Antiangiogenic Gene Therapy With Soluble VEGFR-1, -2, and -3 Reduces the Growth of Solid Human Ovarian Carcinoma in Mice

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1 originl rtile The Amerin Soiety of Gene Therpy Antingiogeni Gene Therpy With Solule VEGFR-, -2, n - Reues the Growth of Soli Humn Ovrin Crinom in Mie Hnn Sllinen, Mrit Anttil, Johnn Nrvinen 4, Jonn Koponen, Kirsi Hmlinen 2,5, Ivn Kholov,2,5, Tommi Heikur, Pyry Toivnen, Veli-Mtti Kosm 2,5, Seppo Heinonen 2,, Kri Alitlo 6 n Seppo Yl-Herttul Deprtment of Moleulr Meiine, A.I. Virtnen Institute, University of Kuopio, Kuopio, Finln; 2 Institute of Clinil Meiine, Gyneology, n Pthology n Forensi Meiine, University of Kuopio, Kuopio, Finln; Deprtment of Gyneology, Kuopio University Hospitl, Kuopio, Finln; 4 Ntionl BIO-NMR Fility, A.I. Virtnen Institute, University of Kuopio, Kuopio, Finln; 5 Deprtment of Pthology, Kuopio University Hospitl, Kuopio, Finln; 6 Moleulr/Cner Biology Lortory Biomeium Helsinki n Hrtmn Institute, University of Helsinki, Helsinki, Finln We stuie ntingiogeni n ntilymphngiogeni effets of svegfr- (sflt-), svegfr-2 (sflk-/kdr), n svegfr- (sflt-4) gene trnsfers n their omintions in intrperitonel ovrin ner xenogrft mie (Bl/ -Anu, n = 55). Gene therpy ws initite when the presene of sizle tumors ws onfirme in mgneti resonne imging (). Aenovirus- meite gene trnsfer ws performe intrvenously vi til vein s follows: A s ontrol (group I), AsFlt- (group II), AsKDR (group III), AsFlt-4 (group IV) n two omintion groups of AsFlt- n AsFlt-4 (group V) n AsFlt-, AsKDR, n AsFlt-4 (group VI). Antitumor effetiveness ws ssesse y sequentil, immunohistohemistry, mirovessel ensity, overll tumor growth, n survivl time. In omintion group VI, intrperitonel tumors were signifintly smller thn in the ontrol group t the en of the follow-up (P < 0.00). Furthermore, in group VI the mirovessel ensity (mirovessels/mm 2 ) in tumor tissue n the totl re of tumors overe y mirovessels were signifintly smller thn in the ontrols. One mouse in group V ws ure. The omine ntingiogeni gene therpy with solule VEGFRs reue tumor growth, tumor vsulrity, n sites formtion in ovrin ner xenogrfts. The results suggest tht the omine ntingiogeni gene therpy is potentil pproh for the tretment of ovrin ner ptients. Reeive 2 April 2008; epte 24 Otoer 2008; pulishe online 2 Deemer oi:0.08/mt Introution Ovrin ner is the most lethl of the gyneologil mlignnies. Two-thirs of the ptients with ovrin rinom hve wiely isseminte isese with intrperitonel rinosis n sites t the time of ignosis. Despite optiml surgery n hemotherpy, the prognosis of ptients remins poor n new tretments re urgently neee. To this en, phse I gene therpy stuies employing ifferent strtegies, suh s suiie genes, trgeting onogenes, or restoring tumor suppressor genes, hve shown the fesiility of suh tretments, ut the effetiveness to suppress tumor growth hs een very limite. The only phse III tril rrie out so fr ws lose fter interim nlysis euse it showe no linil enefit in ptients reeiving the omintion of ropltin n plitxel with intrperitonel p5 gene therpy s ompre to stnr therpy. 2 Angiogenesis, efine s new vessel formtion, is ruil for tumor growth n metstsis. VEGF (vsulr enothelil growth ftor) is potent ngiogeni ftor, whih hs een foun to e overexpresse in vrious tumors, 4 inluing ovrin tumors, 5 n ssoite with poor prognosis. In ition to VEGF, lso VEGF-B, -C, n -D hve een suggeste to ply signifint roles in ovrin tumorigenesis. 