGF-b blockade almost completely abolished ascites

formation. TGF-b blockade significantly inhibited the expression of VEGF, which is the major contributor

to ascites formation. At the same time, TGF-b blockade prevent ‘abnormalization’ of diaphragm lymphatic

vessels and improved ascites drainage.

Conclusions: TGF-b blockade decreased ascites by both inhibiting ascites formation and improving

ascites drainage. Based on our finding, it is reasonable to consider the use of TGF-b blockade as a palliative

treatment for symptomatic ascites. Clin Cancer Res; 17(6); 1415–24. �2011 AACR.

Introduction

Ovarian cancer is characterized by rapid growth ofperitoneal tumors and accumulation of ascites (1). Whenpresent in large amounts, ascites increases abdominalpressure and leads to pain, loss of appetite, nausea,and reduced mobility. In addition to tumor eradication,symptomatic relief from ascites becomes a primary ther-apeutic goal for many patients. Therapeutic options arelimited to paracentesis and diuretics followed by perito-neovenous shunts, diet measures and other modalitieslike systemic or intraperitoneal chemotherapy (2). How-ever, these treatments only temporarily alleviate thesymptoms and can induce adverse effects and discomfort.

In contrast to the treatment of underlying cancer, so farthere is no generally accepted evidence-based guidelinefor the management of malignant ascites.

The ascites results from excessive production andimpaired drainage of intraperitoneal fluid (3, 4). VascularEndothelial Growth Factor/Vascular Permeability Factor(VEGF/VPF) is crucial for the production of malignantascites (3). Avastin, a recombinant humanizedmonoclonalantibody to VEGF, has been shown to reduce ascites (5).However, it only inhibits the production of peritoneal fluidbut does not affect ascites drainage. Lymphatic vessels inthe diaphragm drain peritoneal fluid (6). We have pre-viously shown that lymphatic vessels in hyperplastic, dys-plastic, and neoplastic lesions are compressed andnonfunctional (7). Indeed, relieving the compressivemechanical stress opens up lymphatic vessels, however,these vessels still remain nonfunctional, presumably due toirreversible damage in the lymphatic valves (8, 9). We andothers have been shown both preclinically and clinicallythat antiangiogenic therapy can "normalize" tumor bloodvessels (10–12). However, there are no studies on how tonormalize lymphatic vessels. Here, we show that TGF-bblockade inhibits ascites production (via inhibition ofVEGF production) and prevents ‘abnormalization’ oflymphatic vessel function, resulting in almost complete

Authors' Affiliations: 1Edwin L. Steele Laboratory, Department of Radia-tion Oncology, Massachusetts General Hospital, Boston, Massachusetts;2Department of Breast Surgery, the Second Xiangya Hospital of CentralSouth University, Changsha, Hunan, China; and 3College of Arts andSciences, New York University, New York, New York

Corresponding Author: Lei Xu, Massachusetts General Hospital, ELSteele Lab, 100 Blossom Street, Cox 734, Boston, Massachusetts02114. Phone: 617-7261962; Fax: 617-7261962.E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-2429

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 1415

on March 25, 2020. © 2011 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 28, 2011; DOI: 10.1158/1078-0432.CCR-10-2429

http://clincancerres.aacrjournals.org/

control of malignant ascites. Our findings suggest TGF-bblockade should be explored as a palliative option in end-stage ovarian carcinoma patients with symptomaticascites.

Methods

Cell linesSKOV3 ip1 and Hey-A8 cells were gifts from Dr. Isaiah J.

Fidler (M.D. Anderson Cancer Center, Houston, TX).Mv1Lu cells were obtained from ATCC.

Plasmid constructionMouse TGF-b receptor II extracellular domain was ampli-

fied from a mouse heart cDNA library and cloned intopeak13CD5 vector, which contains a CD5 leader upstreamof the human IgG1 hinge region sequences (a gift from Dr.Brian Seed, Center for Computational and IntegrativeBiology, Massachusetts General Hospital).

