Peptide-conjugated antisense oligonucleotides for targeted inhibition of a transcriptional regulator in vivo

Peptide-conjugated antisense oligonucleotides fortargeted inhibition of a transcriptional regulator in vivoErik Henke1, Jonathan Perk1, Jelena Vider2, Paola de Candia1, Yvette Chin1, David B Solit3,4,Vladimir Ponomarev2, Luca Cartegni4, Katia Manova5, Neal Rosen3,4 & Robert Benezra1

Transcription factors are important targets for the treatment of a variety of malignancies but are extremely difficult to inhibit,

as they are located in the cell’s nucleus and act mainly by protein-DNA and protein-protein interactions. The transcriptional

regulators Id1 and Id3 are attractive targets for cancer therapy as they are required for tumor invasiveness, metastasis and

angiogenesis. We report here the development of an antitumor agent that downregulates Id1 effectively in tumor endothelial

cells in vivo. Efficient delivery and substantial reduction of Id1 protein levels in the tumor endothelium were effected by fusing

an antisense molecule to a peptide known to home specifically to tumor neovessels. In two different tumor models, systemic

delivery of this drug led to enhanced hemorrhage, hypoxia and inhibition of primary tumor growth and metastasis, similar to

what is observed in Id1 knockout mice. Combination with the Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin

yielded virtually complete growth suppression of aggressive breast tumors.

Tumors often depend on deregulation of transcription factor activityto maintain the transformed state by growth factor–independentproliferation, suppression of apoptosis, self-renewal and initiation ofangiogenesis. Whereas direct activation of oncogenic transcriptionfactors such as c-myc1 and c-myb2 is observed in some cancers, mostalterations in transcription factor activity result from events upstreamin the cell signaling cascade such as deregulation of tyrosine kinase orG-protein activity3. Thus, transcription factors can act as critical focalpoints in a number of oncogenic pathways4. Although transcriptionfactors have emerged as important targets for cancer therapy, they areextremely difficult to inhibit by conventional means. Their intra-nuclear localization precludes the use of therapeutic antibodies. Also,transcription factors (other than steroid hormone receptors, SHRs)lack binding sites for small molecules, which makes development ofsmall-molecule inhibitors difficult. Nonetheless, inhibiting transcrip-tion factors has been attempted with varying success; approachesinclude blocking DNA-binding5, using peptidomimetics6 andG-quartet oligonucleotides7, and inhibiting transcription factorexpression by antisense oligonucleotides8. Clinical development ofthese approaches has remained problematic, however, and non-SHRtranscription factors are widely considered undruggable.

Here we report the development of a targeted antisense approach toinhibit the dominant negative transcription factor Id1 in the tumorendothelium of living animals. This test system was chosen for severalreasons. Whereas involvement of Id proteins in tumor cell aggressive-ness9–11 and metastatic behavior12,13 has been suggested from data incultured cells, their role in tumor angiogenesis in vivo is wellestablished. Vascular defects after Id loss have been observed in a

variety of murine models14–17 and Id upregulation is observed in theendothelium of all solid human tumors examined to date11,18. Evenpartial loss of Id1 activity by genetic manipulation in mice has beenshown to inhibit tumor angiogenesis, and subsequently the growth ofprimary tumors and metastases14,15. We could, therefore, set a reason-able goal of phenocopying Id1+/– mice using our targeted therapy. Id1is specifically upregulated in tumor endothelial cells11,15–17, so targetedinhibition is likely to be nontoxic. Finally, Id1 is downstream of pro-angiogenic factors VEGF-A19, bFGF, IGF-1 (ref. 20) and EGF21 so lossof Id1 activity could short-circuit all of these pathways, an importantconsideration as tumors can escape mono-directed, antiangiogenictherapy by upregulation of alternate growth factors22,23.