6 8 VEGF fmily memers meitte their effets through VEGF reeptors, 2, n, lso known s Flt-, KDR/Flk-, n Flt-4, respetively. 9,0 VEGFR-, whih ins VEGF, VEGF-B, n plent growth ftor, is foun in oth vsulr enothelil ells n mrophges. VEGFR- is positive regultor of monoyte/mrophge migrtion n inflmmtion, n it stimultes ngiogenesis, tumor growth n metstsis. Bining of VEGFR-2 y VEGF n proesse forms of VEGF-C n -D is onsiere to e the min meitor of ngiogenesis, vsulogenesis, n vsulr permeility, wheres VEGFR- with high ffinity to VEGF-C n -D, is require for lymphti vessel evelopment n funtioning, 9,0 n is essentil for tumor spre vi lymphti vessels. 2 Beuse VEGFs ply key roles in tumor iology, VEGFs n their reeptors re potentil trgets for ner therpy. Solule VEGF reeptors use in this stuy lk trnsmemrne omin n intrellulr tyrosine kinse-ontining prts n therefore they o not initite signl trnsution. All onstruts ontin n immunogloulin F omin to ensure effiient imeriztion of the solule reeptors. Both svegfr- n svegfr-2 Corresponene: Seppo Yl-Herttul, A.I. Virtnen Institute, University of Kuopio, P.O. Box 627, Kuopio, FIN-702, Finln. E-mil: Seppo.Ylherttul@uku.fi vol. 7 no. 2, fe. 2009

2 The Amerin Soiety of Gene Therpy Antingiogeni Gene Therpy for Ovrin Cner sequester VEGF ligns n lso form intive heteroimers with trnsmemrne reeptors. 5 svegfr- hs shown to in VEGF-C n VEGF-D with the sme effiieny s the full-length reeptor n therefore ompetes with the ining of the ligns to their ntive reeptors. 6 The im of this stuy ws to omine ntingiogeni gene therpy in n ovrin ner xenogrft moel tht losely reflets the omplexity of tumor progression, mimiks the iversity of humn ovrin ner, n shows very ggressive ehvior. 7 Furthermore, we hve pplie mgneti resonne imging () for the timing of gene therpy to tret sizle tumors, not mirometstti isese, n to follow tumor progression noninvsively in vivo. Results Trnsgene expression Outline of the stuy is shown in Figure. Western lotting in vitro showe similr expression of svegfr-, -2, n - in the meium of SKOV-m ells fter enovirus trnsutions (Figure ). Plsm svegfr- n svegfr-2 levels were highest t y fter the gene therpy n the levels were higher thn ng/ml (ref. 8) throughout the follow-up. Plsm level of svegfr-2 ws higher thn 8,400 ng/ml t eh time point. In the ontrol group I, no signls were etete for the solule reeptors t ny time point y enzyme-linke immunosorent ssy (Figure ). Reverse trnsription PCR with 5 yles showe mrna expression of ll trnsgenes in liver smples 6 ys fter the gene trnsfer (Figure ). Intrperitonel tumor growth All mie evelope intrperitonel tumors within weeks (6 2 ys) fter SKOV-m ell inoultion. ws repete weekly fter the gene trnsfer. In the first n seon, there were no signifint ifferenes in tumor volumes etween the ontrol A group n the gene therpy groups. At the time of the thir (2 weeks fter the gene therpy), the men tumor volume in group VI ws signifintly smller (P = 0.05) thn tht of the ontrol mie (866 ± 270 mm versus 2,026 ± 69 mm ) (Figure 2,). The finl men tumor weights t the en of the follow-up were lso signifintly smller (P < 0.00) in the gene therpy group VI (2. ± 0.8 g), s ompre to the ontrol mie (4.5 ± 0.42 g) (Figure ). There ws sttistilly