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VEGFR3and Metastasis inProstate Cancer

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Angiogenesis (bloodvessel growth), lymphangiogenesis (lymph system growth) are allintrinsicallyconnected with lymphedemaand share many of the same genes. Wehave many severalpages on both processes.

PatO'Connor

May23, 2008

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Expressionof Vascular Endothelial Growth Factor Receptor-3 by LymphaticEndothelial Cells Is Associated with Lymph Node Metastasis in ProstateCancer

Yiping Zeng1,2, KennethOpeskin3,4,Megan E. Baldwin5, LisaG. Horvath6,7,Marc G. Achen5, StevenA. Stacker5,Robert L. Sutherland7and Elizabeth D. Williams1,2

1 Bernard O’BrienInstitute of Microsurgery,Melbourne, Australia; Departments of 2 Surgeryand 3Pathology, University of Melbourne, Melbourne, Australia; 4Anatomical Pathology, St. Vincent’s Hospital, Fitzroy, Australia; 5Ludwig Institute for Cancer Research, Parkville, Australia; 6SydneyCancer Centre, Royal Prince Alfred Hospital, Camperdown, Australia; and7Garvan Institute of Medical Research, St. Vincent’s Hospital,Darlinghurst,Australia
  ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALSAND METHODS
RESULTS
DISCUSSION
REFERENCES
 
Purpose: The molecular mechanisms underlying lymphnode metastasis arepoorly understood, despite the well-established clinical importanceof lymph node status in many human cancers. Recently, vascularendothelial growth factor (VEGF)-C and VEGF-D have beenimplicated inthe regulation of tumor lymphangiogenesis andenhancement oflymphatic invasion via activation of VEGF receptor-3.The purpose ofthis study was to determine the expression patternof the VEGF-C/VEGF-D/VEGFreceptor-3 axis in prostate cancer and itsrelationship with lymphnode metastasis.

Experimental Design: Theexpression pattern of VEGF-C, VEGF-D, andVEGF receptor-3 in localized prostate cancer specimens (n=37) was determined using immunohistochemistry.

Results: Widespread, heterogeneous staining for VEGF-C and VEGF-Dwas observed in all cancer specimens. Intensity of VEGF-Cstaining waslower in benign prostate epithelium than in adjacent carcinoma,whereasno difference between benign epithelium and carcinoma wasobservedfor VEGF-D staining. VEGF receptor-3 immunostaining wasdetected inendothelial cells of lymphatic vessels in 18 of37 tissue samples.The presence of VEGF receptor-3-positive vesselswas associated withlymph node metastasis (P = 0.0002), Gleasongrade (P< 0.0001), extracapsular extension (P =0.0382), andsurgical margin status (P = 0.0069). In addition,VEGFreceptor-3 staining highlighted lymphatic invasion byVEGF-C-positive/VEGF-D-positivecarcinoma cells.

Conclusions: Together, these results suggest that paracrine activationof lymphatic endothelial cell VEGF receptor-3 by VEGF-Cand/or VEGF-Dmay be involved in lymphatic metastasis. Thusthe VEGF-C/VEGF-D/VEGFreceptor-3 signaling pathway may provide atarget forantilymphangiogenic therapy in prostate cancer.
  INTRODUCTION
Top
ABSTRACT
INTRODUCTION
MATERIALSAND METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lymph node status provides important information in both diagnosisandtreatment of prostate cancer. The presence of lymph node metastasisis a poor prognostic sign for patients with prostate cancer(1). In addition, lymph node status influences clinical managementbecause a curative treatment approach (such as perineal prostatectomyor brachytherapy) has a low probability of success inprostate cancerpatients with lymph node metastasis (2). Althoughcombined use of clinical data such as tumor stage, serumprostate-specific antigen level, and Gleason grade can helppredictthe risk of having lymph node metastasis, there areno noninvasivetechniques to accurately predict the presence oflymph nodemetastasis, in particular, microscopic metastasis (1). The emphasis on earlier detection and the need for more effectivetreatment of prostate cancer patients with lymph node metastasisrequire a better understanding of the molecular mechanisms involved.

Themolecular mechanisms involved in lymph node metastasis are poorlyunderstood, partly because of the lack of specific lymphaticendothelialmarkers and specific lymphatic growth factors. This situationhasimproved somewhat since vascular endothelial growth factor(VEGF)receptor-3 was described (3) . Inembryos, VEGF receptor-3is initially expressed in venous vasculature (4), butin adults, it is absent in endothelia of all large blood vesselsandis generally restricted to lymphatic endothelial cellsand a subsetof capillary endothelia (5 , 6) . VEGFreceptor-3 is reactivated in the blood vesselendothelium in sometumors, and the up-regulation of its twoligands, VEGF-C and VEGF-D(7) , may accompanythis (8, 9,10) .

