Vascular endothelial growth factor acts in an autocrine manner in rhabdomyosarcoma cell lines and...

Vascular endothelial growth factor acts in an autocrine manner in

rhabdomyosarcoma cell lines and can be inhibited with all-trans-retinoicacid

Matthew FW Gee1,2, Rika Tsuchida1, Claudia Eichler-Jonsson1, Bikul Das1, Sylvain Baruchel1

and David Malkin*,1,2

1Division of Haematology/Oncology, Department of Paediatrics, Hospital for Sick Children, University of Toronto, Toronto, Ontario,Canada; 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

Vascular endothelial growth factor (VEGF) is a potentsignalling molecule that acts through two tyrosine kinasereceptors, VEGFR1 and VEGFR2. The upregulation ofVEGF and its receptors is important in tumour-associatedangiogenesis; however, recent studies suggest that severaltumour cells express VEGF receptors and may beinfluenced by autocrine VEGF signalling. Rhabdomyo-sarcoma (RMS) is the most common paediatric soft-tissuesarcoma, and is dependent on autocrine signalling for itsgrowth. The alveolar subtype of RMS is often character-ized by the presence of a PAX3-FKHR translocation, andwhen introduced into non-RMS cells, the resultant fusionprotein induces expression of VEGFR1. In our study, weexamined the expression of VEGF and its receptors inRMS, and autocrine effects of VEGF on cell growth.VEGF and receptor mRNA and protein were found to beexpressed in RMS cells. Exogenous VEGF additionresulted in extracellular signal-regulated kinase-1/2 phos-phorylation and cell proliferation, and both were reducedby VEGFR1 blockade. Growth was also slowed byVEGFR1 inhibitor alone. Treatment of RMS cells withall-trans-retinoic acid decreased VEGF secretion andslowed cell growth, which was rescued by VEGF. Thesedata suggest that autocrine VEGF signalling likelyinfluences RMS growth and its inhibition may be aneffective treatment for RMS.Oncogene (2005) 24, 8025–8037. doi:10.1038/sj.onc.1208939;published online 22 August 2005

Keywords: autocrine signalling; vascular endothelialgrowth factor; rhabdomyosarcoma; all-trans-retinoicacid

Introduction

Vascular endothelial growth factor (VEGF) is anessential regulator of vasculogenesis and haematopoiesis

during embryonic development (Fong et al., 1995;Shalaby et al., 1995; Ferrara, 1996; Damert et al.,2002; Risau, 1997). In adulthood, it drives angiogenesisin pregnancy and wound healing, as well as inpathological conditions including cancer, rheumatoidarthritis, ocular neovascular disorders and cardiovascu-lar disease (Folkman, 1995; Isner and Losordo, 1999;Zachary et al., 2000). VEGF promotes vascular perme-ability (Keck et al., 1989), and stimulates endothelial cellmigration, proliferation and survival of newly formedblood vessels (Neufeld et al., 1999; Zachary and Gliki,2001; Claesson-Welsh, 2003). The effects of VEGF aretransduced through two tyrosine kinase receptors,VEGFR1 (or fms-like tyrosine kinase receptor (Flt1))(de Vries et al., 1992) and VEGFR2 (or kinase insertdomain-containing receptor (KDR); Flk1 in mice)(Quinn et al., 1993). A third tyrosine kinase receptor,VEGFR3, does not bind VEGF, but instead bindsVEGF-C and VEGF-D (Joukov et al., 1996; Orlandiniet al., 1996). In addition, the effects of VEGF throughVEGFRs may be modulated by neuropilin-1 (NRP1)and neuropilin-2 (NRP2), which act as coreceptors tothe VEGFRs (Tammela et al., 2005). In addition tobinding class 3 semaphorins, NRPs also bind the VEGFfamily ligands VEGF (NRP1/2), VEGF-B (NRP1),VEGF-C (NRP2) and placenta growth factor (PlGF;NRP1/2). NRP1 enhances VEGFR2 signalling andforms complexes with VEGFR1, and NRP2 is oftencoexpressed with VEGFR3 (Tammela et al., 2005).

VEGFR1 is likely constitutively inactive in endothe-lial cells (Gille et al., 2000), suggesting that VEGFR2 isthe primary transducer of VEGF signalling duringphysiological angiogenesis (Waltenberger et al., 1994).In tumours, however, angiogenesis appears to bedependent on both VEGFR2 (Millauer et al., 1996)and VEGFR1 (Itokawa et al., 2002; Luttun et al., 2002),and tumour-associated angiogenesis is more potentlyinhibited by blocking both receptors rather thaninterrupting either alone (Lu et al., 2001). Owing tothe role of VEGF in tumour angiogenesis, severalapproaches have been developed to interrupt its signal-ling. These include neutralizing antibodies to VEGF(Manley et al., 2004) and its receptors (Lu et al., 2001),soluble receptors to sequester VEGF (Holash et al.,

Received 1 March 2005; revised 10 June 2005; accepted 10 June 2005;published online 22 August 2005

*Correspondence: D Malkin, Division of Haematology/Oncology,Department of Paediatrics, Hospital for Sick Children, University ofToronto, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8;E-mail: [email protected]

Oncogene (2005) 24, 8025–8037& 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00

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2002), ribozymes to degrade transcripts of VEGF (Keet al., 1998; Manley et al., 2004) and its receptors (Parryet al., 1999), compounds to decrease VEGF andreceptor expression (Broggini et al., 2003), and smallmolecular kinase inhibitors (Manley et al., 2004).

As VEGF is a key mediator of tumour angiogenesis,most studies of VEGF and its receptors have primarilyfocused on endothelial expression of VEGFR1 andVEGFR2. However, growing evidence suggests thatmalignant cells also express VEGF receptors, and arelikely responsive to VEGF signalling (Dias et al., 2001;Broggini et al., 2003; Narendran et al., 2003). Inleukaemia and malignant myeloma cells, VEGF auto-crine loops likely aid in growth and survival (Dias et al.,2000, 2001, 2002; Podar et al., 2001, 2004). Similarly,some solid tumours may express both VEGF and itsreceptors, and their growth and survival may bepartially regulated by VEGF autocrine signalling (Bateset al., 2003; Wey et al., 2004). Recently, evidence hasbeen presented for VEGF autocrine signalling throughVEGFR1 in colon cancer (Bates et al., 2003). Bates et al.discovered that the colon cancer cell line LIM 1863upregulates VEGF and VEGFR1 as cells progress to aninvasive phenotype. Treatment with a monoclonalantibody against VEGFR1 led to diminished cellsurvival via an increase in apoptosis (Bates et al.,2003). Interestingly, VEGFR1 expression has beenshown to be increased in cells transfected with PAX3-FKHR, a translocation characteristic of alveolar rhab-domyosarcoma (ARMS) (Barber et al., 2002).