Purification and activity of the sTbRIIThe sTbRII constructs were transfected into 293 cells by

Lipofectamine 2000 (Invitrogen). Following overnightincubation, cells were washed with PBS and changed tofresh medium containing 0% FBS. After 3 days of incuba-tion, the supernatant was collected and centrifuged;recombinant sTbRII was purified with Protein A Sephar-ose chromatography in accordance with manufacturer’sprotocol (Chemicon International). To determine theactivity of sTbRII, serial dilutions of sTbRII was incubatedfor 1 hour with 0.1 ng/mL TGF-b1, 0.5 ng/mL TGF-b2,

and 0.05 mg/mL TGF-b3 (R&D Systems), and then addedto Mv1Lu cells (13). Cell proliferation was determined by[3H]TdR incorporation assay (14).

Orthotopic implantationSKOV3ip1 and Hey-A8 tumor cells were injected i.p.

into female nude mice (1 � 106 cells/mice). Intraperito-neal injection of tumor cells produced solid tumorsgrown on the surface of the peritoneal organs and tumorsinvaded into the diaphragm. Mice bearing SKOV3ip1tumors also produced large amount of ascites. Mice weresacrificed 35 days later. Peritoneal tumors were excisedand weighed. Malignant ascites were aspirated andmeasured (14).

Northern blot analysisNorthern blot was performed as described previously

(15). cDNA probes were synthesized by PCR, using thefollowing primers: interlukin (IL)-8F: 5’-CGG ACA GACAGA CAG ACA CC-3’; IL-8R: 5’-AAG AAA ACT GGG TGCAGAG-3’. VEGF-F: 5’-AAGGAGGAGGGCAGAATC AT-3’;VEGF-R: 5’-AAA AAC GAA AGC GCA AGA AA-3’.

ELISATumor tissue was lysed to extract protein, and IL-8 and

VEGF were quantified using Quantikine ELISA kit (R&DSystems). IL-8 and VEGF levels (pg/mL) were normalizedby the amount of total protein measured using Dc ProteinAssay (Bio-Rad; mg/mL; ref. 16).

Western blot analysisCells or tumor tissue was lysed to extract protein (16). A

total of 30 mg of protein per sample was separated on SDS-polyacrylamide gels (17). Phosphorylation status of SMADwas detected by phospho-specific antibodies (Cell Signal-ing, Danvers, MA).

ImmunohistochemistryTissue sections (5-mm-thick) of formalin-fixed, paraffin-

embedded tumor xenografts were deparaffinized in xyleneand rehydrated in graded alcohol. The endogenous perox-idase was blocked with the addition of 3% hydrogenperoxide in PBS for 12 minutes. The samples were incu-bated for 20 minutes at room temperature with protein-blocking solution [PBS (pH 7.5) containing 5% normalhorse serum and 1% normal goat serum] followed byincubation at 4�C with primary antibodies (anti-CD31,1:800; BD Biosciences; anti-Proliferating Cell Nuclear Anti-gen (PCNA), 1:50; Dako Corporation; F4/80, 1:10; Serotec,Raleigh; LYVE-1 and a-SMA). The samples were then rinsedand incubated for 1 hour at room temperature with aperoxidase-conjugated anti-rabbit IgG. The slides wererinsed with PBS and incubated for 5 minutes with diami-nobenzidine. The sections were then washed 3 times withdistilled water, counterstained with Mayer’s hematoxylin,washed once with distilled water and once more with PBS.The slides were mounted with a universal mount andexamined with a bright-field microscope (18).

Translational Relevance

Ovarian cancer is characterized by the rapid growthof solid intraperitoneal tumors and accumulation ofascites, the ascites being the clinical presentation ofend-stage disease. Whereas the role of TGF-b in tumorangiogenesis and progression is well understood, itsrole in lymphatic vessel function remains far fromunderstood. To this end, we dissected the potentialrole of TGF-b blockade in controlling ascites usinghuman ovarian cancer xenografts in mice. We foundthat blocking tumor and host TGF-b signaling cansignificantly inhibit the growth of both VEGF- andinterlukin-8–dependent human ovarian tumors. Moreimportantly, we discovered that TGF-b blockade sig-nificantly decreases the volume of ascites by bothinhibiting ascites formation and preventing impair-ment of lymphatic vessel drainage, thus demonstratingits potential as a promising new treatment for malig-nant ascites. Because a number of agents that target theTGF-b-pathway are in clinical trials, the results of theproposed study could be rapidly translated to theclinic.