VEGF-Cand VEGF-D belong to the VEGF family (11,12) , and like all VEGF familymembers, they contain a centralregion called the VEGF homology domain (11 ,12) . They have NH2-terminaland COOH-terminalpropeptides and can bind VEGF receptor-2 and VEGFreceptor-3 (10, 13 ,14) . Recent studies in tumormodels have provided direct evidence thatVEGF-C and VEGF-D caninduce lymphangiogenesis through VEGFreceptor-3 and/or promoteangiogenesis through VEGF receptor-2 (15, 16,17, 18,19) and thatinduction of lymphangiogenesis is oftenassociated with lymph nodemetastasis in these model systems. In breastcarcinoma models, VEGF-Cinduced only lymphangiogenesis (15 ,16 , 19) , and thiswas associated with lymphnode metastasis in two studies (15, 19) .In contrast, in melanoma and epithelioid-like(293EBNA cell line)tumor models transfected with VEGF-C andVEGF-D, respectively, bothlymphangiogenesis and angiogenesis wereobserved (17, 18) , and lymph node metastasiswas observedonly in the 293EBNA model. The differences in these resultsmostlikely reflect variations in the relative expression ofthe VEGF-C/VEGF-Dreceptors VEGF receptor-2 and VEGF receptor-3 inthe model systems.

Studiesin clinical specimens have shown that VEGF-C expression ispositively associated with lymph node metastasis in several tumortypes. In prostate cancer, Tsurusaki et al. (20) foundthat VEGF-C mRNA levels were significantly higher in lymphnode-positivetumors and that VEGF receptor-3-positive vessels wereincreased inthe stroma of VEGF-C-positive tumors. Moreover, VEGF receptor-3expressionwas correlated with Gleason score, preoperative prostate-specificantigenlevels, and lymph node metastasis in prostate cancer (21). However, there are no reports documenting VEGF-C protein localizationin human prostate cancer. Recent reports in breast andovarian cancersuggest that VEGF-D positivity is a prognostic factor(22,23, 24); however, VEGF-D localization inprostate cancer has not been describedpreviously. In the presentstudy, the protein expression patterns ofVEGF-C, VEGF-D, and VEGFreceptor-3 were examined by immunohistochemistry to explorewhetherthis signaling axis is associated with lymph node status inprimaryprostate cancer.
  MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS ANDMETHODS
RESULTS
DISCUSSION
REFERENCES
 
Prostate Tissue Samples and Patient Characteristics.
Immunohistochemical examination was performed on 37 prostatecancerradical prostatectomy specimens obtained from men treated ateitherSt. Vincent’s Hospital, Melbourne between 2001 and2003 or StVincent’s Hospital, Sydney between 1990 and1998. The pathologicalcharacteristics of the resected primary tumorsand regional lymphnodes are summarized in Table 1. Sections that contained areas ofadenocarcinoma with adjacent benigntissue were selected to allow comparative evaluation. Serial4-µmsections were cut from formalin-fixed, paraffin-embedded tissueandmounted on slides for immunohistochemical detection ofVEGF-C, VEGF-D,and VEGF receptor-3 as described below. The studieswere conductedwith ethical approval of the St. Vincent’s HospitalHuman EthicsCommittee and were in accordance with AustralianNational Health andMedical Research Council Guidelines.

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Table 1Relationships between the presence of VEGFR-3-positive vessels andclinicopathological parameters in prostate adenocarcinoma (n= 37)

 

 
Antibodies.
Goat polyclonal anti-VEGF-C (AF752), goat polyclonal anti-VEGFreceptor-3(AF349), and mouse monoclonal anti-VEGF-D (MAB 286) werepurchasedfrom R&D Systems (Minneapolis, MN) and used forimmunohistochemistry at the following concentrations: anti-VEGF-C,2.5µg ml–1; anti-VEGF receptor-3, 1.67 µgml–1;and anti-VEGF-D, 8.33 µg ml–1. Mouse monoclonalantibody D2-40, which reacts with an O-linkedsialoglycoprotein foundon lymphatic endothelium, was purchased from Signet Laboratories(Dedham,MA) and used at a dilution of 1:100.

Immunoprecipitation,Western Blotting, and Analysis of AntibodySpecificity.
A plasmid encoding FLAG-tagged, full-length VEGF-D and transfectioninto293EBNA cell have been described previously (14). For VEGF-C,an expression plasmid encoding full-length VEGF-C tagged atthe COOHterminus with three Myc tags (single Myc epitope, EQKLISEEDL)wasgenerated. 293EBNA cells were transiently transfected withthisplasmid using FuGENE 6 transfection reagent (Roche MolecularBiochemicals, Indianapolis, IN) according to the manufacturer’sinstructions.VEGF-C and VEGF-D were purified from conditioned mediumcollectedover 24–48 h. A c-Myc antibody (9E10; ZymedLaboratories Inc., SanFrancisco, CA) coupled to cyanogen bromide-activatedSepharose (AmershamBiosciences, Castle Hill, New South Wales,Australia) and M2 beads(anti-FLAG M2-Agarose mouse; Sigma-Aldrich, St.Louis, MO) were usedto pull down VEGF-C and VEGF-D, respectively.

ForWestern blotting, the proteins were transferred to a nitrocellulosemembrane(Hybond-C Super; Amersham Biosciences, Buckinghamshire, UnitedKingdom) and probed with primary antibody against VEGF-C orVEGF-D,followed by the appropriate secondary antibody. Signal wasdevelopedwith SuperSignal West Femto Maximum Sensitivity Substrate(PierceBiotechnology, Rockford, IL).