Rhabdomyosarcoma (RMS) is the most commonpaediatric soft-tissue sarcoma, with an annual incidenceof four to seven cases per million children under 16 yearsof age (Young et al., 1986). RMS is part of the largerfamily of ‘small round blue cell tumours’ of childhood,and histologically resembles rhabdomyoblasts, the pre-cursors of striated muscle (Merlino and Helman, 1999).There are two main subtypes of RMS, embryonal(ERMS) and alveolar (ARMS). ERMS tumours canbe characterized by a loss of heterozygosity at 11p15.5(Koufos et al., 1985), associated with a loss of maternalchromosomal material and duplication of paternalchromosomal material (Merlino and Helman, 1999).The majority of ARMS tumours have characteristicchromosomal rearrangements that translocate the PAX3gene at 2q35 to FKHR at 13q14 (t(2;13)(q35;q14))(Barr et al., 1993), or less frequently contain at(1;13)(p36;q14) translocation that fuses PAX7 toFKHR (Davis et al., 1994). RMS tumours are knownto express many autocrine signalling pathways, includ-ing insulin-like growth factor-II (IGF-II) (El-Badryet al., 1990; De Giovanni et al., 1995), basic fibroblastgrowth factor (bFGF) (Schweigerer et al., 1987; DeGiovanni et al., 1995), epidermal growth factor (EGF)(De Giovanni et al., 1995), transforming growth factor-b (TGF-b) (De Giovanni et al., 1995) and myostatin(Ricaud et al., 2003). By blocking these pathways, RMScells exhibit growth inhibition and occasionally areinduced to differentiate (Schweigerer et al., 1987; El-Badry et al., 1990; De Giovanni et al., 1995; Ricaudet al., 2003).

In the present study, we report the first evidence ofVEGF receptors in RMS. We found that VEGFR1 isexpressed more frequently than VEGFR2 andVEGFR3. We also present evidence that RMS cell linesproduce VEGF, and that application of exogenousVEGF further increases cell growth. Treatment with ablocking antibody for VEGFR1 inhibits VEGF signal-ling and slows RMS proliferation. All-trans-retinoicacid (AtRA), previously shown to slow RMS growth(Crouch and Helman, 1991), decreases VEGF secretionfrom RMS cells. Furthermore, we show that this slowedgrowth of RMS cells upon AtRA treatment can berescued by supplementing cells with VEGF. These datasuggest that RMS cell growth is dependent on autocrinesignalling, which includes VEGF. By interrupting thesepathways, with VEGFR1 blocking antibodies andAtRA, RMS cell growth is reduced. Thus, the VEGFautocrine signalling pathway may represent noveltherapeutic target for the treatment of RMS.

Results

RMS cell lines express VEGF, VEGFR and NRPmRNAs

RT–PCR analysis revealed that VEGF family receptormRNAs are expressed in RMS cell lines. VEGFR1mRNA could be detected in four of the six RMS linesassayed (RH4, RH6, RH18 and RD). RH28 and RH30were the only lines in which VEGFR1 mRNA could notbe detected (Figure 1a). This primer set amplified aregion corresponding to VEGFR1 exons 6–8, a regionthat encodes the extracellular region of the VEGFR1protein. By using a second primer set, which amplified aregion corresponding to the transmembrane region ofthe protein, we confirmed that VEGFR1 mRNA ispresent in four of the six RMS lines (data not shown). Inaddition, expression of VEGFR2 mRNA was detectedin RH28 cells (Figure 1b), and VEGFR3 mRNA wasdetected in RH4 and RH18 cell lines (Figure 1c).Additional RT–PCR reactions, using different primersand an increased number of cycles, confirmed thatVEGFR2 was expressed only in RH28 cells (data notshown). The RMS cells lines also expressed mRNAscorresponding to the non-tyrosine kinase VEGF cor-eceptors, NRPs. NRP1 mRNA was expressed in fourRMS lines (RH4, RH6, RH18 and RH30), and NRP2mRNA was detected in all lines except for RH18(Figure 1c).

VEGF mRNA was expressed in all six RMS cell lines(Figure 1d). Using a previously published primer set(Suzuki et al., 1996), VEGF121, VEGF145 and VEGF165

mRNAs were clearly expressed (Figure 1d, upper panel).Additionally, we designed primers to amplify transcriptscontaining exons 6A and 7, which in theory are specificto VEGF183, VEGF189 and VEGF206. Upon amplifica-tion, only expressions of VEGF183 and VEGF189

mRNAs were seen. A similar transcript expressionpattern was seen in all six RMS lines (Figure 1d, middlepanel).

VEGF autocrine signalling in rhabdomyosarcomaMFW Gee et al

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Figure 1 Expression of VEGF and receptors in RMS cells. (a) RT–PCR for VEGFR1 demonstrated mRNA expression in four of sixRMS lines (two alveolar, two embryonal). (b) RT–PCR for VEGFR2 detected mRNA in one of six RMS lines. (c) RT–PCR forVEGFR3 detected mRNA in two of six RMS lines. NRP1 mRNA was detected in four of six RMS lines, and NRP2 mRNA wasdetected in five of six RMS lines. (d) RT–PCR for VEGF detected uniform expression of multiple VEGF transcripts across all RMSlines. (e) Membrane expression of VEGFR1 was assessed by Western blot. Protein was extracted as described in Materials andmethods. Protein (150mg) was separated by SDS–PAGE (8%), followed by transfer to nitrocellulose. The blot was probed withmonoclonal antibody for VEGFR1, which demonstrated the presence of the receptor in three RMS cell lines. (f) Supernatant VEGF165

levels (after growth for 2 days) were quantified by ELISA. Results from ELISA were normalized by cell numbers as determined byCoulter counter. Data were assayed in triplicate and results are representative of three independent experiments


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RMS cell lines express VEGFR1 protein

RH4, RH18, RH30 and RD were analysed by Westernblot for the membrane expression of VEGFR1(Figure 1e). Similar to results by RT–PCR, RH4,RH18 and RD expressed VEGFR1, and RH30 did not.

RMS cell lines secrete VEGF protein

All six RMS cell lines were analysed by ELISA for theproduction of VEGF165 (Figure 1f). Confirming theRT–PCR results, all six RMS lines produced VEGF165.However, baseline levels varied among the lines, withRH4 secreting the most VEGF165 (158 pg/ml/105 cells)and RD producing the least (25 pg/ml/105 cells).

