Enhancement of Angiogenesis, Tumor Growth, and Metastasis by … · Neovascularization is a critical requirement for tumor growth and metastasis formation. Folkman et al. (1, 2) demon-strated

Enhancement of Angiogenesis, Tumor Growth, and Metastasis byTransfection of Vascular Endothelial Growth Factor into LoVoHuman Colon Cancer Cell Line

Yoko Kondo,1 Shigeki Arii, Akira Mori,Masaharu Furutani, Tsutomu Chiba, andMasayuki ImamuraDivision of Gastroenterology and Hepatology, Department of InternalMedicine [Y. K., T. C.] and Department of Surgery and SurgicalBasic Science [Y. K., S. A., A. M., M. F., M. I.], Graduate School ofMedicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan

ABSTRACTThe expression of vascular endothelial growth factor

(VEGF), a highly potent angiogenic molecule, is thought tobe correlated with the development of colon cancer; how-ever, direct evidence for a role of VEGF in metastasis islacking. This study was designed to more directly establishthe role of VEGF in the growth and metastasis of humancolon cancer using a genetically engineered cancer cell line.A stable VEGF gene-transfected human colon cancer cellline, LoVo, was made by genetic manipulation using eukary-otic expression constructs designed to express the completeVEGF121 cDNA in the sense orientation. Transfected cloneswere screened for VEGF121 mRNA expression by Northernblot analysis and for VEGF121 protein expression by West-ern blot analysis. Consequently, we obtained S17 cells thatexpressed a high level of both VEGF mRNA and VEGFprotein. A vector-transfected clone (V7 cell) was used as acontrol. The experiment with the dorsal air sac methodrevealed that S17 cells elicited a stronger directional out-growth of capillaries than V7 cells. S17 cells formed faster-growing tumors than did V7 cells when xenografted s.c. intonude mice, although there was no significant difference intheir in vitro proliferation. Tumors derived from S17 cellshad more vascularity, as assessed by counting capillaryvessels after staining with factor VIII, than did tumorsderived from V7 cells (P < 0.05). With regard to the meta-static potential, S17 cells exhibited a higher capacity for bothhepatic metastasis after the splenic portal inoculation andperitoneal dissemination after i.p. injection than did V7cells. However, S17 cells showed no apparent metastasis,despite their rapid growth after orthotopic implantation. In

conclusion, the present study showed clearly that VEGFplays an important role in cancer growth due to stimulationof angiogenesis by accelerating cell growth after reachingthe target organs.

INTRODUCTIONNeovascularization is a critical requirement for tumor

growth and metastasis formation. Folkmanet al. (1, 2) demon-strated in ground-breaking studies that solid tumors establishtheir own blood supplies by encouraging the growth of newblood vessels into the tumor tissue to extend beyond the smallsize of approximately 1 mm3. Once the tumor vasculature isestablished, solid tumors acquire the nutrients that enable themto expand exponentially and ultimately to metastasize (2–4). Acommonly cited model of metastasis holds that once neoplasticcells leave a primary tumor and reach the circulation, a series ofhurdles must be overcome to establish a metastatic deposit.Numerous angiogenic factors that regulate this process havebeen identified (5). Among theses factors is VEGF,2 which hasbeen implicated in the neovascularization of a wide variety oftumors (6–10). VEGF, also known as vascular permeabilityfactor, is aMr 36,000–45,000 dimeric glycoprotein that hasbeen identified in conditioned media from numerous cell linesand is expressed in various normal tissues (11) and most humanand animal tumors (11, 12), including ascites tumors (6–19).

Recently, several studies suggested that VEGF is the an-giogenic factor associated most closely with induction andmaintenance of the neovasculature in human tumors (7, 17–19),and clinical studies have implicated VEGF expression in tumorprogression and metastasis. Furthermore, using a murine modelsystem for colon cancer, Warrenet al. (20) demonstrated that amonoclonal antibody against VEGF inhibits s.c. tumor forma-tion in a dose- and time-dependent manner and reduces thenumber and size of liver metastases. There have also been a fewstudies focusing on the suppression of tumor growth usingVEGF antisense or c-src antisense expression vectors (20–23).These studies have thus been aimed at blocking VEGF, and theyhave resulted in the fairly general agreement that VEGF is oneof the molecules that can be targeted to stop tumor growth andmetastasis (24–28).