Liao et al.

Clin Cancer Res; 17(6) March 15, 2011 Clinical Cancer Research1416




Terminal deoxynucleotidyl transferase–mediateddUTP nick end labelingFor terminal deoxynucleotidyl transferase–mediated

dUTP nick end labeling (TUNEL) staining, tissuesamples were fixed in 4% (w/v) paraformaldehyde inphosphate-buffered saline. Paraffin-embedded sections(5 mm) were used for in situ detection of apoptotic cells.After deparaffinization and rehydration, tissue sectionswere stained with terminal deoxynucleotidyl transferaseand incubated with diaminobenzidine. The sections werecounterstained with Methyl Green, and the percentage ofTUNEL-positive cells was quantified (Millipore; ref. 19).

Functional assays for lymphatic vessel drainageFour weeks after cell implantation, mice were injected i.

p. with fluorescent beads (0.2 mL, 1 mm in diameter). Twohours later, diaphragms were harvested and fixed with 4%paraformaldehyde for fluorescent microscopy. Claudalmediastinal lymph nodes (CMLN) were harvested andhomogenized to measure fluorescence intensity with fluor-escent plate reader (Perkin Elmer).

Diphtheria toxin treatmentWhen SKOV3ip1 tumor–bearing mice developed visible

ascites, diphtheria toxin (1 mg) or PBS was injected i.p. (8).Fivedays later,miceweresacrificed totest lymphatic function.

Data analysis and interpretationAll data are presented as mean � SD. The significance of

differences between 2 groups was analyzed using theStudent’s t test (2-tailed)orMann–WhitneyU test (2-tailed).

Result

Characterization of sTbRIIWe used 2 methods to block tumor and host TGF-b

signaling. First we stably transfected the sTbRII constructinto SKOV3ip1 and Hey-A8 cells. These transfected cellsconstitutively secreted large quantities of sTbRII protein(Fig. 1A and B). Second, we used purified recombinantsTbRII protein as a therapeutic agent. To test the functionof the purified sTbRII protein, we treated Mv1Lu cells withrecombinant TGF-b1, -b2, and -b3 in the presence orabsence of purified sTbRII. Our purified sTbRII success-fully blocked TGF-b1 and -b3 but not TGF-b2–mediatedinhibition of cell proliferation (data not shown). It alsoblocked TGF-b1 induced phosphorylation of Smad2(Fig. 1C).

Blocking tumor and host TGF-b signaling inhibitsovarian cancer growth and ascites formation

In the first group, we orthotopically implanted par-ental, mock- and sTbRII-transfected SKOV3ip1 (SKOV-sTbRII) and Hey-A8 (Hey-sTbRII) cells i.p. into nudemice. We examined peritoneal tumor weight at day35. Transfection of sTbRII decreased tumor weight inboth models (P < 0.01). Mice implanted with SKOV3ip1and mock-transfected cells formed large amountsof bloody ascites, whereas transfection of sTbRIIalmost completely abolished ascites formation (Fig. 2Aand B).

In the second group, we implanted parental SKOV3ip1and Hey-A8 cells i.p. into nude mice. Seven days after

Figure 1. Characterization ofsoluble TGF-b receptor type II.A, transfected cells overexpresssTbRII mRNA. Northern blotanalysis of mRNA from parentaland sTbRII transfected SKOV3ip1(SKOV-STbRII) and Hey-A8 (Hey-STbRII) cells. B, transfected cellsoverexpress sTbRII protein.Supernatant collected fromtransfected SKOV3ip1 (SKT) andHey (HT) cells was subjected to12% SDS-gel electrophoresis andWestern blot analysis. Differentamount of recombinant humanIgG region was used as standard.C, sTbRII blocks TGF-b inducedSmad2 phosphorylation.SKOV3ip1 cells were treated with10 ng/mL TGF-b1 alone or withTGF-b1 plus sTbRII (10 mg/mL).Proteins were obtained andanalyzed by Western blot. Themembrane was probed withantibody against phospho-Smad2or total Smad2.