Immunohistochemistry.
Tissue sections were deparaffinized in shellex, followed by rehydrationin graded ethanol. After quenching of endogenous peroxidaseactivity,antigen retrieval (citrate buffer, pH 6.0), andprotein blocking (DAKOprotein block serum free; DAKO Corp., Carpinteria,CA), tissuesections were incubated overnight at 4°C withthe specified primaryantibody. The tissue sections were thenincubated with theappropriate biotinylated secondary rabbitantigoat or antimouseantibodies (DAKO, Glostrup, Denmark) andstreptavidin-biotin-peroxidase complex (DAKO). Peroxidase reactivitywas visualized using 3,3'-diaminobenzidine (DAKO). Thesections werecounterstained with hematoxylin and coverslipped. Negativecontrolsincluded immunostaining of normal human cerebrum (25)and substitution of normal goat IgG (R&D Systems) ormouse IgG1 (DAKO)for the primary antibodies. Normal human heartwas used as a positivecontrol tissue for VEGF-C (11)and VEGF-D (24)staining, human melanoma was used as a positive controltissue forVEGF receptor-3 (26) staining,and normal lymphnode was used as a positive control for D2-40 staining. Twotrainedobservers (K. O. and E. D. W.) evaluated and interpreted theresultsof immunohistochemical staining without knowledge ofthe clinicaldata of each patient. Staining was scored by bothobserverssimultaneously, using a multihead microscope. Stainingofadenocarcinoma, benign epithelia, stroma, lymphatic vessels,vascularendothelial cells, and smooth muscle was recorded asstrong, weak, ornegative and recorded on standardized data sheets.Primary andsecondary Gleason grades were assigned by apathologist (K. O.).

Statistics.
The relationships between the presence of VEGF receptor-3-positivevesselsand clinicopathological parameters were evaluated by Fisher’sexacttest or {chi}2 test asindicated. P < 0.05 wasconsidered statisticallysignificant. All calculations were performedusing the statisticalcomputer program Prism+Instat bundle (GraphPadSoftware, San Diego,CA).
  RESULTS
Top
ABSTRACT
INTRODUCTION
MATERIALSAND METHODS
RESULTS
DISCUSSION
REFERENCES
 
Antibody Specificity.
Western blotting demonstrated specific detection of VEGF-C andVEGF-Dusing the anti-VEGF-C and anti-VEGF-D antibodies, respectively(Fig.1A) , and proteinsof the expected size were detected (13 ,14) . There was no cross-reactivity between either theanti-VEGF-Cantibody and VEGF-D or the anti-VEGF-D antibody andVEGF-C (Fig. 1A). The VEGF-C/VEGF-D-positive control tissue, normalhuman heart (11), exhibited strong granular staining in allcardiac muscle cells forboth VEGF-C and VEGF-D (Fig. 1B). The VEGF receptor-3-positive control tissue, human melanoma(26), showed strong staining for VEGF receptor-3 in the endothelialcellsof all lymphatic and some blood vessels in the tumor and adjacentdermis (Fig. 1B) .Lymphatic vessels in normal lymph node showedstrong positivestaining using the lymphatic endothelial markerD2-40, with nostaining in other tissue components (Fig. 1B). Normal human brain, the negative control tissue for the fourantibodies (25) , did not showany staining (Fig. 1B). Substitution of isotype-matched controls forthe primary antibodyalso showed no immunostaining in each specimen (data notshown).


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Fig. 1.Analysis of anti-VEGF-C and anti-VEGF-D specificity by Western blotting(A) and immunohistochemistry (B).A: VEGF-D, protein purified fromVEGF-D-expressing 293EBNA cell conditioned medium; VEGF-C,protein purified from VEGF-C-expressing 293EBNA cell conditionedmedium. VEGF-C and VEGF-D forms corresponding to each band areindicated. Std, MagicMark Western protein standard;VHD, VEGF homology domain; N-pro,NH2-terminal propeptide; C-pro,COOH-terminal propeptide. B, immunohistochemicalstaining of control tissue using the anti-VEGF-C, anti-VEGF-D,anti-VEGF receptor-3, and anti-D2-40 antibodies in the indicatedpositive control tissues and negative control tissue brain.Magnification, x400.

 

 
Localization of Vascular Endothelial Growth Factor C inHuman ProstateTissue.
Positive staining for VEGF-C was observed in prostate cancercells inall samples (37 of 37 samples). Tumor nests showed heterogeneousgranular cytoplasmic staining (Fig. 2A)and membrane-associated staining (Fig. 2B). By comparison, benign prostate glands adjacent totumor nestsdemonstrated weak or no cytoplasmic staining forVEGF-C in epithelialcells (Fig. 2C) .VEGF-C was also detected at low levels in somestromal cells, insmooth muscle cells of arteries, andoccasionally in blood vesselendothelial cells. This is consistent with theprevious demonstrationof VEGF-C protein expression in smooth muscleand endothelial cellsin normal fetal and adult tissues (6) .