But hurdles for targeting Id1 are substantial. In addition to thedifficulties of targeting transcription factors as outlined above, inhibit-ing Id1 is complicated even more by the structural similarities of theIds and their bHLH binding partners. Drugs targeting Id proteins mustbe selective for the Id-bHLH interaction and not affect bHLH-bHLH-dimerization. We have developed an antisense approach to inhibit Id1.To circumvent poor pharmacokinetic properties of antisense oligonu-cleotides, we covalently coupled them to an address-peptide thattargets tumor endothelial cells. This peptide, fragment F3 of the highmobility group protein (HMG)N2, homes to neo-vessels in xenografttumors and localizes in nuclei of endothelial cells24. Coupling to F3enhanced the effectiveness of antisense oligonucleotides by increasinglocal concentrations in the target cells and facilitating uptake into thecorrect cellular compartment. Properties of the resulting peptide-conjugated antisense oligonucleotide (Id1-PCAO) and its effects ontumor angiogenesis and tumor growth are reported here.

Received 20 September 2007; accepted 19 November 2007; published online 6 January 2008; doi:10.1038/nbt1366

1Department of Cancer Biology and Genetics, 2Department of Radiology, 3Department of Medicine and 4Department of Molecular Pharmacology and Chemistry,5Molecular Cytology Core Facility, Memorial Sloan-Kettering Cancer Center, 1270 York Ave., New York, New York 10021, USA. Correspondence should be addressedto ([email protected]).

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RESULTS

Coupling of oligonucleotides to peptides

We first identified an antisense oligonucleotide that inhibitedexpression of both murine and human Id1. Fully phosphorothioatedantisense oligonucleotides displayed high toxicity in transfectionexperiments (Supplementary Fig. 1 online). We therefore developeda gap-mer of the selected antisense oligonucleotide, which is an oligo-nucleotide consisting of five non-phosphorothioated 2-O¢-methylRNA bases at both the 5¢- and 3¢- end and a central 13-mer stretchof phosphorothioated DNA (referred to as Id1-AO). Id1-AO butnot a reverse complementary control (rcId1-AO) substantiallyreduced Id1 protein levels after standard lipid transfection in twoendothelial cell types derived from human, human umbilical veinendothelial cells (HUVEC), and mouse (MS-1) (Fig. 1a,b). Id3protein levels were unaffected and served as a control for specifi-city as its gene sequence shows only four and five mismatches

(mId3 and hId3, respectively) with the chosen Id1-AO (Supple-mentary Fig. 2 online). To couple this antisense oligonucleotideto the F3-peptide, we modified it with a C6-amino-linker atthe 5¢-end. F3-peptide was coupled by an N-terminal cysteine(Fig. 1c). Conjugation was performed with a hetero-bifunctionallinker (GMBS), in a chemo- and regiospecific way. The productwas verified by mass spectroscopy and digestion with proteinase K(Fig. 1d).

Id1-PCAOs showed remarkable stability in plasma relative tounmodified antisense oligonucleotides, similar to the gap-mer alone(Fig. 1e). Indeed, lability of the PCAO is due primarily to degradationof the peptide (Fig. 1e). At 37 1C little degradation was observed after24 h, suggesting suitability for in vivo experiments. No degradationwas observed in buffered saline after 28 d at 37 1C (Fig. 1f) making itpossible to administer the drug through subcutaneously implantedpumps (see below).

0 control rcld1-AO Id1-AO

24 48 72 24 48 72 24 48 72 h rcld

1-A

OId

1-A

O HUVEC

hld1

hld3

β-Actin

β-Actin

a d e f

b

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0 control rcld1-AO Id1-AO

24 48 72 24 48 72 24 48 72 h

MS1

mld1

mld3

GMBSO

N N

OO

O

OH2N

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2NH2

OO O

OO O O

O

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+

OMe

OMe

N

N

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N

R′

1. TrisHCl, 200 mM,pH 8.4, 20% MeCN

90 min, 25 °C

2. Precipitationwith MeCN

NH

NP

OMe

N

N N

N

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POMeR ′Amino-modified

Oligonucleotide

R ′′′′

O

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NH

S NNH

OO

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NN

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ON

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O

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O OMeO OP R ′

Peptide-conjugated antisense-oligonucleotide (PCAO)