ERK-1/2 are phosphorylated in response to VEGF165

As extracellular signal-regulated kinase (ERK)-1 andERK-2 are phosphorylated following VEGF- andPlGF-induced activation of VEGFR1 (Podar et al.,2001; Selvaraj et al., 2003), we examined their phos-phorylation states after a brief exposure of cells to the

growth factor. ERK-1/2 phosphorylation is an indirectmeasure of receptor activity following VEGF165 stimu-lation. To demonstrate VEGFR1 activation in RMScells, RH4 and RH30 were used as representativeVEGFR1-positive and VEGFR1-negative cell lines,respectively. HeLa cells served as a negative control.After serum starvation for 24 h, with frequent washingand medium changes to reduce baseline activation ofERK-1/2, HeLa, RH4 and RH30 cells either remainedin serum-free media, or were stimulated for 3min with10% fetal bovine serum (FBS) or 200 ng/ml VEGF165.This amount of VEGF165 used was shown to producethe strongest ERK-1/2 activation in preliminary experi-ments, and compares to similar concentrations of VEGFand PlGF used previously to activate VEGFR1 (Podaret al., 2001; Selvaraj et al., 2003). As shown in Figure 2,ERK-1/2 was not activated in HeLa, RH4 or RH30 cellsin serum-free conditions, and all cell lines were positivefor phospho-ERK-1/2 when stimulated with 10% FBS.More importantly, RH4 cells demonstrated ERK-1/2activation in response to VEGF165 stimulation, whereasthe proteins remained unphosphorylated in VEGFR1-

Figure 2 VEGF induces ERK-1/2 phosphorylation in VEGFR1-expressing cells. After reaching 70% confluence, cells were serum starvedand repeatedly washed over a 24h period. Cells remained untreated or treated with either 10% FBS or VEGF (200ng/ml) for 3min. Whereindicated, cells were preincubated with goat polyclonal anti-VEGFR1 blocking antibody (Ab; 20mg/ml) for 1h prior to VEGF treatment.Cells were immediately lysed and 50mg of protein was subjected to SDS–PAGE (12.5%). ERK-1 and ERK-2 activities were analysed byWestern blot using a rabbit polyclonal anti-phospho-MAPK (Thr202/Tyr204) antibody. To account for loading differences, a monoclonalanti-vinculin antibody was also used. Densitometric analysis was performed to determine differences in phospho-ERK-2 levels relative tovinculin between treatments in RH4 cells. Values are expressed relative to VEGF-treated cells (100%)


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negative HeLa and RH30 cells. Moreover, VEGF-induced ERK-1/2 phosphorylation was decreased by36% when RH4 cells were preincubated with a blockingantibody for VEGFR1. This assay demonstrates thatVEGFR1 is activated upon VEGF165 stimulation, andthat this activation can be inhibited with the addition ofa blocking antibody for the receptor. Vinculin was usedto normalize the bands instead of total ERK, becausemembranes were developed by AP staining. With thismethod, membranes cannot be stripped and reprobed.As a result, measurement of total ERK would haverequired separate lanes and would not have reflectedloading differences of the phospho-ERK lanes.

Addition of VEGF165 increases RMS proliferation

Addition of exogenous VEGF165 to RMS cell linesincreased the proliferation of VEGFR1-positive lines(RH4, RH18 and RD) and promoted the survival ofVEGFR2-positive RH28 cells, in a dose-dependentmanner, as determined by counting total cells by CoulterCounter at treatment days 0 and 4 (Figure 3a). RH4,RH18 and RD cell numbers increased with VEGF165

treatment from day 0 to 4. In contrast, the RH28 cellshad decreased in number by day 4; however, thisdecrease was mitigated by VEGF165. At the highestdoses of VEGF165, cell numbers of all four RMS lineswere significantly greater than those of nontreated cells(RH4: 205%, Po0.001; RH18: 82%, Po0.001; RH28:131%, Po0.001; RD: 56%, Po0.01). This increase ingrowth could be blocked by the application of ablocking antibody for VEGFR1, as determined byAlamar Blue assay (Figure 3b and c). A 20 mg portionof anti-VEGFR1 antibody prevented the proliferationof VEGF165-stimulated RH4 cells by up to 97%(Po0.01).

VEGFR1 antibody treatment of RH4 cells decreasesproliferation

As RH4 cells were shown to produce a large amount ofVEGF165 compared to the other RMS cell lines, wehypothesized that ablation of autocrine VEGF bindingto VEGFR1 may inhibit cell growth. Inhibition ofautocrine VEGF signalling was assayed by treating RH4cells with various doses of VEGFR1 blocking antibody,waiting 4 days, followed by counting cells by CoulterCounter (Figure 4). At the lowest doses of the antibody,compared to nontreated cells, RH4 growth increased byup to 17% (Po0.05). At higher antibody concentra-tions, however, RH4 growth decreased by up to 23%(Po0.01).

AtRA decreases VEGF165 production by RH4 cells

Retinoic acid has been previously shown to reduceVEGF production by various tumour types (Weningeret al., 1998; Kini et al., 2001) and slow RMS growth(Crouch and Helman, 1991; Brodowicz et al., 1999). Wehypothesized, therefore, that VEGF production byRMS cells may be decreased with treatment by 5 mM

AtRA, a dose that has previously been shown to arrestRMS cell growth (Crouch and Helman, 1991). AnELISA assay was used to compare VEGF165 productionin AtRA-treated RH4 cells versus control cells treatedwith an equivalent volume of ethanol (Figure 5a). AtRAtreatment decreased VEGF165 secretion by more than50% (Po0.05).

Addition of VEGF165 to AtRA-treated RH4 cells rescuesgrowth

As VEGF165 production by RH4 cells was shown to bedecreased by AtRA treatment, and this cell line appearsto be supported by autocrine VEGF signalling, wereasoned that the decrease of this growth factor byAtRA may contribute to the slowing of RH4 cellgrowth. To examine this possibility, 200 ng VEGF165

was added to AtRA-treated RH4 cells and compared tocells treated solely with AtRA or an equivalent volumeof ethanol. Using Alamar Blue to measure cellularmetabolic activity, viability of RH4 cells in the threetreatment conditions was examined over multiple timepoints (Figure 5b). After 1 day, compared to ethanoltreatment, AtRA reduced the number of viable cells by23% (Po0.001). After 6 days, there were 61% fewerviable AtRA-treated RH4 cells than ethanol-treatedcells (Po0.001). VEGF165 supplementation rescued thenumber of viable AtRA-treated cells by 29% (Po0.05)at day 2, and at day 6, the percent rescue was 74%(Po0.001).