signifint orreltion etween tumor volumes t the time of thir n the finl tumor weights (r = 0.8, P < 0.005). Formtion of sites Formtion of sites ws ompletely loke in mie reeiving svegfr-2 gene therpy n the ifferene ws signifint s ompre to the A ontrol group (.9 ± 0.42 ml), P = (Figure ). In the other gene therpy groups, there were no signifint ifferenes in the mount of sites s ompre to the ontrols. However, in the omintion group VI istint teneny towr lesser formtion of sites (0.85 ± 0.44 ml) ws note (Figure ). Mirovessel mesurements To etet the effet of gene therpy on intrtumorl mirovessels, mirovessel ensity (MVD) n tumor vsulr re (TVA) svegfr-, -2, n - (ng/ml plsm) Inoultion of tumor ells 48 k 98 k 20, Tumor growth 6 2 ys I GT II neg.. III 6 Survivl Dys fter gene trnsfer IV V Survivl Weeks fter the gene trnsfer, svegfr-2 svegfr- svegfr- MW MW VI 500 p Figure Protool of the stuy n expressions of solule VEGFRs. () Outline of the stuy. Tumors evelope within weeks fter the inoultion of the tumor ells. The presene of ll tumors ws verifie y efore strting gene therpy. Tumors were oserve weekly until the eth of the mie. GT, gene therpy;, mgneti resonne imging. () Expression levels of svegfr-, svegfr-2, n svegfr- fter enovirl trnsution in SKOV-m ells were similr s mesure y western lotting. () svegfr-, svegfr-2, n sveg- FR- levels in plsm s etermine y enzyme-linke immunosorent ssys in omintion group VI (svegfr-, svegfr-2, n svegfr-). The trnsgene expression profiles were ientil in the other groups. svegfr-, svegfr-2, n svegfr- were not etete in A ontrol mie t ny time point. Error rs = SEM. () Reverse trnsription (RT)-PCR ws use to onfirm the expression of svegfr-, -2, n - in mouse liver smples. Lnes : svegfr- RT-PCR. : liver smple 6 ys fter AsFlt- gene trnsfer, : liver smple 6 ys fter A gene trnsfer, : positive ontrol, n : no RT-ontrol. Lnes 2 : svegfr- RT-PCR. 2: Liver smple 6 ys fter AsFlt-4 gene trnsfer, 2: liver smple 6 ys fter A gene trnsfer, 2: positive ontrol n 2: no RT-ontrol. Lnes : svegfr-2 RT-PCR. : liver smple 6 ys fter AsKDR gene trnsfer, : liver smple 6 ys fter A gene trnsfer, : positive ontrol, n : no RT-ontrol. were mesure. MVD (42. ± 6.4) n TVA (0.8 ± 0.4%) of tumors in group VI were signifintly smller thn those of the ontrols (86. ± 6.5, P = n 2.6 ± 0.24%, P = 0.005) (Figure,). An exmple of the smller TVA in the omintion gene therpy group VI (Figure 4, right pnel) s ompre to the ontrols is shown in Figure 4, left pnel. The mjority of intrtumorl lymphti vessels in the ontrol group, stine with LYVE-, were foun t the eges of the tumors n only 5% in the entrl prt (Figure 4, left pnel). In group VI, LYVE- positive lymphti vessels were not etete (Figure 4, right Moleulr Therpy vol. 7 no. 2 fe

3 Antingiogeni Gene Therpy for Ovrin Cner The Amerin Soiety of Gene Therpy pnel), n in svegfr--trete mie, only 2 lymphti vessels per setion were foun (t not shown). Histology The tumors were poorly ifferentite (gre ) serous ystenorinoms with vrile size of nuleus n limite strom (Figure 4, left pnel). In the omintion tretment group VI tumor tissue ws prtly reple y onnetive tissue n morphology of the tumors ws isture t the en of the volumes (mm ) 2,000,500, follow-up (Figure 4, right pnel). In the omintion gene therpy group ell prolifertion inex mesure y Ki-67 stining ws 5 40% ompre to 70 80% in the