HSH

R ′′′′

Cystein-modifiedpeptide

100 mMNa-Phosphate,400 mM NaCl

pH 7.024 h, 25 °C

1 2 3

1: Id1-AO2: Id1-PCAO3: Id1-PCAO

+ ProtK0 2 4 8 24 48 h

Incubation time in mouseplasma, 37 °C

Id1-AO(2′OMe/PT modified)

Id1-AO-DNA(non-modified)

Id1-PCAO

Id1-PCAO

Id1-AO

0 d 7 d 14 d 21 d 28 d Id1-AOId1-PCAO

Incubation timein TBS, 37 °C

Figure 1 Characterization of Id1-antisense oligonucleotides and Id1-PCAOs. (a,b) Western blot analysis of the effect of the selected antisense sequence

toward the human (a, HUVEC) and the murine form (b, MS1 cells) of Id1. Transfection with 200 nM Id1-AO or rcId1-AO every 24 h led to substantially

reduced levels of Id1, but has no effect on expression levels of the homologous Id3 proteins. (c) Schematic description of the synthesis of Id1-PCAOs.

(d) Agarose gel analysis of proteinase K digests of Id1-PCAO. (e) Agarose gel analysis of Id1-PCAO after incubation in mouse plasma at 37 1C for up to

48 h. A nonphosphorothioated DNA oligonucleotide (Id1-AO-DNA) is completely degraded within 4 h. (f) Agarose gel analysis of Id1-PCAO after incubation

in buffered saline at 37 1C for up to 28 d.

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Uptake of Id1-PCAOs by endothelial cells

To determine if the Id1-PCAO retained the homing specificity of theF3-peptide and could be taken up by cells in the absence of lipidcarrier, we supplied fluorescence-labeled Id1-PCAOs to different celllines at a concentration of 200 nM. Confocal microscopy showeduptake by endothelial cells (HUVECs and murine endothelioma cells,EOMA (Fig. 2a and Supplementary Fig. 3 online)), whereas all othertested tumor cell lines and normal murine embryonic fibroblasts(MEFs) and human dermal fibroblasts were negative. No endothelialcell internalization was observed using non-peptide-conjugatedfluorescence-labeled Id1-AOs (see below). Similar results wereobtained with fluorescein and PCAOs labeled with tetra-methyl-rhodamine-red. Epifluorescence live imaging on viable HUVECs andHeLa cells yielded similar results thus ruling out fixation artifacts(Supplementary Fig. 4 online).

Downregulation of Id1 with PCAOs in vitro

Exponentially growing HUVECs were incubated with standard growthmedium (EGM-2) supplemented with Id1-PCAOs in the absence oflipophilic transfection reagents (Fig. 2b,c). This treatment resulted in

a dose-dependent downregulation of Id1 expression. Treatment wasrepeated every 24 h for at least two consecutive days to yield asubstantial effect on Id1 levels. Near complete inhibition of Id1expression was reached after 3 d with a dosage of 200 nMId1-PCAO, conditions under which unconjugated Id1-AOs showedonly minor Id1 inhibition (Fig. 2b). Moreover, Id1-PCAO treatmentresulted in upregulation of p16ink4a and downregulation of MMP-2,known Id1 targets25–27. Id1-PCAO concentrations as high as 1 mM for5 d did not affect HUVEC (Fig. 2d) or tumor cell proliferation(Supplementary Fig. 5 online). However, 200 nM Id1-PCAO over3 d blocked tube formation of HUVECs on Matrigel and substantiallyinhibited cell migration (Fig. 2e,f and Table 1). Reverse complementrcId1-PCAO or Id1-AO plus the F-peptide in unconjugated formhad no effect.