Discussion

Initially, research on VEGF signalling focused almostexclusively on its mitogenic and survival functionstowards endothelial cells. More recently, however,mounting evidence suggests that malignant cells withincertain tumour types also express VEGF receptorsand respond to VEGF signalling (Dias et al., 2001;Broggini et al., 2003; Narendran et al., 2003).Both VEGFR1 and VEGFR2 have been detected inleukaemia and malignant myeloma, and their growthand survival are decreased by inhibiting autocrineVEGF signalling (Dias et al., 2000, 2001, 2002;Podar et al., 2001, 2004). Tumour cell expression ofVEGF receptors has also been seen in some solidtumours (Bates et al., 2003; Wey et al., 2004), and recentreports suggest that autocrine VEGF signallingmay also influence solid tumour growth and survival.For example, our laboratory has recently shownthat VEGFR1 signalling participates in hypoxia-mediated drug resistance in neuroblastoma (Das et al.,in press).

Using the VEGF- and VEGFR1-expressing coloncancer cell line LIM 1863, Bates et al. (2003) treatedcells with a neutralizing antibody against VEGFR1 thatled to widespread apoptosis. This result was supportedby a report by Fan et al. (2005), who demonstratedthat the activation of VEGFR1 by VEGF or VEGF-Benhances colorectal cancer cell invasion. Interestingly,


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the VEGF-sequestering agent Avastint (Genentech) hasbeen very successful at treating colorectal cancer(Brower, 2003; Kabbinavar et al., 2003; Willett et al.,2004), and perhaps this high degree of efficacy resultspartially from interfering with autocrine VEGF signal-ling (Mercurio et al., 2004). This suggests that anti-VEGF therapy may be successful at treating othercancers in which the tumour cells express VEGFreceptors.

RMS might be such a tumour that responds well toanti-VEGF therapy. RMS xenografts were used in theearliest published mouse studies demonstrating theeffectiveness of both Avastint (Kim et al., 1993;Borgstrom et al., 1996) and the more recent angiogenesisblocker VEGF Trap (Regeneron; Holash et al., 2002).Furthermore, a recent report hinted that RMS mightexpress VEGF receptors. Barber et al. (2002) found thatP19, HeLa and NIH3T3 cells transfected with PAX3-


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FKHR had induced expression of several genes, includ-ing VEGFR1. As PAX3-FKHR is the characteristictranslocation of ARMS, we reasoned that RMStumours may be supported by autocrine VEGF signal-ling. To address this, we characterized six RMS cell lines(four PAX3-FKHR-positive ARMS lines and twoERMS lines) by RT–PCR and ELISA and we havedemonstrated that all six cell lines express VEGF. Thisresult is perhaps not surprising based on previousreports demonstrating that angiogenesis is promoted inRMS xenografts in mice via VEGF (Kim et al., 1993;Borgstrom et al., 1996). Our study, however, also showsthat all six RMS lines studied express one or morereceptors for VEGF (VEGFRs and/or NRPs). To thebest of our knowledge, this is the first time that tumourcell expression of VEGF receptors has been seen inRMS.

Figure 4 VEGFR1 blockade induces changes in RH4 growth.RH4 cells were seeded in 24-well plates and allowed to recover.Fresh DMEM containing 2% FBS was added and cells were eitherleft untreated or incubated in the presence of increasing concentra-tions of anti-VEGFR1 polyclonal antibody (1, 2, 5, 10, 20 and40mg/ml). After 4 days, cells were trypsinized and counted byCoulter counter. At low doses of the VEGFR1 blocking antibody,cell numbers were higher than the control, whereas at high doses,cell numbers were lower than untreated cells. Data shown are themean7s.d. of experiments performed in triplicate, and results arerepresentative of three independent experiments. Asterisks indicatestatistically significant differences between treatment groups(*Po0.05; **Po0.01)

Figure 5 VEGF rescues growth of AtRA-treated RH4 cells. Inseparate experiments, RH4 cells were seeded at a density of5� 105 cells/well in a 24-well plate in DMEM containing 10% FBS.(a) After 24 h, fresh DMEM containing 2% FBS and 5.0 mm AtRAor an equivalent volume of ethanol was added to the cells, andDMEM and treatments were refreshed after 48 h. After anadditional 48 h, cells were counted with a Coulter counter andsupernatants were collected to detect VEGF165 by ELISA. Cellstreated with AtRA produced less VEGF165 than ethanol-treatedcontrols (*Po0.05). (b) After 24 h, fresh serum-free mediumcontaining DMEM was added to the wells in addition to treatmentconditions: 5.0mm AtRA (’), 200 ng VEGF165 to AtRA-treatedcells (E), or an equivalent volume of ethanol (EtOH; m), withrefreshment 24 h post-treatment. At various time points (0¼ initialtreatment), 10% Alamar Blue was added to each well andfluorescence was measured following 5 h of incubation. Treatmentwith 5.0mm AtRA decreased the number of viable cells, ascompared to ethanol-treated controls at all time points(Po0.001). Addition of 200 ng VEGF to AtRA-treated cellsrescued the number of viable cells by day 2 (*Po0.05;**Po0.005; ***Po0.001). Data shown are the mean7s.d. ofexperiments performed in triplicate, and results are representativeof three independent experiments

Figure 3 VEGF signals through VEGFR1 to promote RMS growth. (a) VEGF promotes RMS cell growth in a dose-dependentmanner. RMS cell lines RH4, RH18, RH28 and RD were plated in 24-well plates, allowed to recover and then serum starved (DMEM,0% FBS) for 24 h. DMEM was then replaced and cells were either left untreated or incubated in the presence of VEGF165 (2, 5, 10, 50or 100 ng/ml) and refreshed 48 h later. At 96 h post-treatment, cells were counted by Coulter counter. Data shown are the mean7s.d. ofexperiments performed in triplicate, and are representative of three independent experiments for each cell line. The asterisks indicatesignificant differences (*Po0.05; **Po0.01; ***Po0.005; ****Po0.001) between treatments and untreated controls. (b, c) VEGFR1blockade of RH4 cells abolishes VEGF165-induced proliferation. A total of 3.6� 104 RH4 cells were plated in a 24-well plate, allowedto recover and then serum starved (DMEM, 0% FBS) for 24 h. Medium was changed and cells were either left untreated (m) orincubated with 100 ng/ml of VEGF165 (E) or 100 ng/ml of VEGF165 plus 20 mg of anti-VEGFR1 blocking antibody (Ab; ’) withrefreshment at 48 and 72 h post-treatment (b). Alternatively, treatments were 20 mg of nonspecific goat IgG, 100 ng/ml of VEGF165 plusgoat IgG or 100ng/ml of VEGF165 plus 20mg of anti-VEGFR1 blocking antibody (Ab), with refreshment at 48 h (c). The number ofviable cells was quantified by Alamar Blue assay at various time points. Asterisks indicate significant differences (*Po0.05; **Po0.01)between VEGF-treated and nontreated cells, and number signs show significant differences (#Po0.05; ##Po0.01) between VEGF-treated cells plus or minus VEGFR1 Ab (b). Alternatively, comparison groups showed significance (*Po0.001) or were notsignificantly (NS) different (c). Data shown are the mean7s.d. of experiments performed in triplicate