To our knowledge, however, there have been no studiesthat attempted to analyze the changes in malignant potentialinduced by high expression of VEGF. Considering this back-ground, the current study was designed to investigate the effectof high constitutive expression of VEGF on tumor growth andmetastasis, thereby establishing the role of VEGF in colon

Received 8/9/99; revised 11/1/99; accepted 11/15/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 To whom requests for reprints should be addressed, at Department ofSurgery and Surgical Basic Science, Graduate School of Medicine,Kyoto University of Medicine, Kyoto University, 54-Shogoin Kawara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751-3445; Fax:81-75-751-3219; E-mail: [email protected].

2 The abbreviations used are: VEGF, vascular endothelial growth factor;BSD, blasticidin S deaminase.

622 Vol. 6, 622–630, February 2000 Clinical Cancer Research

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cancer, based on our previous reports that VEGF mRNA ex-pression was closely correlated with highly malignant potentialin the human colon cancer.

MATERIALS AND METHODSCell Line and Culture. LoVo, a human colon cancer cell

line that expresses a low level of VEGF mRNA was obtainedfrom the Health Science Research Bank and maintained inRPMI 1640 supplemented with 10% fetal bovine serum (Bio-Whittaker), penicillin (100 units/ml), and streptomycin (100mg/ml) in a humidified atmosphere of 5% CO2. The crudewild-type LoVo cells were cloned by the limiting dilutionmethod.

Subcloning of VEGF121 into pCAG-BSD and DNATransfection. The full-length cDNA for VEGF121 was madefrom reverse transcription-PCR products of human colon cancerspecimens. We used the following PCR primers, which werebased on the human VEGF cDNA sequence: (a) sense primer,59-CCTCCGAAACCATGAACTTT-39; and (b) antisenseprimer, 59-AGAGATCTGGTTCCCGAAAC-39. The humanVEGF121 cDNA was subcloned into pBluescript II SK(2), andwe confirmed the sequence using an ALF red DNA Sequencer(Pharmacia Biotech, Uppsala, Sweden). A eukaryotic expres-sion vector, pCAG-BSD, was newly constructed with two partsof pCAGGS (a kind gift from Dr. J. Miyazaki, Osaka Univer-sity, Osaka, Japan; Ref. 29) and pMAM2-BSD (Kaken, Tokyo,Japan). The human VEGF121 cDNA was cloned into theXhoIrestriction enzyme site of pCAG-BSD to create the plasmidpCAG-BSD-VEGF, in which the transcription of VEGF wasconstitutively driven by the CAG enhancer promoter, and thedrug-resistant selection gene BSD was present (30). The orien-tation of the VEGF insert was confirmed by restriction mapping.

The cloned wild-type LoVo cells were cultured in 6-wellplates. DNA transfections were performed using 4ml of Lipo-fectAMINE (Life Technologies, Inc., Gaithersburg, MD) and 1mg of pCAG-BSD-VEGF or 1mg of pCAG-BSD vector alone(as a control) per well. The cells were cultured in 1 ml ofserum-free OPTI-MEM (Life Technologies, Inc.) medium con-taining the DNA/LipofectAMINE complex for 4 h and thensupplemented with 1 ml of RPMI 1640 containing 20% FBS.After 48 h, the cells in each well were trypsinized and replated1:20 into two 10-cm culture dishes in complete culture mediumcontaining 10mg/ml blasticidin S (Kaken) to select transfec-tants. Some cell death was observed after 3–4 days in culture,and discrete colonies were apparent by 7 days after selection.Twenty-two individual colonies of sense VEGF transfectantsand 10 individual colonies of vector transfectants were pickedand pooled, and cells were maintained in complete culturemedium with 10 mg/ml BSD to obtain stable transfectants.These transfectants were individual clones.

RNA Isolation and Northern Blot Analysis. All trans-fectants were grown to confluence in RPMI 1640 in 10-cm2

Petri dishes, and total cellular RNA was extracted using Trizolregent (Life Technologies, Inc.). RNA samples were resolved byelectrophoresis through 1% agarose-formaldehyde gels andtransferred to Hybond-N1 nylon membrane filters (Amersham,Buckinghamshire, United Kingdom). The probes were labeledwith [a-32P]deoxycytidine 59-triphosphate using the Megaprime