Mechanism of uptake

Id1-PCAOs colocalized with nucleolin in the nucleus of HUVECs(Fig. 3a) in accordance with published data indicating that nucleolinis the cell surface binding partner for F3 and that F3 is transportedwith nucleolin into the cytoplasm and subsequently into the

a

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p16ink4a

MMP2

0.040.370.610.810.761.00

72 h48 h24 h0 h

1.00 0.87 0.71 0.07

Id1-PCAO (200 nM)

120967248240Time (h)

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Cel

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Control200 nM F3 + Id1AO

200 nM Id1-PCAO1 µM F3 + Id1AO

1 µM Id1-PCAO

Id1-PCAOF3 + Id1-AOSaline

rcld1-PCAOId1-PCAOF3 + Id1-AOSaline

Figure 2 In vitro homing and activity of Id1-PCAOs. (a) Standard growth media of exponentially growing cells was supplemented with 200 nM tetra-methyl-

rhodamine-labeled Id1-PCAO (Id1-PCAO-TAMRA). Uptake and nuclear localization is only observed in endothelial cells (HUVEC, EOMA). Laser confocal

images, scale bars: 20 mm. (b) Western blot analysis of Id1 levels in HUVEC after incubation with different concentrations of Id1-PCAO over 72 h withrenewal of Id1-PCAO–supplemented media every 24 h. (c) Western blot analysis of time-dependent Id1 levels in HUVEC after incubation with Id1-PCAO.

Treatment with Id1-PCAOs leads to inhibition of Id1 expression in a time and concentration dependent way. (d) Cell proliferation assay. HUVEC were

incubated with different concentrations of Id1-PCAO over 5 d. Supplemented medium was renewed every 24 h. (e,f) Prolonged exposure of HUVEC to

Id1-PCAOs (72 h, 200 nM Id1-PCAO, exchange of supplemented media every 24 h) inhibits tube formation on Matrigel (e) and migration of HUVEC

in a scratch assay (f). Cells were counterstained with calcein AM for tube analysis. Scale bars, 20 mm; all errors given in ± s.e.m.

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nucleus28. To verify that Id1-PCAO is translocated by this mechanism,we performed a blocking experiment using an antinucleolin antibody(ZN004) that recognized nucleolin on the surface of HUVECs (datanot shown). ZN004 was able to block uptake of fluorescein-labeledId1-PCAO into the nucleus of proliferating HUVECs (Fig. 3b).ZN004 did not block binding of PCAOs to cell surface nucleolin(Fig. 3b, rightmost panel) suggesting that F3 binding to nucleolin isnot sterically hindered by the antibody, rather internalization of theconjugate is. ZN004, but not an IgG control, efficiently inhibiteddownregulation of Id1 protein levels by PCAOs in HUVECs (Fig. 3c).It has been reported that nucleolin is translocated to the cell surface ofendothelial cells after stimulation with vascular endothelial growthfactor (VEGF)29. Consistent with this observation, binding of PCAOto the cell surface and transport into HUVECs is VEGF-A165

dependent (Fig. 3d).

Homing of Id1-PCAOs to tumor endothelium

The ability of PCAOs to accumulate in tumor endothelium was testedin different murine models. MDA-MB-435S xenografts were firsttested because the vasculature of these tumors is efficiently targetedby F3 (ref. 24). Allografts from spontaneous breast tumors arising inId1�/� MMTV-HER2/neu (YD) mice were also tested15. Fluores-cence-labeled Id1-PCAOs were injected systemically into graft-bearingimmunodeficient mice. After 4 h, accumulation of fluorescent dyecould be observed in the endothelium of dissected tumors as verifiedby costaining for the endothelial marker CD31 (Fig. 4a, top twopanels). Unconjugated antisense oligonucleotides were not deliveredinto tumor endothelium (Fig. 4a, bottom panel). Id1-PCAOs were notdetected in most other organs (brain, heart, colon, liver and spleen,

Fig. 4b and Supplementary Fig. 6 online). However, fast uptake ofId1-PCAO into tubular cells of the renal cortex was observed (Fig. 4c,upper left panel). Accumulation in renal cortex was also observed withfluorescence-labeled Id1-AO—that is, the partially phosphorothioatedgap-mer (Fig. 4c, lower left panel) and fully phosphorothioatedantisense oligonucleotides (Supplementary Fig. 7 online)—andFITC-F3 (Supplementary Fig. 8 online). In addition Id1-AO andfully phosphorothioated antisense oligonucleotides also accumulatedin liver (Fig. 4c, lower right panel and Supplementary Fig. 7). This isin accordance with biodistribution studies that showed a preferentialaccumulation of phosphorothioated antisense oligonucleotides in liverand kidney30. Conjugation to F3-peptide seems to block most of theliver accumulation (Fig. 4c, upper right panel).