ontrol group, P = 0.00 (Figure 4). Survivl n sfety The men survivl (ys) in the stuy groups ws s follows: 2 ± 2 in group I, 5 ± 2 in group II, 6 ± 2 in group III, 5 ± 2 in group IV, 55 ± 6 ys in group V n 8 ± 2 in group VI. A tren I II * III IV VI + V + + I II III I II III Group VI + + Group VI + + Group VI + + I II III Group I Group I Group I I 28 fter GT 56 fter GT Group V + Group V + Group V + I 28 fter GT 04 fter GT Group V + Group V + Group V + Figure 2 mesurements of tumor growth. () Mesure y, the men tumor volumes (mm ) were signifintly smller in group VI (svegfr-, -2, n -) versus ontrols 2 weeks fter the gene therpy (III ) *P < () pitures of the evelopment of ovrin tumors in group VI ompre to group I (A). At the time of the first n the seon, there ws no ifferene in tumor volumes, ut in the thir tumors were signifintly smller in group VI. Tumors re mrke with rrows. () pitures of the ure mouse in group V (svegfr- n svegfr-). An intrperitonel ovrin tumor (rrow) ws visile in 8 ys fter the tumor ell injetion. 28 ys fter the gene therpy, the tumor ws shrunken (rrow) n 56 ys fter the gene therpy the tumor ws not visile in. () pitures of the mouse in group V (svegfr- n svegfr-) whih h ormny in tumor growth n notly prolonge survivl. Tumors re mrke with rrows., mgneti resonne imging vol. 7 no. 2 fe. 2009

4 The Amerin Soiety of Gene Therpy Antingiogeni Gene Therpy for Ovrin Cner Tumor weights (g) *** for prolonge survivl in the omintion gene therpy groups ws oserve, ut the ifferene ws not sttistilly signifint. Inee, in group V (svegfr- n svegfr-) the men survivl ws notly longer thn in the ontrol group (55 ± 6 versus 2 ± 2 ys) with one niml eing ompletely ure hving no etetle tumor in 56 ys fter the gene therpy or t 4 Group I () Group VI ( + + ) Asites (ml) 2 HE ** 00 Ki MVD ** * 20 CD-4 TVA 2 ** LYVE- Figure Tumor n sites mesurements. () At the en of the follow-up, the weights of the tumors were signifintly smller in oth omintion groups n in the mie trete with svegfr-. () sveg- FR-2-trete mie i not form ny sites in the peritonel vity. () Mirovessel ensity (MVD: mirovessels/mm 2 ) ws signifintly reue in mie trete with svegfr-2 n with the omintion of svegfr-, -2, n -. () omintion gene therpy with svegfr-, -2, n - signifintly reue the totl re of tumors overe y mirovessels (TVA: tumor vsulr re). *P < 0.05; **P < 0.0; ***P < 0.00 versus, Error rs = SEM. Figure 4 Histology of intrperitonel ovrin tumors. () Hemtoxylin eosin stining of serous enorinom in group I (A, left pnel). Fol nerosis (rrowhe) n onnetive tissue (rrow) were present in the tumor tissue in group VI (svegfr-, -2, n -, right pnel). () more proliferting tumor ells were seen in ontrol group I (left pnel) thn in group VI (right pnel), 70 80% n 5 40%, respetively. Ki-67 stining. () CD-4 positive mirovessels in tumor tissue. In group I (left pnel), totl vsulr re ws higher thn in group VI (right pnel). () LYVE- positive lymphti vessels were seen in the periphery of tumors in group I (left pnel) ontrry to the tretment group VI (right pnel). Mgnifition, 200 (,, n ); 00 (). Br = 00 µm. Tle Clinil hemistry fter the gene trnsfer (men ± SEM) Dy Dy 6 Dy Killing Group ALT Cre ALT Cre ALT Cre ALT Cre I () 90 ± 99 6 ± 2 58 ± 25 ± 6 ± ± 8 40 ± 97 4 ± 2 II () 90 ± 5 2 ± 2 67 ± 6 ± 4 99 ± 48 5 ± ± ± III () 7 ± 6 6 ± 8 24 ± 9 ± 0 ± 6 ± ± ± 2 IV () 2 ± 5 ± 6 2 ± 0 4 ± 2 74 ± 57 8 ± 6 7 ± 5 9 ± 8 V ( + ) 6 ± 8 ± 5 0 ± 5 ± 220 ± ± 2 09 ± 29 4 ± VI ( + + ) 45 ± ± 7 87 ± 64 4 ± ± 79 2 ± ± ± 5 ALT, lnine minotrnsferse (U/l), seline 20 ± 0; Cre, retinine (µmol/l), seline 2 ±. Moleulr Therpy vol. 7 no. 2 fe