Random Primer DNA Labeling Kit (Amersham). Hybridizationwas performed at 65°C for 2 h in Rapid Hybridization Solution(Amersham) with a labeled probe. After hybridization, the fil-ters were washed twice with 23SSC (13SSC5 0.15M NaClplus 0.015M sodium citrate) and 0.1% SDS at room temperaturefor 10 min, washed once with 13 SSC and 0.1% SDS at 65°Cfor 15 min, and then washed twice with 0.13SSC and 0.1%SDS at 65°C for 15 min. Autoradiographs were made using

Fig. 1 Northern blot analysis and Western blot analysis.A, expressionof VEGF mRNA in LoVo cells transfected with VEGF.wild type, crudewild-type LoVo; PC, HT1080 as a positive control;endogeneous, en-dogeneous transcript;transfected, transcript from transfected plasmid;GAPDH, hybridization with a glyceraldehyde-3-phosphate-dehydrogen-ase probe as a control for the quantity of RNA.B, expression andsecretion of VEGF protein in LoVo cells transfected with VEGF.rhVEGF, recombinant human VEGF as a positive control.C, expressionof VEGF mRNA in the s.c. tumors formed by S17 cells and V7 cells.HT1080, positive control;endogeneous, endogeneous transcript;trans-fected, transcript from transfected plasmid;GAPDH, hybridization witha glyceraldehyde-3-phosphate-dehydrogenase probe as the control forthe quantity of RNA.

623Clinical Cancer Research



X-ray film and an imaging plate and analyzed with a BAS 2000Image Analyzer (Fuji, Tokyo, Japan). The membranes wererehybridized with a human glyceraldehyde-3- phosphate dehy-drogenase probe.

Western Blot Analysis. Nearly confluent cells werewashed twice with PBS and replenished with serum-free me-dium. After 24 h, the medium was collected, centrifuged for 10min at 3000 rpm to remove cell debris, and concentrated withacetonic sulfate. The proteins were separated by electrophoresison 12% polyacrylamide gels and transferred to Immobilon-Pmembranes (Millipore Corp., Bedford, MA) by electrotransfer.After blocking with 5% milk in 0.1% Tween 20 in PBS solution,the membrane-bound proteins were probed with the primaryantibody (1:5000 dilution of polyclonal rabbit antihumanVEGF; Santa Cruz Biochemicals, Santa Cruz, CA). The mem-brane was washed, and the secondary antibody labeled withhorseradish peroxidase (1:2000 dilution of goat antirabbit im-munoglobulin; Zymed) was applied. After washing, the boundantibody complexes were detected using an enhanced chemilu-minescence reagent (Amersham) and X-ray film, as describedby the manufacturers.

Determination of in Vitro Cell Proliferation. Into eachwell of a 6-well plate, 13 105 stably transfected LoVo cellswere seeded in RPMI 1640–10% FBS containing 10mg/mlblasticidin S. Cells were fed with fresh medium every other day.On the days indicated, cells were released by exposure totrypsin, pelleted, resuspended in the same medium, and countedusing a hemocytometer.

Detection of Neovascularization with Dorsal Air SacMethod. The dorsal air sac method was used as describedpreviously (28). Sense transfectant (S17) and vector transfectant(V7) cells were washed once with PBS and suspended in PBS ata concentration of 23 106 cells/0.2ml. A Millipore chamber(diameter, 10 mm; filter pore size, 0.45mm) was filled with 0.2ml of the cells and implanted s.c. into the dorsal side of the mice.At 5 days after implantation, the mice were anesthetized andfixed in the prone position. A wide, rectangular incision wasmade in the skin on the dorsal side, and the skin was carefullyablated. To locate the chamber-contacting region, a ring (Mil-lipore) of the same shape as the chamber was placed onto thes.c. tissues adjacent to the chamber region, and the area wasphotographed. For histological analysis, all samples were takenfrom this skin area, embedded in 10% formaldehyde, and slicedinto 4-mm-thick vertical sections, which were then stained withH&E.

s.c. Tumors. Confluent cultures of sense VEGF-trans-fected LoVo cells (S17) and vector-transfected LoVo cells (V7)grown in 10-cm2 Petri dishes were harvested by a brieftrypsinization (0.05% trypsin/0.02% EDTA in Ca21/Mg21-freePBS), washed several times in Ca21/Mg21-free PBS, and re-suspended at a final concentration of 33 106 cells/0.2 ml inPBS. Athymic BALB/c nude mice (4-week-old males obtainedfrom Charles River, Yokohama, Japan) were housed in steril-ized cages and injected s.c. with 33 106 viable tumor cells.Animals were observed daily for tumor growth, and s.c. tumorswere measured every 7 days. Tumor volume was calculated as1/2 3 length3 width2 (length. width).