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VEGFR1 mRNA was detected in only two of fourARMS lines (RH4 and RH18), and this was confirmedby Western blot. It had been expected that all fourPAX3-FKHR-positive lines would express VEGFR1mRNA, as the fusion protein has been shown toincrease VEGFR1 expression (Barber et al., 2002). Ascultured cell lines have an increased rate of geneticinstability, the absence of VEGFR1 mRNA expressionin RH28 and RH30 cell lines may simply be the result ofdeletion of genetic loci encompassing the VEGFR1gene. It is possible, however, that these two cell linesmay represent a distinct subset of ARMS (PAX3-FKHRþ , VEGFR1�). Whether such an RMS sub-classification exists in primary tumours, and its potentialvalue in clinical stratification, remains to be explored.Using a larger sample size, including both RMS celllines and primary tumour samples, we are currentlyexamining this question. Notably, VEGFR1 was alsodetected in both ERMS lines (RH6 and RD). AsVEGFR1 is also a target of PAX3 (Barber et al.,2002), upregulation of PAX3 in ERMS lines (previouslynoted in RD cells; Barr et al., 1999) may lead to anincrease in VEGFR1 expression, which would help toexplain its expression in the ERMS cell lines.

Interestingly, other VEGF receptors were detected inthe RMS lines. All cells that expressed VEGFR1 alsoexpressed either NRP1 or NRP2, and VEGFR3 wascoexpressed with NRP2 in RH4 cells. Coexpression ofNRPs and VEGFRs has been previously reported inendothelial cells (Karkkainen et al., 2001) and neuro-blastoma cell lines (Beierle et al., 2003, 2004; Das et al.,in press). The detection of both NRPs and VEGFRs inRMS cell lines suggests that VEGFR expression in thistumour type likely has clinical relevance and is notmerely a tissue culture artefact.

As the majority of RMS cell lines expressed VEGFRs,we assessed the responsiveness of these lines toexogenous VEGF165. Stimulating RH4 cells withVEGF165 induced ERK-1/2 phosphorylation, whichcould be decreased by 36% when cells were preincu-bated with a blocking antibody for VEGFR1. Thisdemonstrates that VEGFR1 in RH4 cells (and possiblyother RMS lines) is activated in response to addition ofVEGF165. However, the antibody was unable tocompletely abolish VEGF-induced ERK activation. Incontrast, Selvaraj et al. (2003), using the same antibody(albeit in a different cell system (monocytes)), demon-strated that ERK activation following stimulation ofcells with PlGF could be reduced by approximately 75%with VEGFR1 blockade. We used 20 mg of antibody toblock signalling from 200 ng of exogenously addedVEGF165, while Selvaraj et al. used only 5 mg ofantibody to block 250 ng of PlGF from binding toVEGFR1. This suggests that while VEGFR1 is likelythe primary VEGF signalling molecule in RH4 cells,treatment with the blocking antibody is only partiallyeffective. Perhaps the antibody may not block VEGFbinding as effectively as it blocks PlGF. This is possible,as the effectiveness of this antibody was validated, interms of its ability to inhibit VEGFR1 signalling, usingPlGF, not VEGF.

The presence of NRPs may also help to explain thelow rate of inhibition with blocking antibody treatment.NRP1 and NRP2 have been shown to bind to bothVEGF and VEGFR1 (Fuh et al., 2000). We demon-strated that RH4 cells express both NRP1 and NRP2, soit is possible that the antibody may fail to disruptVEGFR1–ligand binding when in complex with NRPs.This may explain why the ERK phosphorylation wasonly inhibited by 36% with pretreatment with VEGFR1blocking antibody. Furthermore, we showed that bothRH4 and RH30 cells express NRP1 and NRP2;however, upon VEGF stimulation, only RH4 cellsexhibited ERK phosphorylation. Thus, it is not likelythat the NRPs independently contribute to ERKphosphorylation in RMS, which is not expected sincethey have short intracellular domains that are unlikelyto transduce signals (Neufeld et al., 2002). This wasconfirmed in a separate but related experiment. Wetreated RH4 cells with a VEGFR tyrosine kinaseinhibitor (5 mM; Calbiochem, San Diego, CA, USA),which resulted in an 82% reduction in ERK-2 phos-phorylation following VEGF stimulation (data notshown). This suggests that VEGFR1 is likely thereceptor transducing the VEGF signal in RH4 cells.

By treating RH4 cells with a VEGFR1 blockingpolyclonal antibody in the absence of additional growthfactors, cell proliferation was reduced compared to thenontreated control. This suggests that autocrine VEGFsignalling in RMS can be inhibited by blockingVEGFR1. At the highest doses, VEGFR1 blockingantibody inhibited growth of RH4 cells. As noexogenous growth factors were added, the blockage ofVEGFR1 appears to have decreased autocrine signallingthrough the receptor. It is possible that RH4-producedPlGF also acts as an autocrine factor through VEGFR1,although this was not examined in our study. Thus,presumably signalling by both VEGF and PlGF isdecreased by blocking VEGFR1 (Lyden et al., 2001).

Interestingly, low doses of the VEGFR1 blockingantibody appear to provide RH4 cells with a prolif-erative advantage, as the addition of 1 or 2 mg ofantibody yields higher cell numbers than nontreatedcells. While this effect appears to be counterintuitive, thebiological nature of VEGFR1 may provide someinsight. Aside from the membrane-spanning version ofVEGFR1, alternative splicing yields soluble VEGFR1(sVEGFR1). This soluble form competes with mem-brane-bound VEGFR1 for both VEGF and PlGF, andit has been suggested that the soluble receptor may actas a sink for excess ligand (Kendall and Thomas, 1993;Kendall et al., 1996). Perhaps, in a manner similar towhat has been suggested for soluble Her2 receptor (Kitaet al., 1996), VEGFR1 at the cell surface may havelimited accessibility of epitopes compared to its freemolecule in solution. As a result, the VEGFR1 blockingantibody may preferentially bind to sVEGFR1, and atlow antibody doses, antibody binding to sVEGFR1 maycause displacement of ligand. If so, low antibody dosesmay cause additional VEGF to be available to bind tomembrane-spanning VEGFR1, resulting in higher cellnumbers versus nontreated cells. When present in excess,


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antibody would likely bind both forms of VEGFR1resulting in decreased signalling through the receptor.Alternatively, sVEGFR1 may bind to membrane-span-ning receptors and act in a dominant negative fashion(Kendall et al., 1996); therefore, low doses of theantibody may disrupt this interaction thereby relievingthis intrinsic repression of VEGFR1 signalling. Theseresults raise a troubling issue. If receptor blockingantibodies are used as therapeutics, it is possible thatinadequate doses may be fostering tumour growthrather than inhibiting it. We are currently working toelucidate this paradoxical effect and attempting todetermine if blocking antibodies are effective agents inRMS treatment.