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Fig. 2.Localization of VEGF-C (A–C) and VEGF-D (D–H)in human prostate cancer. For VEGF-C, cancer cells showed either strongstaining (A) or weak cytoplasmic and strongmembrane-associated staining (B); benign prostateglands showed weak or no staining (C). For VEGF-D,strong cytoplasmic staining in cancer was observed (D),benign prostate glands stained heterogeneously (E),stromal cells showed positive staining (F), smoothmuscle cells of vessels showed strong staining (G),and vascular endothelial cells stained positively (H).t, tumor; b, benignprostate gland; s, stromal cells; m,smooth muscle cells; v, vessel. Magnification: x400(A, B, G, and F); x200 (Dand H); and x100 (C and E).

 

 
Localization of Vascular Endothelial Growth Factor D inHuman ProstateTissue.
Carcinoma cells in all specimens stained positively for VEGF-D(37 of37 specimens). Heterogeneous cytoplasmic staining was observedincancer cells (Fig. 2, D–F), with membrane-associated staining detected insome cancer cells(Fig. 2F) .Furthermore, staining was much stronger at theedges of tumor neststhan in the central parts in most samples (datanot shown).Heterogenous expression of VEGF-D was alsonoted in the benignglandular epithelial cells adjacent to tumorareas in all 37 cases(Fig. 2E). The staining pattern of the hypertrophic glands and atrophicglandswas similar to that of prostate cancer glands, showing heterogeneitywithin each sample.

Astrong and consistent pattern of VEGF-D expression was observedwithinthe fibromuscular stroma of all prostate cancer specimens (37of 37specimens; Fig. 2F). The smooth muscle cells of blood vessels inand adjacent to areasof tumor expressed VEGF-D strongly (37 of 37samples; Fig. 2G). In contrast, microvessel endothelial cellsshowed inconsistentstaining. Vascular endothelia adjacent tocarcinoma showed positivestaining in some specimens (Fig. 2H).

Localizationof Vascular Endothelial Growth Factor Receptor-3 inProstate Tissue.
VEGF receptor-3 immunostaining decorated the endothelial cellslininga subpopulation of vessels in 18 of 37 specimens. The mediannumberof VEGF receptor-3-positive cross-sections of vesselswas 45 pertissue section (range, 5–519 per tissue section).The vessels werethin-walled and devoid of erythrocytes andneutrophils, suggesting alymphatic nature (Fig. 3, A–D andF). Furthermore, the vessels stained positively using the lymphaticendothelium marker D2-40 (Fig. 3E). The adjacent blood vessels were alwaysnegative for VEGF receptor-3(Fig. 3A). VEGF receptor-3-positive vessels were located within the tumornests, at the tumor periphery, and in benign tissue. Usually,intratumoralVEGF receptor-3-positive vessels were very small orcollapsed (Fig.3, B and C), although large intratumoral VEGFreceptor-3-positive vessels wereobserved occasionally (Fig. 3D). In five cases, four of which had known clinical lymphnodemetastasis, cancer cells were visible within the lumenofD2-40-positive (Fig. 3E), VEGF receptor-3-positive (Fig. 3F)vessels adjacent to tumor areas. The cancer cells werepositive forVEGF-C (Fig. 3G)and VEGF-D (Fig. 3H). The vessels containing the cancer cells werethin-walled, devoidof erythrocytes, and D2-40 positive, indicating lymphaticinvasion.


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Fig. 3.Immunohistochemical staining of VEGF receptor-3 in human prostatecancer. Lymphatic vessels stained positively for VEGF receptor-3,whereas blood vessels stained negatively (A).Positive staining was observed in peritumoral lymphatics (A),small intratumoral vessels (B), collapsedintratumoral vessels (C), and large intratumoralvessels (D). In serial sections (E–H),endothelial cells of vessels were positive for lymphatic marker D2-40 (E)and VEGF receptor-3 (F), and tumor cells inside thevessels showed positive staining for VEGF-C (G) andVEGF-D (H). Arrows, lymphaticvessels; arrowheads, blood vessels; t,tumor. Magnification, x400.

 

 
VEGF receptor-3 expression was not detected in either epithelialcomponentsor stromal cells in the majority of the prostate cancerspecimens.Only 3 of 37 samples (8%) showed focal positive stainingfor VEGFreceptor-3 (data not shown) in cytoplasm of cancercells.

Correlationbetween Vascular Endothelial Growth Factor Receptor-3Expression and Clinicopathological Factors.
As shown in Table 1 , thepresence of VEGF receptor-3-positive vessels(one or more) wassignificantly associated with Gleason grade (P< 0.0001),surgical margin status (P = 0.0069), andlymph node status (P= 0.0002). Consistent with our results, Li etal. (21)found a significant correlation between VEGF receptor-3expressionand lymph node metastasis.
  DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALSAND METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lymphatic invasion is one of the major routes for prostate cancercelldissemination, and pelvic lymph node involvement is the firstsign ofmetastasis in many prostate cancers (2, 27). However, studies on the role of tumorlymphangiogenesis inlymphatic spread have been overshadowed by thefocus on tumorangiogenesis. Recent work on theVEGF-C/VEGF-D/VEGF receptor-3 axishas begun to elucidate the molecular mechanismsinvolved inlymphangiogenesis and lymphatic metastasis. Ourstudy has revealedthe presence of VEGF-C, VEGF-D, and VEGFreceptor-3 protein in humanprostate cancer and suggests that together,these molecules may playa role in the formation of prostate cancer lymph nodemetastasis.