Homing studies were also performed in MMTV-HER2/neu (YD)and PTEN+/�animals bearing tumors. Accumulation of Id1-PCAOs intumor endothelium was observed in these models, showing that thehoming properties are maintained in spontaneous tumor models(Supplementary Fig. 6).

To test PCAO activity in vivo we injected tumor-bearing transgenicMMTV-HER2/neu (YD) Id1+/� mice intravenously with Id1-PCAOs.Because repeated application of the drug was necessary to yieldsignificant downregulation in vitro, animals were treated with15 nmol/d of Id1-PCAO or Id1-AO for three consecutive days. Over80% of tumor vessels in animals treated with Id1-PCAO werecompletely negative for Id1 expression by immunohistochemistry(Fig. 4d). The Id1-AO alone showed no detectable downregulationof Id1 in tumor vessels.

Single agent and combination therapy

Short-term treatment of established allograft tumors with Id1-PCAOled to drastically increased hemorrhage and hypoxia and a moderatebut substantial growth suppression (Supplementary Fig. 9 online). Todetermine if longer treatment leads to stronger growth suppression,we implanted animals with osmotic pumps that delivered Id1-PCAOcontinuously over 21 d. Id1-PCAO was also combined with theHsp90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) as genetic Id-loss in combination with 17-AAG is more effectivethan either alone in reducing tumor burden15, perhaps owing to therequirement of Hsp90 for maintaining Hif1a or HER2/neu stability31.

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3334

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Figure 3 The uptake of Id1-PCAOs is nucleolin dependent and can be stimulated by VEGF-A. (a) Laser confocal images of HUVEC after incubation with

Id1-PCAO or Id1-AO for 4 h. Medium was supplemented with 200 nM FITC-labeled Id1-PCAO (Id1-PCAO-FAM) or Id1-AO-FAM. Subsequently cells were

immunofluorescence stained for nucleolin. Id1-PCAO colocalize with nucleolin in the nucleus, Id1-AO is not taken up by HUVEC. (b) Exponentially growing

HUVECs were treated with anti-nucleolin AB ZN004 for 2 h before media was supplemented with fluorescein-labeled PCAO (Id1-PCAO-FAM, 200 nM).

Antibody ZN004 blocked uptake of the PCAO completely. (c) HUVEC were treated for 72 h with Id1-PCAO together with addition of antinucleolin or control

antibodies. ZN004 treatment resulted in inhibition of the Id1-downregulation, with H-250 and IgG-control this effect was not observed. (d) HUVEC were

serum- and GF-starved for 24 h and incubated with varying amounts of VEGF-A165 (0, 2, 5 20 ng/ml) for 8 h. Subsequently the cells were treated with200 nM of Id1-PCAO-FAM plus VEGF-A165 for an additional 2 h, showing that Id1-PCAO uptake is VEGF dependent. Results were similar if the cells

were plated on standard culture slides or culture slides coated with fibronectin. All scale bars, 50 mm; all errors given in ± s.e.m.

Table 1 HUVEC tube formation on Matrigel after treatment with

Id1-PCAO

Total tube length mm/mm2 Branching points/mm2

Saline 8.13 ± 0.74 270.7 ± 32.18

F3 + AO 8.40 ± 0.43 322.9 ± 21.09

Id1-PCAO 3.51 ± 0.66 P ¼ 0.004 116.8 ± 24.88, P ¼ 0.02

rcId1-PCAO 8.10 ± 1.05 278.2 ± 42.99

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Id1-negative cells from a spontaneous tumor formed in a MMTV-HER2/neu (YD) Id�/� animal were used to ensure that observedeffects were caused by Id1 inhibition in the microenvironment,presumably the endothelium, and not the tumor cells.