Previously, reports have demonstrated that, likely viadecreases in AP1 activity (Diaz et al., 2000), retinoidtreatment of various tumour types results in decreases inVEGF production (Weninger et al., 1998; Kini et al.,2001). Thus, we treated RH4 cells with AtRA to lookfor similar effects and to further investigate autocrinesignalling in RMS. Using an ELISA, we demonstratedthat AtRA treatment of RH4 cells decreased VEGF165

secretion by approximately half of baseline levels. Wereasoned that the decline in growth and increase inapoptosis of RMS cells treated with AtRA may be aresult of a reduction in VEGF secretion. To verify this,we supplemented AtRA proliferation-inhibited RH4cells with exogenous VEGF165 to see if growth could berescued. It should be noted that we used 5 mM AtRA toinduce growth arrest in RH4 cells, a dose that may notbe physiologically attainable. However, as cell linesinherently have resistance to pharmacologic doses oftherapeutic agents, this dose was deemed appropriatefor our studies. Moreover, this dose has been previouslyestablished as one that effectively inhibits RMS growthin vitro (Crouch and Helman, 1991; Brodowicz et al.,1999).

Previous experiments demonstrated that treatmentwith AtRA resulted in significantly decreased growthversus ethanol-treated control cells (Crouch and Hel-man, 1991; Brodowicz et al., 1999). We confirmed thisfinding in our experiment. However, upon addition ofVEGF165, cells demonstrated significantly increasedgrowth compared to cells treated solely with AtRA.To our knowledge, this experiment represents the firstevidence that the growth of AtRA-treated RMS cellscan be rescued upon the addition of exogenous growthfactor.

This rescue experiment highlights two importantpoints. First, the results further support our claim thatRMS cell lines are responsive to changes in the VEGFsignalling pathway. By reducing the amount of VEGFnormally produced, cellular responses to exogenouslyadded VEGF165 become very evident. Second, thisdemonstrates a potential mechanism by which AtRAinhibits RMS cell growth. Likely, the reduction ofproliferative and antiapoptotic factors, such as auto-crine VEGF signalling, results in decreased RMSgrowth and increased apoptosis. When an autocrinefactor, like VEGF, is returned to the system, the growthof the RMS cells is rescued. This phenomenon is clearly

demonstrable in serum-free conditions where there is anabsence of growth-promoting factors.

While our data show that the growth of AtRA-treatedRMS cells is rescued with supplementation with VEGF,this rescue is not complete. This suggests that inaddition to VEGF, other prominent RMS autocrinesignalling pathways, such as IGF-II, bFGF, EGF andTGF-b, may be inhibited by AtRA. In an early report ofretinoid use in RMS, Crouch and Helman (1991) usedAtRA to inhibit the growth of RD cells, which areknown to produce IGF-II at a high rate (Zhang et al.,1998) and express IGF-I-R (Kalebic et al., 1994). Theyreasoned that the decrease in RD proliferation may bedue to decreases in IGF-II signalling; however, nogrowth rescue was seen in cells treated with AtRA andIGF-II compared to those treated solely with AtRA(Crouch and Helman, 1991). Recently, however, it wasshown that retinoic acid decreases protein levels andphosphorylation of insulin receptor substrate-1, a keyadapter molecule for IGF-I-R, which serves as adocking module for several SH2 domain-containingproteins (del Rincon et al., 2003). This may help toexplain why Crouch and Helman did not observe anyrestoration in growth with IGF-II treatment, as RAinterference of the IGF-II pathway likely occurs not atthe level of the ligand, but downstream of IGF-I-R. Thissuggests that AtRA may inhibit multiple autocrinesignalling pathways in RMS, such as IGF-II and VEGF,and may be an appropriate treatment when used aloneor in combination with other therapies.

In summary, our experiments have shown thatmultiple RMS cell lines express both VEGF andreceptors, suggesting a possible autocrine loop. Toverify this, we showed that RMS cells are responsiveto VEGF, and signals are likely transduced viaVEGFR1. By blocking VEGFR1, we were able toreduce RMS cell numbers, suggesting that we inhibitedan autocrine VEGF signalling pathway. Furthermore,we reduced VEGF production through AtRA treatmentand saw a reduction in RMS growth. This was likely dueto decreases in autocrine signalling, and as a result ofreintroduction of VEGF, the cells showed rescuedgrowth. Thus, our results demonstrate a novel autocrinesignalling pathway in RMS and suggest that the VEGFpathway may be an appropriate target for the treatmentof RMS.

Materials and methods

Cell lines and culture conditions

The human ARMS lines RH4, RH18, RH28 and RH30, andthe human ERMS line RH6 were provided by Dr Tom Look.RD (human ERMS), SK-N-BE(2) (human neuroblastoma),HeLa (human cervical adenocarcinoma) and HUVECs (hu-man umbilical vein endothelial cells) were obtained fromAmerican Type Culture Collection. The pre-B acute lympho-blastic leukaemia cell line A1 was a gift from Dr MelvinFreedman (Hospital for Sick Children, Toronto, ON, Cana-da). HeLa cells were used as negative controls for expression ofVEGFR1 (Wakiya et al., 1996) and VEGFR2. HUVECs were


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used as positive controls for expression of VEGFR3, NRP1and NRP2 (Beierle et al., 2004). A1 (Narendran et al., 2003)and SK-N-BE(2) (Langer et al., 2000) cells were used aspositive controls for VEGFR1 expression. All RMS cell lines,SK-N-BE(2) and HeLa cells were grown in Dulbecco’smodified Eagle’s medium (DMEM; Wisent Inc., Montreal,QC, Canada) supplemented with 10% FBS (Sigma, St Louis,MO, USA) and 1% penicillin–streptomycin (Invitrogen,Burlington, ON, Canada). A1 cells were grown in suspensionin 10ml OPTI-MEM I (Invitrogen, Burlington, ON, Canada)supplemented with 5% FBS and 1% penicillin–streptomycin.Cells were kept at 371C in a humidified atmosphere containing5% carbon dioxide with free gas exchange.