5 Antingiogeni Gene Therpy for Ovrin Cner The Amerin Soiety of Gene Therpy utopsy (Figure 2) n we note ormny in tumor growth with nother mouse elonging to the sme group (Figure2). Sfety ws juge y the ssessment of histologil smples of liver, spleen, kineys n lungs s well s y the nlysis of plsm lnine minotrnsferse n retinine levels. Histologilly, liver smples of oth trete n ontrol mie were norml 6 ys fter the gene trnsfer. At the en of the follow-up, there ws eviene of regenertive hnges in group VI liver smples n 25% of mie showe mrosopi ltertions in the liver, whih histologilly onsiste of lol nerosis. Plsm lnine minotrnsferse levels were elevte t the lte stges of the isese in oth trete n ontrol groups (Tle ). There were no histologil ltertions in other orgns n, oringly, retinine vlues were within norml rnge (Tle ). Disussion We present here the first ntingiogeni ner gene therpy stuy with omintion of three solule VEGF reeptors VEGFR-, 2, n - whih ompetitively inhiit the ining of ll memers of the VEGF fmily to their reeptors. The im of the stuy ws to ollet prelinil eviene of the effiy n sfety in vne isese, not t mirometstti stte, the ltter eing n unlikely linil senrio for potentil phse I stuies in ovrin ner. Overll, this omine enovirl gene therpy ppere to inhiit, ut not ompletely reverse, the growth of humn intrperitonel ovrin tumors in nue mie with well-estlishe isese, lrge soli tumors n sites. Inee, ntingiogeni therpy reue tumor growth, tumor vsulrity n sites formtion, s ssesse y sequentil, histology, n immunohistohemistry. The most potent effet of gene therpy on tumor growth ws hieve in group VI, in whih the mie reeive omintion of ll three svegfrs. It seems tht omintion therpy hs more powerful ntitumor effet thn single gene therpy. This is lso supporte y the ft tht one mouse in the omintion group V ws ure n nother mouse lso h notly longer survivl thn ny of the ontrol mie. MVD, TVA, n the weights of tumors were lso signifintly reue in the omintion group VI suggesting tht this is omptile with VEGF tivity. However, in mie trete with svegfr-2, the MVD ws signifintly smller thn in the ontrols lthough the tumor weights t the en of the follow-up were not signifintly smller. This my imply tht lso other mehnisms thn the ntingiogeni effet my e involve in mie reeiving triple therpy. As n exmple, reue lymphngiogenesis with n ntitumor effet ws etete in mie trete with svegfr-. VEGF is lso lle s vsulr permeility ftor n it is thought to e essentil for the evelopment of mlignnt sites. 9 When svegfr- hs een use in intrperitonel ovrin ner gene therpy stuies, it hs een reporte to suppress the formtion of sites 8,20 wheres similr results with regr to svegfr-2 re sprse. Wu et l. hve reently use enovirl svegfr-2 (ref. 2), ut in their stuy ovrin ner ells were injete suutneously, n therefore, the mount of sites oul not e evlute. In the urrent moel, svegfr-2 ompletely loke the formtion of sites flui n similr tren showing reue sites ws lso foun in mie trete with svegfr-, whih supports the view tht VEGF plys mjor role in the sites formtion. It hs een shown tht inhiition