Liver Metastases. Sense VEGF transfectants (S17) andvector transfectants (V7) were grown to confluence and har-vested as described above for s.c. injection and resuspended inPBS at a concentration of 23 106 cells/0.2 ml. Athymic micewere anesthetized by ether inhalation and wiped with antisepticsolution, and the spleen was exteriorized through a left-flankincision. Two million cells were slowly injected into the splenicpulp through a 30-gauge needle. All animals were killed on day28. The spleens and livers were excised, and the metastaseswere checked by H&E staining.

i.p. Injection. A single-cell suspension of 23 106 cellswith a viability of .95% was injected into the peritoneal cavityof the mice. The mice were monitored daily for evidence ofdisease (abdominal swelling, hunched posture, and listlessness)and were killed when moribund or approximately 42 days afterthe i.p. injection. All mice were necropsied, and the pattern(discrete solid lesions, carcinomatosis) and extent of abdominaldisease (size and number of lesions, volume of ascites) weredetermined.

Orthotopic Implantation to the Cecum. AthymicBALB/c nude mice (4–5-week-old males) were anesthetized by

Fig. 2 Angiogenesis evaluation by the dorsal air sac method and his-tological analysis of skin vertical sections.A andB, representative viewsfrom the inner side of the dorsal skin of mice 5 days after implantationof a chamber filled with S17 cells.C, dorsal skin of mice 5 days afterimplantation of V7 cells.

Table 1 Characteristics of the sense VEGF transfectants and vector transfectants

Transfectant

In vitro cellgrowth doubling

time (hr)s.c. tumor volume

(mean6 SD)

Liver metastasisratio after spleen

injection

Tumor volume afterinoculation to cecal wall

(mean6 SD)

Peritonealdissemination

ratio

S17 cells 69 52126 1612.04 mm3 (n 5 10) 5/10 (50%) 7436 393.3 mm3 (n 5 7) 9/10 (90%)V7 cells 72 5286 181.78 mm3 (n 5 10) 0/10 (0 %) 14.46 2.79 mm3 (n 5 7) 0/10 (0 %)

624 VEGF Gene Transfection into Colon Cancer Cell



ether inhalation, and the abdomen was prepared for sterilesurgery. A small abdominal incision was made, and the cecumwas identified. Tissue blocks of about 3 mm in diameter of eachs.c. tumor were implanted to the serosal side. The abdomen wasclosed with continuous nylon sutures. The animals were killed12 weeks later, and abdominal organs and the thorax wereexamined for the presence of macroscopic “primary” cecaltumors and metastasis. Organs including the cecum, liver, mes-enteric lymph nodes, and lungs were removed and examinedhistologically as described below.

Histological Examination of Metastases. Specimensfor histological examination were fixed in 4% paraformalde-hyde for 24 h. Representative sections of cecum, lymph nodes,

lung, and liver were also cut and embedded in paraffin.Four-mm sections were then cut and stained with H&E.

Microvessel Staining and Counting. The streptavidin-biotin-peroxidase complex method was performed using astreptavidin-biotin kit. Antihuman von Willebrand factor (factorVIII) rabbit serum (DAKO, Copenhagen, Denmark) was used ata dilution of about 1:100 in BSA. As negative controls forimmunohistochemical staining, tissue sections were treated withnormal rabbit serum instead of primary antibodies. Microvesseldensity was assessed in tumor areas showing the highest densityof staining, as determined by an initial scan with low magnifi-cation (340). For vessel counting, one field magnified 200-fold(high-power field,i.e.,320 objective and310 ocular, 0.739 per

Fig. 3 A andB, H&E staining ofvertical skin sections 5 days afterimplantation of S17 cells (A) andV7 cells (B).




field) in each of three vascularized areas was counted, and theaverage counts were recorded (31).

Statistical Analysis. Microvessel densities in the s.c. tu-mors and cecal primary tumors were compared by the Wil-coxon-Mann-Whitney test. The statistical analysis was doneusing JMP 3.1 software (SAS Institute, Cary, NC), and aP valueof less than 0.05 based on a two-tailed test was considered toindicate statistical significance.