MW (kDa) SKT HT 50 100 200 500 1,000 μμg 216

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TGF-b Blockade Normalization of Lymphatic Vessels

www.aacrjournals.org Clin Cancer Res; 17(6) March 15, 2011 1417




tumor implantation, we began treatment with control IgGor recombinant sTbRII protein (1mg/kg, i.p., every 3 days).Recombinant sTbRII treatment significantly inhibitedtumor growth (P < 0.01). More dramatically, sTbRII treat-ment almost completely abolished ascites formation(Fig. 2C and D, P < 0.001).

Blocking tumor and host TGF-b signaling inhibitstumor cell proliferation and angiogenesis viainhibition of IL-8 and VEGF expression

VEGF and IL-8 are angiogenic and autocrine growthfactors for ovarian tumors (20). In TGF-b–blockedtumors, VEGF and IL-8 mRNA and protein decreasedsignificantly (Fig. 3A and B). As a result, we found thenumber of PCNAþ cells decreased significantly in TGF-bblocked tumors (SKOV3-sTbRII: 36 � 13; Hey-sTbRII: 62� 27) compared with parental tumors (SKOV3ip1: 128 �25; Hey-A8: 142 � 22; data shown as number of PCNAþ

cells per 0.329 mm2 area; P < 0.001). We also foundparental tumors had significantly more CD31þ endothe-lial cells (SKOV3ip1: 62 � 7; Hey-A8: 48 � 4) than sTbRIItumors (SKOV3-sTbRII: 22 � 4; Hey-sTbRII: 16 � 6;

Fig. 3C) (P<0.001, data are shown as number ofCD31þ structures per 1.355 mm2).

Tumor-associated macrophages (TAM) play an impor-tant role in tumor progression (6). We examined the effectof TGF-b blockade on TAM infiltration in peritoneal ovar-ian tumors using the macrophage marker F4/80. A lowerinfiltrating macrophage density was detected in SKOV-sTbRII tumors (52 � 16) than in SKOV3ip1 tumors(145 � 22, P < 0.001, data are shown as number of F4/80þ cells per 1.335 mm2).

Blocking tumor and host TGF-b signaling preventedabnormalization of diaphragm lymphatic vesselnetwork

We examined the diaphragm lymphatics by fluorescencelymphangiography (FITC-dextran, 0.2 mL, 2�106 MW,i.p.). Twenty minutes later, diaphragms were collectedand observed under fluorescence microscopy. In nontu-mor-bearing mice, we observed the distinct outline oforganized lymphatic strips on the peritoneal side of thediaphragm. In mice bearing SKOV3ip1 tumors, the dia-phragms contained lymphatic vessels of increased density

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Figure 2. A, transfection of sTbRII decreased SKOV3ip1 andHey-A8 tumor weight, and (B) volume of ascites in SKOV3ip1 tumor-bearingmice. C, recombinantsTbRII treatment decreased SKOV3ip1 and Hey-A8 tumor weight, and (D) volume of ascites in SKOV3ip1 tumor-bearing mice. n ¼ 10.

Liao et al.





and branching. In SKOV-sTbRII tumor-bearing mice, lym-phatic strips appeared similar to those in normal mice. Onthe pleural side of the diaphragms, the dye showed normallymphatic network in nontumor-bearing mice, an enlargedarrangement of lymphatic vessels in mice bearing SKO-V3ip1 tumors and a ‘‘normalized’’ lymphatic network inmice with SKOV-sTbRII tumors (Fig. 4A). Quantificationdata showed TGF-b blockade decreased diameter of lym-phatic vessels on the pleural side (Fig. 4B).Normal lymphatic vessels have 1-way valves to prevent

fluid from flowing backwards (21). We visualized lympha-tic valves using CD31 and LYVE-1 double staining in wholemounts of the diaphragm. In parental tumors, profoundlymphangiogenesis occurred and lymphatic valve struc-tures disappeared completely-–similar to our previousobservations (9). In contrast, in TGF-b blocked tumors,normal lymphatic network was present and valve structuresremained intact (Fig. 4C).

Blocking tumor and host TGF-b signaling decreaseddiaphragm lymphangiogenesis

In peritoneal tumors attached to the surface of peritonealorgans, we found little LYVE-1 immunostaining (data notshown). However, in tumors invading the diaphragms,LYVE-1 immunostaining showed that lymphatic vesselswere abundant, enlarged and irregularly shaped. In dia-phragms with invading SKOV-sTbRII tumors, lymphaticvessel density (in size-matched tumors in the diaphragm)had decreased (Fig. 5).