of lymphngiogenesis in trnsgeni emryos expressing svegfr- inues lympheem 6 n inrese sites formtion is lso present in genetilly moifie mie hving mlfuntionl VEGFR- (ref. 22). In this stuy, svegfr-, whih ins VEGF-C n VEGF-D, inrese the formtion of sites, whih is lere from the peritoneum vi lymphti hnnels. It is possile tht the triple omintion therpy my hve use more lekiness from the pillries n the ringe pity of the lymphti vessels my hve een reue s ompre to the single svegfr-2 therpy. However, in the omintion tretment groups V n VI the mount of sites ws reue ompre to the ontrols. Survivl ws only slightly prolonge in iniviul gene therpy groups, proly ue to the very ggressive nture of the moel with the first tumors rising within 6 ys fter the engrftment. Previous ntingiogeni stuies hve shown tht omintion of hemotherpy with gene therpy prolongs survivl n suh regimens wrrnt further stuy. Liver toxiity hs een reporte previously when enovirl svegfr- ws injete intrvenously. 2 At the time of the highest svegfr- protein levels in plsm 6 ys fter the gene trnsfer, the histology of liver smples ws norml. However, regenertive hnges were oserve in the liver smples of the group VI t the en of the follow-up ut these mie lso h severe metstsize intrperitonel rinom whih my ontriute to these finings. In this stuy, we hve use the mximum levels of enovirl svegfrs lthough lower levels of solule reeptor expression might reue liver toxiity without ompromising the tretment effet tissue. Tkei et l. 8 hve shown tht peritonel issemintion of ovrin ner in mie is inhiite t the level of ng/ml of svegfr- in irultion, whih ws mintine in our stuy throughout the follow-up perio with ll three solule reeptors. Also, when ws given lone, the very high levels in plsm only inhiite sites (Figure ) ut not the tumor growth. As ovrin ner is n intrperitonel isese usully with numer of ner noules of vrying size, intrvenous elivery of ntingiogeni genes n their prouts is expete to e the est strtegy to trget mlignnt ells or their vsulr supply lthough the risk of systemi sie effets lso eomes relevnt. The trnsgene expression ws in line with previous stuies on enoviruses ut to hieve longer expression times it woul e interesting to omine enovirl tretment with long-term expression vetors. The omintion of svegfr-, svegfr-2, n svegfr- ws more effetive in suppressing tumor growth thn ny of the gents lone. In tht group, the interiniviul vriility ws less thn in the other gene therpy groups, where the hnges in mirovsulr mesurements were not etete in ll tumors. This result is in line with previous prelinil stuies utilizing n ntingiogeni pproh using gene-se therpy ginst VEGF. 20 The present moel showe tht even the omintion of ntingiogeni n ntilymphngiogeni therpy ws insuffiient to inue tumor reggression, even though tumor vsulture ws reue throughout the tumor fter the gene elivery. It is plusile tht the ility of the tumors to espe the tretment is euse of the upregultion of other ngiogeni ftors suh s PDGF-B (pltelet-erive growth ftor), 24 Ang- or Ang-2 (ref. 25). Although ntingiogeni gents lone re unlikely to erite tumors ompletely, similr strtegies vol. 7 no. 2 fe. 2009