RESULTSSelection of LoVo Transfected with VEGF. We first

constructed the eukaryotic expression vectors pCAG-BSD-VEGF for VEGF expression and pCAG-BSD as a control.Because wild-type LoVo human colon cancer cell lines wereheterogeneous even in their morphology as seen by light mi-croscopy, we cloned them to obtain homogeneous parental cellsbefore transfection. Northern blot analysis (Fig. 1A) revealed ahigh level of VEGF mRNA expression in sense VEGF-trans-fected LoVo (S17) cells and very low levels of expression intwo cell lines, parent LoVo cells and vector-transfected LoVo(V7 cells). Two cell lines, S17 and V7, were chosen for furtherstudy. Western blot analysis revealed the presence of VEGFprotein only in the conditioned medium of S17 cells (Fig. 1B).

Proliferation Rate of the Transfectants in Vitro. Therewas no significant difference between the proliferation rates ofS17 cells and V7 cells (Table 1).

Angiogenesis Evaluation by Dorsal Air Sac Method.Representative images of the area adjacent to the s.c. implantedchamber are shown in Fig. 2. On the fifth day after implantationof the S17 cells, the blood vessels had expanded and branchedout from the large blood vessels. In some of the mice injectedwith S17 cells, severe hemorrhaging from these vessels wasobserved. Fig. 3 shows representative views of H&E staining ofskin sections 5 days after implantation. The surface with S17cells attached showed the development of small vessels andlarge hemorrhages. On the other hand, neither markedly in-creased vessels nor hemorrhages were seen among the miceinjected with V7 cells.

Growth Rate of the Transfectants and VEGF mRNAExpression in Vivo. s.c. injection of equal numbers of S17cells and V7 cells into BALB/c nude mice gave rise to tumorsthat grew at the same rate until about 40 days, but thereafter,S17 cells grew faster than V7 cells (Figs. 4 and 5). The sizes oftumors at 90 days were 52156 1612.04 (n5 10) and 5286181.78 mm3 (n 5 10) for S17 and V7 cells, respectively (Table1). VEGF mRNA expression in the s.c. tumors is shown in Fig.1C. s.c. tumors derived from S17 cells had high levels of VEGFmRNA expression for as long as 90 days after the inoculation.

Microvessel Density of s.c. Tumors. The microvesseldensities in S17 and V7 tumors were 6.636 1.08 and 2.2961.08, respectively (P5 0.021).

Hepatic Metastases after Splenic Portal Injection.Among 10 mice injected with S17 cells, macroscopic metastasisand microscopic metastasis in the liver were seen in 5 mice(50%), and the percentage of mice with tumor growth in thespleen was 80%, whereas no mice injected with V7 cells hadeven microscopic metastasis in the liver or growth in the spleen(Fig. 6; Table 1).

Spontaneous Metastasis after Implantation into theCecum. The volumes of the tumors derived from S17 and V7cells were 7436 393.3 (n5 7) and 14.46 2.79 mm3 (n 5 7),respectively (P5 0.02; Table 1). Both types of tumors showedmicrovessel metastasis. However, no obvious metastasis in lung,liver, or mesenteric lymph nodes was observed in either group,except for lymph node and lung metastasis in one of seven miceof the S17-inoculated group (Fig. 6).

Peritoneal Dissemination. Macroscopic observation 28days after inoculation revealed numerous tumors (up to 10 mmin diameter) in the peritoneal cavity in 9 of 10 mice injectedwith S17 cells (Fig. 7). A large number of small white tumornodules were formed in the greater omentum, mesenterium, andgonadal fat. Occasionally, smaller nodules were seen at thesurface of the stomach and intestine. On the other hand, neithermetastasis nor ascites were seen in the mice injected with V7cells (Table 1).

DISCUSSIONWe reported previously that expression of VEGF mRNA

in colon cancer was correlated with tumor progression, in-cluding metastasis (32), and more recently, we obtainedevidence that tumor angiogenesis occurred along with tumordevelopment and was partially associated with VEGF expres-sion in adenomas and the early stages of human colorectalcancer (33). Kumaret al. found increased serum levels ofVEGF in patients with advanced (Dukes’ C) colorectal can-cers compared with patients with early-stage disease (32,34 –38).These findings suggest a potential role of VEGF inthe development and metastasis of colon cancer. However, nodirect evidence has been provided indicating that VEGF isresponsible for the acceleration of the appearance of malig-nant phenotypes in colon cancer. Therefore, we attempted toclarify the role of VEGF in tumor growth, neovasculariza-tion, and metastasis of human colon cancer by using cellstransfected with the VEGF gene.