We further analyzed the number of infiltrating macro-phages in (size-matched) tumors invading the diaphragm.In diaphragms fromnontumor-bearingmice, using stainingwith CD11b (an alternative marker for macrophages), wefound no infiltratingmacrophages. Inmice with SKOV3ip1tumors, we identified a high number ofmacrophages (thesecells being closely associated with LYVE-1–positivelymphatic vessels; Fig. 5). In diaphragms with invading

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Figure 3. Blocking TGF-b signaling decreased VEGF and IL-8 expressions. Parental, and sTbRII-transfected SKOV3ip1 and Hey-A8 cells were culturedunder confluent conditions. A, mRNA was extracted and Northern blot analysis was performed. Representative of 3 independent experiments is shown.B, protein extracts from parental, mock- and sTbRII-transfected tumor tissues were analyzed by human VEGF and IL-8 ELISA. C, frozen sections oftumors obtained from parental and sTbRII-transfected SKOV3ip1 and Hey-A8 tumors were analyzed for PCNA and CD31 levels by immunohistochemistrystaining.






SKOV-sTbRII tumor, we identified the same amount ofpositive CD11b staining, but a reduced number of LYVE-1–positive vessels. This observation indicates that despitethe presence of a large number of macrophage, these cellsstill require the presence of TGF-b to induce lymphangio-genesis.

Blocking tumor and host TGF-b signaling improvedlymphatic vessel function

To study whether TGF-b blockade affects the function ofdiaphragm lymphatic vessels, we injected fluorescent beadsintraperitoneally. In nontumor-bearing mice, few beadswere observed in the diaphragm 2 hours after injection,indicating their clearance through the lymphatic vessels. InSKOV3ip1 tumor-bearing mice, despite the large numberof lymphatic vessels, many beads remained in the dia-phragm, indicating impaired drainage. In SKOV-sTbRIItumor-bearing mice, few beads were present (Fig. 6A).

Diaphragm lymphatic vessels drain into the CMLN. Toconfirm and quantify drainage, we measured fluorescenceintensity of beads drained to CMLN. Compared with mice

with normal drainage, CMLNs from SKOV3ip1 tumor-bearing mice had low fluorescence intensity, indicatingdecreased drainage. CMLNs from SKOV-sTbRII tumor-bearing mice showed high fluorescence intensity(Fig. 6B), indicating TGF-b blockade improved drainageto CMLN.

This functional normalization may be attributed to TGF-b decreasing tumor weight, thus relieving the compressionof lymphatic vessels (8). Indeed, we observed that in micetreated with diphtheria toxin (DT), which is much lesscytotoxic to mouse than to human cells, ascites wereresolved to a similar extent as in sTbRII-treated mice(Control group: 3.5 � 1.7 mL, DT group: 0.1 � 0.3 mL).

Discussion

TGF-b is a multifunctional cytokine. Depending on thecontext, it can act on the tumor cells to increase epithelialto mesenchymal transition (EMT), migration, invasion,and survival. It can act on stromal cells to induce angio-genesis and dampen local immune surveillance (22–24).

SKOV3ip1 tumor-bearing mice SKOV-sTβRII tumor-bearing miceNontumor-bearing miceA

Peritonealside

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Figure 4. A, fluorescent microscopy of diaphragm lymphatic vessels highlighted by injection of FITC-dextran. B, lymphatic vessel diameter in the pleural sideof the diaphragm was quantified using a macro in ImageJ. C, lymphatic vessels valves were visualized by anti-CD31 (green) and anti-LYVE-1 (blue)double staining; arrows point to valves.

Liao et al.