6 The Amerin Soiety of Gene Therpy Antingiogeni Gene Therpy for Ovrin Cner Tle 2 Chrteriztion of the stuy groups Groups I II III IV V VI n Aenovirl vetor sflt- skdr sflt-4 sflt- sflt-4 Volume (µl) Titer (pfu/ml) sflt- skdr sflt-4 hve een pplie in ovrin ner linil trils using ntihumn VEGF monolonl ntioy evizum, 26 solule hyri eoy reeptor VEGF-Trp, 27 tyrosine kinse inhiitor AZD 27 (ref. 28), reominnt humn IL-2 (ref. 29) n thliomie. 0 To onlue, we hieve signifint in vivo ntitumor response y the triple omintion of ntingiogeni svegf reeptors, 2, n in novel mouse moel hving n vne isese stte t the time of the tretment. These results wrrnt further evelopment of the omine ntingiogeni gene therpy to efine the est ose n sheule for suh tretment, n suggest tht this pproh oul e utilize in linis long with other ntiner therpies. Mterils An Methos Cell line. Detile hrteristis of the SKOV-m ell line hve een esrie previously. 7 SKOV-m ells were ulture in MCoy s 5A meium (Sigm, Steinheim, Germny). Before in vivo inoultion the ells were trypsinize n ounte. Virl vetors. Aenovirl vetors enoing humn svegf reeptor--ig fusion protein (AsFlt-), humn svegf reeptor-2-igg fusion protein (AsKDR), 4 mouse svegf reeptor--igg fusion protein (AsFlt-4), 6,5 n (A) s ontrol vetor were use for the stuy. Replition-efiient E-E elete linil GMP-gre enoviruses were proue in 29 ells. Aenoviruses were nlyze to e free from helper viruses, lipopolyshries, n teriologil ontminnts. 6,7 For western lotting, SKOV-m ells were plte on 2-well pltes t ensity of 00,000 ells per well. After 24 hours of ultivtion, enovirl vetors enoing solule VEGFR-F fusion proteins were e to the pltes (multipliity of infetion 200). Culture mei were hnge fter 6 hours n smples were tken 72 hours fter trnsution. Protein expression ws nlyze using immunolotting with ntihumn IgG (F-speifi) ntioy I26 (Sigm-Alrih, St Louis, MO), onkey nti-got IgG-HRP seonry ntioy s-2020 (Snt Cruz Biotehnology, Snt Cruz, CA), SuperSignl West Dur (Thermo Fisher Sientifi, Rokfor, IL) sustrte, n CL-XPosure Films (Thermo Fisher Sientifi, Rokfor, IL). Animl moel. Eight to 0-weeks ol (n = 55) Bl/A-nu femle nue mie were use for the stuies. The mie were kept in pthogen-free isolte unit t the Ntionl Experimentl Animl Center of the University of Kuopio. The mie reeive how n wter liitum. Foo, wter, n swust eing were utolve. Ovrin rinom ws proue y inoulting 0 7 SKOV- m ells into the peritonel vity of the nue mie with 22 G neele. Development of the ovrin rinom tumors ws followe y sequentil (Figure ). When the first soli, mesurle tumor ws etete in, gene trnsfer ws one the following y. The mie were rnomly ivie into six groups: seven nimls reeive AsFlt- ( 0 9 pfu), six nimls reeive AsKDR ( 0 9 pfu), eight nimls reeive AsFlt-4 ( 0 9 pfu), 2 nimls reeive AsFlt- n AsFlt-4 ( 0 9 pfu oth vetors), nine nimls reeive AsFlt-, AsFlt-4, n AsKDR ( pfu eh vetor) n ontrol nimls reeive A (2 0 9 pfu) (Tle 2). Gene trnsfer ws performe intrvenously vi til vein in the finl volume of 200 µl in 0.9% sline. ws one weekly fter gene trnsfer n tumor volumes were ssesse. The overll follow-up time lste until the pperne of signifint symptoms neessitting killing or to the eth (Figure ). At the time of eth, ll tumor tissue, liver, spleen, kineys, n lungs were hrveste n tumor msses were weighe. Asites flui ws ollete with syringe. All niml stuies were epte y the Experimentl Animl Committee of the University of Kuopio. Histology, immunohistohemistry n mirovessel mesurements. Tissue smples were immerse in 4% prformlehye for 4 6 h, followe y overnight immersion in 5% surose. 