Fig. 4 In vivo growth curves of S17 cells and V7 cells.f, S17 (n510); F, V7 (n 5 10).f, F, andbars show the mean values and SDs.




We found that sense VEGF transfectants have a growthadvantagein vivo but not in vitro. The rapidin vivo growthof sense VEGF transfectants was due to vigorous tumorangiogenesis, which was confirmed using the dorsal air sacmethod. Namely, the constitutive expression of VEGF led toexpansion of the blood vessels, the branching out of newlyformed blood vessels from large blood vessels, and occa-sional severe hemorrhaging, possibly due to fragile neovas-culature. In addition, we also evaluated the microvessel den-sity of the s.c. tumors and concluded that the growthadvantage of S17 cells appeared to arise from increasedtumor vascularization induced by VEGF. With regard to therole of VEGF in metastasis, a number of clinical studieshave shown a positive correlation between VEGF expressionand metastatic potential. We also demonstrated that coloncancers with high levels of expression of VEGF mRNAmetastasized to the liver and lymph nodes at significantlyhigher rates than those without high levels of VEGF mRNAexpression (32).

However, those studies did not prove a causative role ofVEGF in metastasis but rather showed an association ofVEGF with metastasis. In the present study, we obtaineddirect evidence that VEGF plays a crucial role in hepaticmetastasis in the intrasplenic portal inoculation model sys-

tem, although it is true that the hepatic metastasis in thisexperimental model system occurs by the forced introductionof the cancer cells into the blood vessels and thus does notcompletely mimic spontaneous metastasis. We also checkedwhether these cells have the ability to complete all stages ofthe metastasis cascade after orthotopic inoculation, and wefound that even S17 cells formed no apparent metastases,despite the rapid tumor growth. Taken together, these find-ings suggested that VEGF contributes predominantly to tu-mor growth, probably due to the induction of angiogenesis,and thereby leads to visible foci rather than dormant tumorsin either the primary site or metastatic lesions. However, itappears that VEGF alone cannot overcome all of the barriersthat must be surmounted for achieving metastasis.

Peritoneal dissemination is also a critical event affectingpatient prognosis. Over the past few years, a considerablenumber of studies have been performed on the correlationbetween VEGF and peritoneal dissemination (39 – 41). Thepresent study showed clearly that VEGF transfectants had agreater potential to induce peritoneal dissemination than didthe controls. Considering that peritoneal dissemination seemsto require lower neovascularization than solid tumor growth,we have not yet clarified why VEGF transfectants showedmassive peritoneal dissemination. One putative mechanism is

Fig. 5 Macroscopic photographs of the s.c. tumors.A, S17 cells;B, V7 cells.C andD, histological examination of tissue sections 6 weeks afterinjection of S17 cells and V7 cells into the s.c. tissue of athymic nude mice. All are stained with H&E. Histological examination revealed moderatelydifferentiated adenocarcinomas.




that VEGF transfectants can be supplied more efficiently thanthe control transfectants with a large quantity of nutrients bydiffusion because VEGF is also able to function as vascularpermeability factor, which may, in turn, occasionally causeascites. Indeed, Kraftet al. (42) reported that VEGF mightplay an important role in tumor progression and the formation

of malignant effusions (43). Mesianoet al. (44) demonstratedthat neutralization of VEGF activity may have clinical appli-cation in inhibiting malignant ascites formation in ovariancancer.

In conclusion, the present study provided clear evidencethat VEGF plays an important role in cancer growth due to

Fig. 6 Liver colonization by derivatives of S17 cells and V7 cells after splenic portal injection. Spleens and livers were harvested from athymic nudemice 4 weeks after splenic portal injection of 23 106 cells.A andC, macroscopic views of S17 cells.B, macroscopic views of V7 cells.D, H&Estaining of the liver metastases from S17 cells. (3400).Arrowheads, the metastasis lesion.




stimulation of neovascularization and subsequent metastasis,which is probably mediated by preventing tumors from enteringa dormant state.

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Fig. 7 Macroscopic views ofthe peritoneal cavity of mice in-oculated i.p. with 23 106 S17cells. Arrowheads, the metasta-sis lesion.




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2000;6:622-630. Clin Cancer Res Yoko Kondo, Shigeki Arii, Akira Mori, et al. LoVo Human Colon Cancer Cell Lineby Transfection of Vascular Endothelial Growth Factor into Enhancement of Angiogenesis, Tumor Growth, and Metastasis

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