Inthis study heterogeneous staining was demonstrated in prostatecancercells for both VEGF-C and VEGF-D. The intensity of VEGF-D stainingwas much stronger on the edge than in the center of thetumor,indicating the heterogeneity of prostate cancer, whichcontainssubpopulations of cells with different biological propertiessuch asinvasive potential (28 , 29) . Indeed, inhuman gastric carcinoma, VEGF-C immunoreactivity was moreintense inthe invasive tumor component compared with in situtumor (30). In several human cancers, including prostate cancer, immunohistochemicalanalysis has shown that expression of genes andproteins associatedwith angiogenesis and invasion is higher inperipheral zones ofcancers than in their centers (28).

Epithelial-stromalinteractions are thought to play a critical rolein the initiation and promotion of carcinogenesis in prostatecancer (31). Expression of several growth factors involved intumor growth,angiogenesis, invasion, and metastasis has beendemonstrated in bothprostate epithelia and stroma (32,33,34) . It has been suggested thatgrowth factors may havea role in inducing epithelial proliferation and prostaticcarcinogenesisvia both autocrine and paracrine pathways (32,33 , 35 , 36) . In thepresent immunohistochemical study, VEGF-C and VEGF-D werelocalizedto both cancer epithelial cells and stromal cells inall prostatecarcinoma specimens. VEGF-C and VEGF-D have beenshown to enhancetumor growth in tumor mouse models (16, 18). It is possible that VEGF-C, which was overexpressed incarcinomacells compared with benign epithelial cells, may contribute toprostate cancer growth. In contrast, VEGF-D was readily detectedinboth epithelial and carcinoma cells. Because VEGF receptor-3wasdetected in carcinoma cells in only 3 of 37 samples, it islikelythat, if VEGF-C and VEGF-D elicit any direct biological actionsontumor cells, this is achieved via other receptor(s). Thismay be VEGFreceptor-2, or alternatively, there might exist asyet unidentifiedspecific receptor(s) for VEGF-C and VEGF-D.

VEGFreceptor-3 is predominantly localized to lymphatic endothelialcellsin adult tissue (4 , 5 , 37), although its up-regulation in angiogenicblood vessel endotheliumhas been detected in breast cancer (9) and somevascular tumors (38) . In thepresent study,expression of VEGF receptor-3 was restricted to a small proportionofvessels that had morphology characteristic of lymphaticvessels andstained positively using the lymphatic vesselmarker D2-40. There wasa strong association between positive stainingof prostatic lymphaticvessels for VEGF receptor-3 and the presence oflymph node metastasis(Table 1) .Occasionally,tumor emboli were detected within peritumoral VEGFreceptor-3-positivevessels. An essential prerequisite for the formation oflymphatic metastasisis the entry of cancer cells into lymphatic vessels (29). Our study suggests that peritumoral lymphatic vessels mayserve asa route for nodal metastasis in human prostate cancer.Similar to ourfindings, tumor invasion into peritumoral lymphaticvessels has beendemonstrated in human colorectal carcinoma (24)and head and neck cancer (39) .Tumor emboli havealso been detected in intratumoral lymphatics of VEGF-C- andVEGF-D-transfected tumor mouse models (15,18) .

VEGF-Chas been shown to be capable of increasing vascular permeabilityandmay enhance cancer cell dissemination via lymphatic vasculatureinsome human tumors (9 , 40 ,41) . Because VEGF-D shares 61% sequencewith VEGF-C (12), it is conceivable that VEGF-D may havesimilar functional roles tothose of VEGF-C. Our study demonstrates thepresence of VEGF-C- andVEGF-D-positive carcinoma cells within VEGFreceptor-3-positivevessels. Moreover, the presence of VEGFreceptor-3-positive vesselswas correlated with lymph node metastasis inprostate cancer. Takentogether, these results suggest that VEGF-C andVEGF-D secreted bycancer cells may activate VEGF receptor-3expressed on theendothelial cells of adjacent lymphatic vesselsvia a paracrinemechanism to induce lymphatic invasion,possibly by modifying vesselpermeability (9, 40 ,41) . This would provide a route for tumor metastasisvia thelymphatic vessels to the lymph nodes.