Id1-PCAO was delivered by osmotic pumps at a rate of 7 nmol/dfor 21 d. 17-AAG was given by intraperitoneal injection on threeconsecutive days per week15 after tumor establishment. As controls,saline or the unconjugated components of the Id1-PCAO (F3-peptideand Id1-AO each at 20 nmol/d) were administered. In Id1-PCAO–treated animals the average tumor volume on day 21 was 40% of thatobserved in untreated tumors (Fig. 5a). The efficacy was comparableto treatment with 17-AAG alone (34% tumor volume).

Combination of both drugs, however, yielded virtually completeinhibition of tumor growth over the treatment period (10% tumorvolume, P ¼ 0.0001, P o 0.0001, P ¼ 0.0002 versus control, 17-AAG,and F3 plus Id1-AO plus 17-AAG, respectively). In contrast admin-istration of Id1-AO and peptide in unconjugated form did not inhibittumor growth and showed no enhancement over the 17-AAG aloneeffect (Fig. 5a,b). To further control for nonspecific effects, werepeated the experiment with a PCAO with the reverse complemen-tary oligonucleotide sequence (rcId1-PCAO, delivered at the same rateas the Id1-PCAO, 7 nmol/d) and F3-peptide without addition ofId1-AO (20 nmol/d) (Supplementary Fig. 10 online). Id1-PCAOadministration in combination with 17-AAG again had a robust effecton growth (P ¼ 0.0079) whereas neither rcId1-PCAO nor F3enhanced 17-AAG efficacy (P ¼ 0.97 and 1.00, respectively). Injectionof Evans blue in selected animals showed massively increased leakagefrom Id1-PCAO–treated tumor blood vessels (Fig. 5c) probablyaccounting for hypoxic stress and sensitivity to 17-AAG.

Treatment with Id1-PCAO or 17-AAG alone resulted in a decreaseof Id1-positive endothelial cells in the tumor (Fig. 5d and Supple-mentary Fig. 11 online). Whereas Id1-PCAO downregulated Id1expression in the cells, 17-AAG led to a decreased vascular density,which resulted in the lower count for Id1-positive cells. Combination

of both drugs further diminished Id1-positive cells. Whereas Id1-PCAO administration caused upregulation of Hif1a, 17-AAG injec-tions counteracted this response (Fig. 5e,f). The hypoxic regions werecharacteristically surrounded by necrotic areas that displayed signsof cystification.

Id1-PCAO treatment did not affect animal weight or wound healing(data not shown). Kidneys were examined after the 3-week treatment,and no gross histological signs of toxicity were observed.

Inhibition of metastatic growth

The antitumor properties of Id1-PCAO as a single agent were furtherexamined in a second tumor model. Lewis lung carcinoma (LLC)allografts were chosen because genetic loss of Id1 alone had beenshown previously to significantly slow tumor growth and metastasis inthis model14. LLC cells form tumors when implanted subcutaneouslyin nonimmunocompromised mice with a pure or partial C57BL/B6background14,25 and formation of metastasis is observed after removalof the primary tumor32.

To follow metastatic spread, we transduced LLC cells beforeimplantation with a retroviral dual-modality reporter vector expres-sing eGFP and firefly luciferase33,34. GFP-positive cells, obtained byfluorescence-activated cell sorting (FACS), were injected into thedorsal flank of male C57/B6 mice. When 7 d after injection tumorsreached an average size of 20 mm3, osmotic pumps were implanteddelivering Id1-PCAO at 20 nmol/d over 14 d. Control animals receivedsaline, rcId1-PCAO (20 nmol/d) or F3 and Id1 AO in nonconjugatedform (75 nmol/d). Primary tumors were surgically removed 14 d afterinjection and animals were monitored for metastatic development byin vivo luciferase imaging (Fig. 6a). Id1-PCAO treatment, althoughstarted in progressed, aggressively growing tumors, resulted in asignificant reduction of primary tumor growth (P ¼ 0.0079), whereasF3 plus Id1-AO or the reverse complimentary rcId1-PCAO controlshad no effect (P ¼ 1.0 and P ¼ 0.4206, Fig. 6b). Id1-PCAO again ledto an increase in Hif1a-positive cells (data not shown), but baseline

HE

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a b c d

Figure 4 In vivo tumor homing and activity of Id1-PCAOs. (a) Id1-PCAOs accumulate in the vasculature of MMTV-HER2/neu (YD) and MDA-MB-435s graft

tumors grown in NudeNCR mice after systemic injection (20 nmol/mouse Id1-PCAO-TAMRA or Id1-AO-TAMRA, dissection of organs 4 h after i.v. injection).