Recent studies also showed TGF-b to affect intratumoraldistribution of chemotherapeutic agents (25, 26). Wechose to study TGF-b because both TGF-b and its recep-tors are overexpressed in ovarian carcinomas, and becausegenes in the TGF-b signaling pathway are dysregulated inovarian carcinomas (27). Several strategies to block TGF-bare being tested in various stages of investigation, fromthe laboratory to phase III clinical trials (28). Thesestrategies fall into 2 categories: direct inhibition ofTGF-b (via TGF-b neutralizing antibodies, soluble TGF-b receptors and decorin) and interference with the down-stream signaling cascades (e.g., interferon-g , peroxisomeproliferator activated receptor-g agonist). Soluble TGF-breceptor II competes with cell-bound TbRII for activeTGF-b ligand, resulting in reduced TGF-b signaling. Wechose to use this strategy because it has been shown thatTGF-b1 and -b3 play a greater role in ovarian cancerprogression than TGF-b2 (29), and sTbRII has beenshown to specifically block the effects of TGF-b1 and-b3 (26).In our study, we utilized a well-established human

ovarian carcinoma orthotopic model in nude mice. Whenhuman ovarian cancer cells were injected orthotopically(intraperitoneal) into nude mice, they produced solidtumors that invaded the surface of peritoneal organs anddiaphragm, and these tumors formed ascites. This growthpattern mimics the growth of human ovarian cancer inpatients. We used 2 methods to block tumor and host TGF-b signaling. First, we established sTbRII-overproducingovarian tumor cells (genetic approach). Second, we treated

tumor-bearing mice with recombinant sTbRII (pharmaco-logic approach). A previous study showed that 5 days aftertumor cell i.p. injection, diaphragmatic lymph vesselsbecome occluded (30, 31). In our study, we also observedsmall tumors established on the diaphragm and in theperitoneal cavity 7 days after tumor implantation. Thistumor burden mimics that in patients after cytoreductivesurgery and adjuvant chemotherapy. Based on these pub-lished studies and our own observations, we started sTbRIItreatment 7 days after tumor implantation, when tumorlesions and obstruction of lymphatics were established.

Targeted therapy often fails because different tumorsdepend on different growth/angiogenic factors for theirprogression. The outcome of a therapy using specific inhi-bitors depends on the expression level of the target in thetumor, which varies significantly between patients. Inaddition, malignant tumors often switch growth factordependence, thus permitting ‘therapeutic escape’ fromspecific, targeted drugs (15). In our study, we used 2 humanovarian carcinoma cell lines to represent cancer heteroge-neity. SKOV3ip1 cells express high levels of VEGF and lowlevels of IL-8. When implanted orthotopically into theperitoneal cavity of nude mice, these cells formed solidtumors with large volumes of ascites. On the other hand,Hey-A8 cells express high levels of IL-8 and low levels ofVEGF; when implanted into the peritoneal cavity, theyformed solid tumors with little ascites (32). Previous studyusing PTK787, a VEGFR2 tyrosine kinase inhibitor, to blockVEGF pathways has shown that inhibition of VEGF wasonly effective in inhibiting the growth of VEGF-dependent

PLPL

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Figure 5. Blocking tumor and host TGF-b signaling decreased diaphragm lymphangiogenesis. We performed LYVE-1/CD31 and LYVE-1/CD11b doublestaining in cross-section of the diaphragms from nontumor-bearing mice, SKOV3ip1 and SKOV-sTbRII tumor-bearing mice. Diaphragm from micebearing size-matched SKOV3ipl and SKOV-sTbRII tumors were used. PL, pleural side; P, peritoneal side. In LYVE-1/CD31 double staining: red,LYVE-1 staining; green, CD31 staining; blue, DAPI. In LYVE-1/CD11b double staining: red, LYVE-1 staining; green, CD11b staining; blue, DAPI.






SKOV3ip1 tumors but not IL-8–dependent Hey-A8 tumors(14). TGF-b stands at the crossroad of diverse signalingpathways, therefore, targeting TGF-b would have theadvantage of bypassing the 2 problems-–pointed out pre-viously—associated with VEGF- or IL-8–targeted therapies.Indeed, in our study, we observed that TGF-b blockadeinhibited the expression of both VEGF and IL-8. As a result,TGF-b blockade effectively inhibited the progression ofboth VEGF-dependent (SKOV3ip1) and IL-8–dependent(Hey-A8) human ovarian tumor xenografts.