Insummary, our results demonstrate that both VEGF-C and VEGF-Darewidely expressed in human prostate carcinoma. VEGF-D, but notVEGF-C,is also abundantly expressed in adjacent benign prostateepithelia.In contrast, VEGF receptor-3 is up-regulated invessels in a subsetof prostate cancers. The demonstration of tumoremboli in VEGFreceptor-3-positive vessels and the significantcorrelation betweenthe presence of prostatic VEGF receptor-3-positivevessels and lymphnode metastasis provide tantalizing evidencefor a role of VEGFreceptor-3 signaling in the development oflymph node metastasis. Ahumanized monoclonal antibody against VEGF-Ahas recently beendeveloped for therapeutic use in metastaticcolorectal cancer (42), and it is possible that VEGF-C or VEGFreceptor-3 may provideuseful clinical targets for developing newtherapeutic agents towardprostate lymph node metastasis. VEGF-D may alsoprovide a potentialtherapeutic target for inhibiting prostatecancer progression, giventhe stronger staining for VEGF-D observed atthe edge of tumor nests;however, the role of VEGF-D in prostate cancer requiresfurther analysis.
  ACKNOWLEDGMENTS
 
We thank Anthony Penington and Wei Chun Wang for statisticaladviceand Annet Hammacher and Erik Thompson for helpful discussionandcritical reading of this manuscript. We also acknowledge theimportant contributions of Sue Henshall, John Grygiel, and PhillipStricker toward the Garvan Institute Prostate Cancer TissueBank andof Jeremy Goad, Pamela Crouch, Bonnie Dopheide, andCourtney Thornelytoward the St. Vincent’s Hospital MelbourneTissue Bank.
  FOOTNOTES
 
Grant support: Y. Zeng is supported by aUniversity of MelbourneInternational Post-Graduate Research Scholarship. S. StackerandM. Achen are supported by a Program Grant from the National Healthand Medical Research Council of Australia and by Senior ResearchFellowships from the Pharmacia Foundation and the National HealthandMedical Research Council of Australia, respectively.

The costs ofpublication of this article were defrayed in part bythe payment of page charges. This article must therefore beherebymarked advertisement in accordance with 18 U.S.C.Section 1734solely to indicate this fact.

Note:M. Baldwin is currently in the Department of Molecular Oncology,Genentech Inc., South San Francisco, California.

Requestsfor reprints: Elizabeth D. Williams, Bernard O’BrienInstituteof Microsurgery, 42 Fitzroy Street, Fitzroy, Victoria 3065,Australia. Phone: 61-3-9288-4018; Fax: 61-3-9416-0926; E-mail:edw@medstv.unimelb.edu.au.

Received10/21/03; revised 4/ 8/04; accepted 4/19/04.
  REFERENCES
Top
ABSTRACT
INTRODUCTION
MATERIALSAND METHODS
RESULTS
DISCUSSION
REFERENCES
 

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  28. Gershenwald JE, Fidler IJ.Cancer targeting lymphatic metastasis. Science (Wash DC), 296:1811-2, 2002. [Abstract/FreeFull Text]
  29. Fidler IJ. The pathogenesis ofcancer metastasis: the "seed and soil" hypothesis revisited. Nat RevCancer, 3: 453-8, 2003.[CrossRef][Medline]
  30. Amioka T, Kitadai Y, Tanaka S,et al Vascular endothelial growth factor-C expression predicts lymphnode metastasis of human gastric carcinomas invading the submucosa. EurJ Cancer, 38: 1413-9, 2002.[CrossRef][Medline]
  31. Cunha GR. Role of stroma incarcinogenesis of the prostate. Differentiation, 70:473-85, 2002. [CrossRef][Medline]
  32. Haughney PC, Hayward SW, DahiyaR, Cunha GR. Speciesspecific detection of growth factor gene expressionin developing murine prostatic tissue. Biol Reprod, 59:93-9, 1998. [Abstract/FreeFull Text]
  33. Hayward SW, Haughney PC, RosenMA, et al Interactions between adult human prostatic epithelium and raturogenital sinus mesenchyme in a tissue recombination model.Differentiation, 63: 131-40, 1998.[CrossRef][Medline]
  34. Tuxhorn JA, McAlhany SJ, YangF, Dang TD, Rowley DR. Inhibition of transforming growth factor-betaactivity decreases angiogenesis in a human prostate cancer-reactivestroma xenograft model. Cancer Res, 62: 6021-5,2002. [Abstract/FreeFull Text]
  35. Taylor TB, Ramsdell JS.Transforming growth factor-alpha and its receptor are expressed in theepithelium of the rat prostate gland. Endocrinology, 133:1306-11, 1993. [Abstract]
  36. Cohen D, Simak R, Fair W, et alExpression of transforming growth factor-alpha and the epidermal growthfactor receptor in human prostate tissues. J Urol, 152:2120-4, 1994. [Medline]
  37. Jussila L, Valtola R, PartanenTA, et al Lymphatic endothelium and Kaposi’s sarcoma spindle cellsdetected by antibodies against the vascular endothelial growth factorreceptor-3. Cancer Res, 58: 1599-604, 1998.[Abstract]
  38. Partanen TA, Alitalo K,Miettinen M. Lack of lymphatic vascular specificity of vascularendothelial growth factor receptor-3 in 185 vascular tumors. Cancer, 86:2406-12, 1999. [CrossRef][Medline]
  39. Beasley NJ, Prevo R, Banerji S,et al Intratumoral lymphangiogenesis and lymph node metastasis in headand neck cancer. Cancer Res, 62: 1315-20, 2002.[Abstract/FreeFull Text]
  40. Yonemura Y, Fushida S, Bando E,et al Lymphangiogenesis and the vascular endothelial growth factorreceptor (VEGFR)-3 in gastric cancer. Eur J Cancer, 37:918-23, 2001. [CrossRef][Medline]
  41. Niki T, Iba S, Tokunou M, et alExpression of vascular endothelial growth factors A, B, C, and D andtheir relationships to lymph node status in lung adenocarcinoma. ClinCancer Res, 6: 2431-9, 2000.[Abstract/FreeFull Text]
  42. Kabbinavar F, Hurwitz HI,Fehrenbacher L, et al Phase II, randomized trial comparing bevacizumabplus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patientswith metastatic colorectal cancer. J Clin Oncol, 21:60-5, 2003. [Abstract/FreeFull Text]