Reagents and treatments

Recombinant human VEGF165 (rhVEGF165), VEGFR1 block-ing antibody (goat polyclonal anti-VEGFR1) and nonspecificgoat IgG (R&D Systems, Minneapolis, MN, USA) werereconstituted according to the manufacturer’s recommenda-tions. Briefly, rhVEGF165 was reconstituted with PBS supple-

mented with 0.1% human serum albumin (Bayer, Toronto,ON, Canada), which yielded a 10 mg/ml stock solution.VEGFR1 blocking antibody and nonspecific goat IgG wereboth reconstituted in PBS, producing 1 mg/ml stock solutions.AtRA (Sigma Chemical, St Louis, MO, USA) was resus-pended in 95% filter-sterilized ethanol at a stock concentrationof 5mM.

RT–PCR analysis

RMS cell lines were grown as monolayers in Falcon 10 cmdishes (Becton Dickinson, Franklin Lakes, NJ, USA). At 80%confluence, total RNA was isolated using Trizol (Invitrogen,Burlington, ON, Canada), according to the manufacturer’sprotocol. Bone marrow-derived stromal cell RNA (positivecontrol during the amplification of VEGFR2) was a gift fromDr Aru Narendran (Alberta Children’s Hospital, Calgary, AB,Canada). RNA samples (250 ng) were incubated at roomtemperature for 10min prior to being placed in a 96-wellGeneAmp PCR System 9700 (Applied Biosystems, FosterCity, CA, USA) at which point RNA was reverse transcribed

Table 1 RT–PCR primer sequences, cycling conditions and product sizes

Primer set Sense (S) and antisense (A) sequences Cycling conditions Product size (bp)

VEGFR1 S: 50-AAATAAGCACACCACGC-30

A: 50-ACCTGCTGTTTTCGATGTTTC-3030 cycles94.01C for 60 s54.11C for 60 s72.01C for 60 s

331

VEGFR2 S: 50-AAAACCTTTTGTTGCTTTTGGA-30

A: 50-GAAATGGGATTGGTAAGGATGA-3035 cycles94.01C for 30 s56.01C for 30 s72.01C for 55 s

236

VEGFR3 S: 50-CAGGATGAAGACATTTGAGG-30

A: 50-AAGAAAATGCTGACGTATGC-3035 cycles94.01C for 60 s52.01C for 60 s72.01C for 120 s

193

NRP1 S: 50-AGGACAGAGACTGCAAGTATGAC-30

A: 50-AACATTCAGGACCTCTCTTGA-3035 cycles94.01C for 60 s60.01C for 60 s72.01C for 120 s

209

NRP2 S: 50-AGCACTAATGGAGAGGACTG-30

A: 50-CCGTTTAGGCTGTAGGAGAC-3035 cycles94.01C for 60 s56.01C for 60 s72.01C for 120 s

481

VEGF set #1 S: 50-CGAAGTGGTGAAGTTCATGGATG-30

A: 50-TTCTGTATCAGTCTTTCCTGGT-3040 cycles94.01C for 70 s58.91C for 70 s72.01C for 70 s

404 – VEGF121

475 – VEGF145

535 – VEGF165

VEGF set #2 S: 50-TCAGTTCGAGGAAAGGGAAAG-30

A: 50-CATTTACACGTCTGCGGATCT-3040 cycles94.01C for 70 s57.81C for 70 s72.01C for 70 s

116 – VEGF183

134 – VEGF189

185 – VEGF206

b-Actin S: 50-ACCATGGATGATGATATCGCC-30

A: 50-GTGCCAGATTTTCTCCATGTC-3030 cycles94.01C for 60 s56.01C for 60 s72.01C for 60 s

263

GAPDH S: 50-AACGGGAAGCTCACTGGCATG-30

A: 50-TCCACCACCCTGTTGCTGTAG-3030 cycles94.01C for 60 s56.01C for 60 s72.01C for 60 s

304


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at 421C for 15min, denatured at 991C for 5min and thencooled at 51C for 5min. All reagents for reverse transcriptionwere obtained from Applied Biosystems (Foster City, CA,USA). PCR reactions were performed on a TGradientThermocycler (Whatman Biometra, Goettingen, Germany)using AmpliTaq Gold DNA Polymerase (Applied Biosystems,Foster City, CA, USA) and gene-specific oligonucleotideprimers (see Table 1 for cycling conditions and primersequences). Primers for VEGF set #1 (Suzuki et al., 1996),VEGFR2 (Ziegler et al., 1999), VEGFR3, NRP1 and NRP2(Beierle et al., 2004) have been previously published. All PCRreactions were preceded by a 10min hot start at 951C, andwere followed by a 10min final extension step at 721C. A 5 mlportion of reaction products (15 ml for VEGF) was run onagarose gels (4% for VEGF; 1% for all others) and visualizedusing ethidium bromide staining.

Western blot analysis

For ERK-1/2, cells (cultured in six-well plates) were washedtwice with cold PBS and lysed with 200 ml of cold lysisbuffer (50mM Tris/HCl (pH 7.4), 5mM EDTA (pH 7.4),150mM NaCl, 1% Triton X-100) containing complete, Miniprotease inhibitor cocktail tablets (Roche, Penzberg,Germany). Lysates were placed on ice for 30min and clarifiedby microcentrifugation (14 000 r.p.m., 10min, 41C). A 12.5%SDS–PAGE gel was used to separate 50 mg/sample of protein,followed by transfer onto nitrocellulose membranes. Vinculinand phosphorylated forms of ERK-1/2 were detected byimmunoblotting using a mouse monoclonal anti-vinculin(Upstate, Charlottesville, VA, USA), rabbit polyclonal anti-phospho-p44/42 MAP kinase (Cell Signaling Technology,Beverly, MA, USA) and appropriate alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearchLaboratories, West Grove, PA, USA). An alkaline phospha-tase staining solution (1M Tris/HCl (pH 9.5), 1M NaCl, 50mM