Id1-AO alone does not home to the tumor (lowest panel). (b,c) Organ sections of animals bearing MMTV-HER2/neu (YD) allografts 4 h after i.v. injection of

20 nmol Id1-PCAO-TAMRA. The same dose does not lead to accumulation in other organs (b), with the exception of the renal cortex (c). Unconjugated Id1-

AO accumulates in the renal cortex and in the liver (lower panel). All images in b and c merged channel confocal images (green: CD31 immunofluorescence

staining, red: Id1-PCAO-TAMRA or Id1-AO-TAMRA and DAPI counterstain). (d) IHC staining of tumor sections for Id1 after treatment with Id1-AO or

Id1-PCAO. Systemic treatment with Id1-PCAO (15 nmol/d i.v.) over 3 d leads to loss of Id1 expression in endothelial cells (arrows) in spontaneous MMTV-

HER2/neu (YD) driven tumors in an Id1+/� background. All scale bars, 50 mm.

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Hif1a levels in treatment-naive tumors were strongly elevated relativeto the HER2/neu allografts.

Histological analysis of primary tumors showed a decrease in Id1levels in the endothelium (data not shown) and enhanced hemorrhageafter Id1-PCAO treatment (Fig. 6), indicating a similar therapeuticresponse as seen in the other models described above. After removalof the primary tumor, all animals developed metastasis to the lungand occasionally to intestines and aggressive invasive local secondaryregrowth (Fig. 6c). Median survival, free of secondary tumors, wasprolonged by the Id1-PCAO treatment from 3 to 27 d(P ¼ 0.0191), whereas tumor-free survival in the F3 plus Id1-AOand rcID1-PCAO groups was only 12 d, which is not statisticallysignificant (P ¼ 0.483 versus saline-treated animals). Effects of thePCAO on both tumor growth and hemorrhage recapitulated resultsobtained in Id1+/� animals (compare Fig. 6b,d with SupplementaryFig. 12 online) consistent with partial inhibition of Id1 protein levelsrevealed by immunohistochemistry.

DISCUSSION

The Id proteins have attractive characteristics as targets forantiangiogenic tumor therapy. They are essential for the mobilizationof endothelial progenitors from the bone marrow to the tumor17,25,are not expressed in normal adult vasculature and lead to severeperturbations in vascular integrity when partially inhibited

genetically14–17. But inhibiting the activity of the Id proteins is difficultbecause they work by blocking the DNA binding activity of transcrip-tion factors by direct physical association (reviewed in refs. 35,36). Inan attempt to inhibit expression of Id1 protein in tumor endothelialcells, we have developed an antisense targeting strategy wherebyintroduction of the antisense moiety into endothelial cells is facilitatedby fusion with a peptide (F3) that binds tumor endothelial cellsspecifically. The resulting PCAO retains its homing specificity andability to inhibit Id1 protein expression both in vitro and in vivo. Theuptake is VEGF-A dependent and can be blocked with antinucleolinantibodies, indicating that the mechanism of uptake is active and inaccordance with the mechanism proposed previously for F3 (ref. 28).The dependence on endothelial cell stimulation by VEGF-A andpresumably other angiogenic growth factors29 explains the selectivityfor the tumor vasculature versus resting blood vessels. Whereas it hasbeen shown that F3 can transport a payload like fluorophores ornanoparticles into the tumor vasculature24,37,38, it was anticipated thatthe homing potential of the highly basic F3-peptide might be affectedby conjugation to the anionic oligonucleotide. However, the PCAOseems to show higher selectivity for endothelial cells than F3 itself,which is also taken up by tumor cells in vitro24. This might indicatethat the binding affinity of F3 for nucleolin is reduced by the attachedanionic antisense moiety and we could, therefore, observe uptake onlyin cells that have the highest surface concentration of the receptor,

a d

e

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c

2220181614121086420–2

Days post tumor implantation

100

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Id1-PCAO + 17-AAG (n = 5)Id1-PCAO (n = 5)F3 + Id1-AO +17-AAG (n = 5)F3 + Id1-AO (n = 5)Saline + 17-AAG (n = 5)Saline (n = 5) b