AmericanAssociation for Cancer Research

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LymphedemaPeople Angiogenesis Related Pages:

Angiogenesis

http://www.lymphedemapeople.com/thesite/angiogenesis.htm

Angiogenesis and Cancer

http://www.lymphedemapeople.com/thesite/angiogenesis_and_cancer.htm

Angiogenesis and CancerControl

http://www.lymphedemapeople.com/thesite/angiogenesis_and_cancer_control.htm

Angiogenesis Inhibitorsand Cancer

http://www.lymphedemapeople.com/thesite/angiogenesis_inhibitors_and_cancer.htm

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LymphedemaPeople Lymphangiogenesis Related Pages:

The Formation ofLymphatic Vessels and Its Importance in the Setting of Malignancy

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_formation_of_lymphatic_vessels_and_malignancy.htm

LymphangiogenesisLymphedema and Cancer

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_lymphedema_and_cancer.htm

Lymphangiogenesis andGastric Cancer

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_and_gastric_cancer.htm

Lymphangiogenesis in Headand Neck Cancer

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_in_head_and_neck_cancer.htm

Lymphangiogenesis andKaposi's Sarcoma VEGF-C

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_and_kaposis_sarcoma_vegfc.htm

Lymphangiogenesis inWound Healing

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_in_wound_healing.htm

A model for gene therapyof human hereditarylymphedema

http://www.lymphedemapeople.com/thesite/a_model_for_gene_therapy_of_human_lymphedema.htm

VEGFR-3 Ligands andLymphangiogenesis (1)

http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_1.htm

VEGFR-3 Ligands andLymphangiogenesis (2)

http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_2.htm

VEGFR-3 Ligands andLymphangiogenesis (3)

http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_3.htm

Vascular EndothelialGrowth Factor; VEGF

http://www.lymphedemapeople/thesite/vascular_endothelial_growth_factor_VEGF.htm

VEGF-D is the strongestangiogenic andlymphangiogenic effector

http://www.lymphedemapeople.com/thesite/VEGFD_Angiogenic_Lymphangiogenic_Effector.htm

Inhibition of LymphaticRegeneration by VEGFR3

http://www.lymphedemapeople.com/thesite/Inhibition_of_Lymphatic_Regeneration_by_VEGFR3.htm

VEGFR3 and Metastasis inProstate Cancer

http://www.lymphedemapeople.com/thesite/VEGFR3_and_Metastasis_in_Prostate_Cancer.htm

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LymphedemaPeople Genetics,Research, Lymphangiogenesis, Angiogenesis Forum

http://www.lymphedemapeople.com/phpBB2/viewforum.php?f=16

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Joinus as we work for lymphedema patients everywehere:

Advocatesfor Lymphedema

Dedicatedto be an advocacy group forlymphedema patients. Working towards education, legal reform, changinginsurancepractices, promoting research, reaching for a cure.

http://health.groups.yahoo.com/group/AdvocatesforLymphedema/

Subscribe:AdvocatesforLymphedema-subscribe@yahoogroups.com

PatO'Connor

LymphedemaPeople / Advocates forLymphedema

===========================

For information aboutLymphedema

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema\

For Information aboutLymphedema Complications

http://www.lymphedemapeople.com/wiki/doku.php?id=complications_of_lymphedema

For Lymphedema PersonalStories

http://www.lymphedemapeople.com/phpBB2/viewforum.php?f=3

For information about Howto Treat a Lymphedema Wound

http://www.lymphedemapeople.com/wiki/doku.php?id=how_to_treat_a_lymphedema_wound

For information aboutLymphedema Treatment 

http://www.lymphedemapeople.com/wiki/doku.php?id=treatment

For information aboutExercises for Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=exercises_for_lymphedema

For information on InfectionsAssociated with Lymphedema

http://www.lymphedemapeople.com/wiki/doku.php?id=infections_associated_with_lymphedema

Forinformation on Lymphedema inChildren

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_in_children

LymphedemaGlossary

http://www.lymphedemapeople.com/wiki/doku.php?id=glossary:listing

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Lymphedema People - SupportGroups

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Childrenwith Lymphedema

The time has come for families, parents, caregivers to have a supportgroup oftheir own. Support group for parents, families and caregivers ofchilren withlymphedema. Sharing information on coping, diagnosis, treatment andprognosis.Sponsored by Lymphedema People.

http://health.groups.yahoo.com/group/childrenwithlymphedema/

Subscribe: childrenwithlymphedema-subscribe@yahoogroups.com

......................