MgCl2) containing 5-bromo-4-chloro-3-indolyl phosphate andnitro blue tetrazolium (Pierce Biotechnology, Rockford, IL,USA) was used to visualize the protein. Band intensity ofimmunoblots was quantified using a FluorChem 8000 ImagingSystem and FluorChem 2.00 software (Alpha InnotechCorporation, San Leandro, CA, USA) and normalized tointensities of corresponding vinculin bands.For VEGFR1, cells (cultured in 10 cm dishes) were washed

twice with cold PBS and homogenized with 500 ml of coldlysis buffer (10mM Tris/HCl (pH 7.4), 2mM EDTA (pH 7.4),250mM sucrose) containing complete, Mini protease inhibitorcocktail tablets (Roche, Penzberg, Germany) using delicatesyringing with a 29 gauge needle. Lysates were centrifugedat 41C and 900 g for 10min, and supernatants were thencollected and ultracentrifuged at 41C and 32 000 r.p.m. for80min. Pellets were collected and dissolved in 100ml ofsolubilization buffer (10mM Tris/HCl (pH 7.4), 2mM ETDA,0.5% Triton X-100, 0.5% sodium deoxycholate) containingcomplete, Mini protease inhibitor cocktail tablets (Roche,Penzberg, Germany). An 8% SDS–PAGE gel was used toseparate 150 mg/sample of protein, followed by transfer ontonitrocellulose membranes. VEGFR1 and b-actin were detectedby immunoblotting using a mouse monoclonal anti-VEGFR1(Chemicon, Temecula, CA, USA) or mouse monoclonal anti-b-actin (Sigma-Aldrich, Oakville, ON, USA) and a horse-radish peroxidase-conjugated rabbit anti-mouse secondaryantibody (Pierce Biotechnology, Rockford, IL, USA). Mem-branes were developed using the ECL chemiluminescencesystem (Amersham, Piscataway, NJ, USA). Ponceau stainingof membranes was performed to confirm equal loading ofprotein.

ELISA analysis

To assay VEGF165 secretion, cells were seeded in 24-well plateswith DMEM containing 10% FBS. After 24 h, medium wasreplaced by DMEM supplemented with 2% FBS. Formeasurement of baseline VEGF165, supernatants were col-lected 24 h later. For measurement of VEGF165 followingAtRA treatment (initial seeding density of 7.5� 105 RH4cells), medium was supplemented with either ethanol or 5mMAtRA at the switch to low serum. After 24 h, mediumcontaining treatment conditions was refreshed, and super-natants were collected 48 h later. Secreted VEGF165 wasmeasured using the Quantikine Human VEGF Immunoassay(R&D Systems, Minneapolis, MN, USA) following themanufacturer’s protocol. The optical density of each wellwas detected by means of a microplate reader detectingabsorbance at 450 nm with a wavelength correction of 540 nm.Results were normalized by the number of cells per well, asmeasured by Coulter Counter (Coulter, Hialeah, FL, USA).

Cell counting

Growth of RH4, RH18, RH28 and RD cells in response toaddition of rhVEGF165 or growth of RH4 cells in response toVEGFR1 blocking antibody was determined by counting totalcells with a Coulter Counter (Coulter, Hialeah, FL, USA). Toexamine growth in response to rhVEGF165, RMS cells wereseeded in Costar 24-well plates (Corning, Corning, NY, USA)with DMEM containing 10% FBS. The RMS lines requireddifferent initial densities (3.4� 104 RH4, 2.2� 104 RH18,9.0� 104 RH28 and 2.0� 104 RD) to survive 4 days inserum-free medium and be responsive to exogenous growthfactor. After cells had adhered to the wells (24 h), cells wereserum starved by adding serum-free medium. A further 24 hlater, rhVEGF165 was added to fresh serum-free medium atvarious doses. Media and rhVEGF165 were refreshed 48 hlater, and cells were trypsinized and counted after 4 days fromthe point of initial cytokine addition. To examine growth inresponse to anti-VEGFR1 blocking antibody, 5.3� 104 RH4cells were seeded in 24-well plates with DMEM containing10% FBS. After 24 h, medium was removed and replaced with500 ml of DMEM containing 2% FBS, supplemented withVEGFR1 antibody at various doses. Cells were counted after6 days.

Alamar Blue assay

The Alamar Blue assay was used to determine the cell viabilityof RH4 cells at different time points under different treatmentconditions. To examine growth of RH4 cells over a 6-dayperiod, 3.6� 104 RH4 cells were seeded in 24-well plates withDMEM containing 10% FBS. After recovering (24 h), cellswere serum starved for 24 h. At this point (day 0), serum-freemedium was refreshed and treatments (100 ng rhVEGF165

alone or in combination with 20mg VEGFR1 blockingantibody) were applied. Medium and treatments wererefreshed at days 2 and 4. In a separate experiment, cells weretreated with 20 mg nonspecific goat IgG, 100 ng rhVEGF165

plus goat IgG or rhVEGF165 plus 20mg VEGFR1 blockingantibody, and viability was measured at day 4. To determine ifVEGF rescues growth of AtRA-treated RH4 cells, 5.0� 104

cells were seeded in 24-well plates with DMEM containing10% FBS. Cells were allowed to recover for 24 h, and then 1mlof DMEM was added to each of the wells. Cells were treatedwith 5 mM AtRA, 5mM AtRA plus 200 ng rhVEGF165, ornontreatment represented by an equivalent volume of ethanol.Medium and treatment conditions were refreshed 24 h laterand every 2 days thereafter for the course of the experiment.


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Alamar Blue was added to the wells (rhVEGF165 addition timecourse: days 0, 2, 4, 6; rhVEGF165 addition end point: day 4;AtRA rescue: days 0, 1, 2, 3, 4, 5, 6) at a final concentration of10%, and after 5 h of incubation, fluorescence (excitation:540 nm; emission: 590 nm; auto-cutoff: 570 nm) was detectedusing a microplate reader.

Statistical analysis

Significance between treatment conditions was determined bytwo-tailed, unpaired Student’s t-test. A P-value of 0.05 or lesswas considered to be statistically significant.

Abbreviations

VEGF, vascular endothelial growth factor; rhVEGF165,recombinant human vascular endothelial growth factor (165amino-acid isoform); PlGF, placenta growth factor; VEGFR,vascular endothelial growth factor receptor; flt1, fms-liketyrosine kinase receptor (or VEGFR1); KDR, kinase insert

domain-containing receptor (or VEGFR2); NRP, neuropilin;RMS, rhabdomyosarcoma; ARMS, alveolar rhabdomyosar-coma; ERMS, embryonal rhabdomyosarcoma; AtRA, all-trans-retinoic acid; IGF-II, insulin-like growth factor-II;bFGF, basic fibroblast growth factor; EGF, epidermal growthfactor; TGF-b, transforming growth factor; ERK, extracel-lular signal-regulated kinase.

AcknowledgementsThis work was funded by the Andrew Mizzoni CancerResearch Fund and the James Birrell Research Fund, andthrough studentships of the Ontario Student OpportunityTrust Fund – Hospital for Sick Children Foundation StudentScholarship Program (MG, BD) and the Brainchild Founda-tion (BD). RT is supported in part by the ScotiabankClinician-Scientist Training Program Fund, and the ResearchTraining Centre, Hospital for Sick Children, Toronto. DM is aResearch Scientist of the National Cancer Institute of Canada/Canadian Cancer Society. We are grateful for helpfuldiscussions with Drs Herman Yeger and Meredith Irwin.

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