8 The speimens were emee in prffin n 5 µm thik setions were proesse for hemtoxylin eosin, Ki-67 (DkoCytomtion, Glostrup, Denmrk), CD-4 (HyCult iotehnology.v., AA Uen, The Netherlns) n LYVE- (ReliTeh, Brunshweig, Germny) stinings. Photogrphs of histologil setions were tken n proesse using n Olympus AX70 mirosope (Olympus Optil, Tokyo, Jpn), n nlysis (Soft Imging System, Münster, Germny) n PhotoShop (Aoe) softwre. Men mirovessel re (μm 2 ), MVD, n totl mirovsulr re (%) of the tumors (TVA) were mesure from CD4- immunostine setions using nlysis softwre t 00 mgnifition in line mnner. Ten ifferent fiels whih represente mximum mirovessel res were selete from eh tumor. Neroti res were voie. The totl numer of LYVE- positive lymphti vessels per setion ws ounte. Mens ± SEM of the mesurements re reporte.. To follow the evelopment of ovrin rinom n to mesure tumor volumes, 9.4 T vertil mgnet (Oxfor Instruments, Oxfor, UK) equippe with tively shiele fiel grients (Mgnex Sientifi, Aington, UK) interfe to n s.m.i.s. onsole (Surrey Meil Imging Systems, Guolfor, UK) ws use. The etils of imging hve een esrie previously. ws performe weekly fter the first tumors were etetle. Reverse trnsription PCR. Reverse trnsription PCR ws use to onfirm the trnsgene expression in mouse liver smples. The liver tissue ws snp-frozen t the sixth y fter the gene trnsfer in liqui nitrogen n store t 70 C for reverse trnsription PCR nlysis. Totl RNA ws extrte using Trizol Regent (Gio BRL, Grn Isln, NE) oring to mnufturer s instrutions. Totl RNA ws trete with DNseI (Promeg, Mison, WI) to remove ny ontminting DNA n DNA synthesis ws performe with 2 μg of RNA with rnom hexmers. Primers for the mplifition of svegfr- DNA were forwr: 5 -AGG CCA GAC ACT GCA TCT CC- n reverse: 5 -GCT TCA CAG GTC AGA AGC CC-, for svegfr- DNA forwr: 5 -TGA AGG CAC AGA AGC TAG GCC- n reverse: 5 -ACC TGA GTC GAA CTC AGC CC- n for svegfr-2 forwr: 5 -ACA GAG GGA CTT GGA CTG GC- n reverse: 5 -TTC ACA GAA GAC CAT GCC AGC-, with mplion sizes of 500 p, 50 p, n 480 p, respetively. PCR mixtures onsiste of 20 pmol of eh primer, 0.2 mmol/l NTPs (Promeg,),.5 mmol/l MgCl 2, Dynzyme EXT PCR uffer,.5 U of Dynzyme EXT DNA polymerse (Finnzymes, Helsinki, Finln) n 500 ng of DNA smple. Amplifitions Moleulr Therpy vol. 7 no. 2 fe

7 Antingiogeni Gene Therpy for Ovrin Cner The Amerin Soiety of Gene Therpy were rrie out with the following onitions: the first enturtion step t 95 C for minutes, followe y 5 yles with 45 seons t 95 C, 45 seons t 60 C for sflt- n skdr or t 62 C for sflt-4, 45 seons t 72 C with the finl step t 72 C for 5 minutes. Clinil hemistry n enzyme-linke immunosorent ssy nlyses from plsm smples. Plsm smples were ollete t y, 6, n fter the gene trnsfer n when the mie were kille. Alnine minotrnsferse n retinine were monitore using routine linil hemistry ssys t Kuopio University Hospitl Centrl Lortory. Enzyme-linke immunosorent ssys (Quntikine; R&D Systems, Minnepolis, MN) were use to etet the presene of humn solule VEGFRs in plsm smples. Sttistil nlyses. Sttistil signifine ws evlute using Kruskll Wllis test, followe y Mnn Whitney U-test with pproprite orretion for multiple omprisons. Results re expresse s men ± SEM. A vlue of P < 0.05 ws onsiere s sttistilly signifint. 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