LipedemaLipodema Lipoedema

No matter how you spell it, this is another very little understood andtotallyfrustrating conditions out there. This will be a support group forthosesuffering with lipedema/lipodema. A place for information, sharingexperiences,exploring treatment options and coping.

Come join, be a part of the family!

http://health.groups.yahoo.com/group/lipedema_lipodema_lipoedema/?yguid=209645515

Subscribe: lipedema_lipodema_lipoedema-subscribe@yahoogroups.com

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MENWITH LYMPHEDEMA

If you are aman with lymphedema; a man with a loved one with lymphedema who you aretryingto help and understand come join us and discover what it is to be themasterinstead of the sufferer of lymphedema.

http://health.groups.yahoo.com/group/menwithlymphedema/

Subscribe: menwithlymphedema-subscribe@yahoogroups.com

......................

AllAbout Lymphangiectasia

Support group for parents, patients, children who suffer from all formsoflymphangiectasia. This condition is caused by dilation of thelymphatics. It canaffect the intestinal tract, lungs and other critical body areas.

http://health.groups.yahoo.com/group/allaboutlymphangiectasia/

Subscribe: allaboutlymphangiectasia-subscribe@yahoogroups.com

......................

LymphaticDisorders Support Group @ Yahoo Groups

While we have a numberof support groups for lymphedema... there is nothing out there forotherlymphatic disorders. Because we have one of the most comprehensiveinformationsites on all lymphatic disorders, I thought perhaps, it is time thatone beoffered.

DISCRIPTION

Information and support for rare and unusual disorders affecting thelymphsystem. Includes lymphangiomas, lymphatic malformations,telangiectasia,hennekam's syndrome, distichiasis, Figueroa
syndrome, ptosis syndrome, plus many more. Extensive database ofinformationavailable through sister site Lymphedema People.

http://health.groups.yahoo.com/group/lymphaticdisorders/

Subscribe: lymphaticdisorders-subscribe@yahoogroups.com

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LymphedemaPeople New Wiki Pages

Haveyou seen our new “Wiki”pages yet?  Listedbelow are just asample of the more than 140 pages now listed in our Wiki section. Weare alsoworking on hundred more.  Comeandtake a stroll! 

LymphedemaGlossary 

http://www.lymphedemapeople.com/wiki/doku.php?id=glossary:listing 

Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema 

ArmLymphedema  

http://www.lymphedemapeople.com/wiki/doku.php?id=arm_lymphedema 

LegLymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=leg_lymphedema 

AcuteLymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=acute_lymphedema 

TheLymphedema Diet 

http://www.lymphedemapeople.com/wiki/doku.php?id=the_lymphedema_diet 

Exercisesfor Lymphedema  

http://www.lymphedemapeople.com/wiki/doku.php?id=exercises_for_lymphedema 

Diureticsare not for Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=diuretics_are_not_for_lymphedema 

LymphedemaPeople Online SupportGroups 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_people_online_support_groups 

Lipedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=lipedema 

Treatment 

http://www.lymphedemapeople.com/wiki/doku.php?id=treatment 

Lymphedemaand Pain Management 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pain_management 

ManualLymphatic Drainage (MLD) and Complex Decongestive Therapy (CDT)

http://www.lymphedemapeople.com/wiki/doku.php?id=manual_lymphatic_drainage_mld_complex_decongestive_therapy_cdt 

InfectionsAssociated with Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=infections_associated_with_lymphedema 

Howto Treat a Lymphedema Wound 

http://www.lymphedemapeople.com/wiki/doku.php?id=how_to_treat_a_lymphedema_wound 

FungalInfections Associated withLymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=fungal_infections_associated_with_lymphedema 

Lymphedemain Children 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_in_children 

Lymphoscintigraphy 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphoscintigraphy 

MagneticResonance Imaging 

http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging 

Extraperitonealpara-aortic lymph node dissection (EPLND) 

http://www.lymphedemapeople.com/wiki/doku.php?id=extraperitoneal_para-aortic_lymph_node_dissection_eplnd 

Axillarynode biopsy 

http://www.lymphedemapeople.com/wiki/doku.php?id=axillary_node_biopsy

SentinelNode Biopsy 

http://www.lymphedemapeople.com/wiki/doku.php?id=sentinel_node_biopsy

 SmallNeedle Biopsy - Fine Needle Aspiration 

http://www.lymphedemapeople.com/wiki/doku.php?id=small_needle_biopsy 

MagneticResonance Imaging 

http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging 

LymphedemaGene FOXC2

 http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_foxc2

 LymphedemaGene VEGFC

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_vegfc

 LymphedemaGene SOX18

 http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_sox18

 Lymphedemaand Pregnancy

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pregnancy

Home page:Lymphedema People

http://www.lymphedemapeople.com

Page Updated:Dec. 17, 2011