The pathogenesis of malignant ascites is well elucidated.Increased fluid production and decreased lymphaticabsorption are identified as contributing factors of ascitesformation. In our study, we administered sTbRII intraper-itoneally and showed a dual effect on ascites productionand drainage. Tumors exhibit extensive angiogenesis, buttumor blood vessels are structurally and functionallyabnormal (11). These vascular abnormalities contributeto high vessel permeability. Decreasing permeability oftumor vessels by inhibiting VEGF signaling using VEGFtrap (soluble decoy receptor) or a VEGF neutralizing anti-body has been shown to inhibit the formation of malig-

nant ascites (33–35). TGF-b blockade has been shown torecruit pericyte and induce blood vessel ‘‘normalization’’(26); this, combined with our data showing TGF-b block-ade inhibits VEGF production, provides a mechanism fordecreased ascites production.

Furthermore, we studied the effect of TGF-b blockade onascites drainage by examining vessel morphology andfunction of diaphragm lymphatics. The function of TGF-b on tumor growth and progression has been studiedextensively (36), however, its effect on lymphangiogenesiswas only recently studied (6, 37, 38). Therefore, its effectand underlying mechanisms on lymphatic vessel functionremain largely unknown. Recent studies showed TGF-b candirectly block lymphatic regeneration (in wound healing)and signal transduction in lymphatic endothelial cells (37,38). TGF-b can also increase the secretion of lymphangio-genic factors, thus indirectly enhancing lymphangiogenesis(6). The net result depends on the balance of the responseto TGF-b. In our study, we observed that peritoneal ovariantumors induced profound lymphangiogenesis in the dia-phragm. However, these newly formed lymphatic vesselsare not functional. Based on previous studies, which

SKOV3ip1 tumor-bearing mice SKOV-sTβRII tumor-bearing miceNontumor-bearing mice

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Figure 6. A, fluorescent microscopy of the fluorescent beads in diaphragm. B, CMLNs were collected and homogenized. Fluorescent intensity of the lysateswas determined using a fluorescent microplate reader.

Liao et al.





showed diaphragmatic lymph vessels becoming occluded5 days after intraperitoneal injection of tumor cells (30,31), we administered i.p. sTbRII 7 days after tumor implan-tation. We showed TGF-b blockade decreased lymphangio-genesis, reduced tumor burden in the diaphragm andmaintained the normal lymphatic vessel morphologyand valve structure. As a result, it improved the drainagefunction of diaphragm lymphatic vessels. This dual effect ofTGF-b blockade on ascites production and drainageexplains why it is a more efficient strategy than blockingVEGF alone (e.g., treatment with PTK787 leads to a 50%decrease in ascites volume, whereas sTbRII decreasedascites volume by 98%; ref. 39).In summary, our study shows that by blocking tumor

and host TGF-b signaling we can significantly inhibit thegrowth of both VEGF- and IL-8–dependent human ovar-ian tumors. More importantly, we have shown that TGF-b blockade significantly decreases the volume of ascitesby both inhibiting ascites formation and preventingimpairment of lymphatic vessel drainage, thus demon-strating its potential as a new treatment for malignantascites.

Disclosure of Potential Conflicts of Interest

R.K. Jain, consultant, Genzyme and Noxxon. Neither reagent nor finan-cial support from these two companies was used in the current study.

Acknowledgments

We thank Dr. T.P. Padera for critical and helpful comments on themanuscript and C. Smith for her expert technical assistant and Dr. P. Huangfor the assistant with experimental animals.

Grant Support

This work was supported in part by Clafflin Distinguished Scholar Award,Harvard Medical School (L. Xu), Program Project Grant P01-CA80124 from NCI(to R.K. Jain) and Federal Share/NCI Proton Beam Program (R.K. Jain) andTosteson Postdoctoral Fellowship from the Massachusetts Biomedical ResearchCorporation (S. Liao.).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 9, 2010; revised November 23, 2010; acceptedNovember 30, 2010; published OnlineFirst January 28, 2011.

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Liao et al.





2011;17:1415-1424. Published OnlineFirst January 28, 2011.Clin Cancer Res Shan Liao, Jieqiong Liu, Peichun Lin, et al. ModelsLymphatic Vessels in Orthotopic Human Ovarian Carcinoma

Blockade Controls Ascites by Preventing Abnormalization ofβTGF-

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