17-AAG

17-AAG+ F3

+ Id1-AO

17-AAG+ Id1-PCAO

Saline F3 +Id1-AO

Id1-PCAO

P = 0.0034 P = 0.00224

3

2

1

0

CD

31 p

os. a

rea

(%)

Saline

F3 +

Id1-

AO

Id1-

PCAO

Saline

+ 1

7-AAG

F3 +

Id1-

AO + 1

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Id1-

PCAO + 1

7-AAG

750

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0

Id1-pos. cells/m

m2

CD31 positive area (%) Id1-positive cells/mm2

P = 0.04

1,000

800

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0Hif1

α po

s. c

ells

/mm

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Saline

17-A

AG

F3 +

Id1A

O

F3 +

Id1A

O + 1

7AAG

Id1-

PCAO

Id1-

PCAO + 1

7-AAG

Anti-Hif1α IHC

SalineId1-PCAO +

17-AAG

Id1-PCAO Id1-PCAO

Figure 5 Combination therapy with 17-AAG affects tumor growth and vascular integrity.

(a) Immunodeficient mice were subcutaneously implanted with osmotic pumps delivering

7 nmol/d Id1-PCAO or 20 nmol/d F3-peptide and Id1AO (red line, working period of pumps).

Animals received 17-AAG by i.p. injection on three consecutive days/week (black arrows).

(b) Animals receiving 17-AAG with or without Id1-PCAO 14 d after tumor implantation

(tumors: dotted outline). (c) Epifluorescence images of whole tumor sections from animals

in different treatment arms. Animals were injected with Evans blue 4 h before being killed.The red fluorescent Evans blue-albumin complex indicates vessel leakiness in the Id1-PCAO

treated tumors. (d) Tumor sections were evaluated for area percentage positive staining

for CD31 and for the density of Id1-positive cells (positive cells/mm2) by IHC. Both values

for different treatment arms are normalized to the untreated specimen. Treatment with

17-AAG leads to decreased vascularization, treatment with Id1-PCAOs to decreased Id1

expression in remaining endothelial cells. (e) Tumor sections were stained for Hif1aexpression. Treatment with 17-AAG reduces Hif1a levels, whereas Id1-PCAO leads to

increased hypoxia. (f) Id1-PCAO–treated tumors stain highly positive for Hif1a (scale

bars, 500 mm). Hypoxic areas surround necrotic regions showing signs of cystic lesions.

Combination with 17-AAG suppresses the hypoxic response while the large necrotic areas

remain (black arrow). Error bars, ± s.e.m.

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thereby increasing the selectivity of the PCAO compared to F3. Thevastly improved potency of PCAOs when compared to antisenseoligonucleotides might in part be due to enhanced hybridization tothe RNA target, as it has been shown previously that conjugation tolysine-rich peptides can accelerate hybridization39. However, thehigher local concentration of the oligonucleotide in the endotheliumis likely to be more important, as the unconjugated antisense oligo-nucleotide could not be detected in the tumor vasculature.In vivo, the action of PCAOs closely recapitulates the effects of Id1

loss observed in genetically manipulated mice, which strongly sup-ports the idea that we have effectively hit the intended target. As asingle agent Id1-PCAO was able to reduce the growth rate ofexperimental breast tumors and the antitumor effect was enhancedwhen combined with the Hsp90 inhibitor 17-AAG. Also growthof highly aggressive Lewis Lung carcinomas was significantly impededby Id1-PCAO alone. Moreover, after treatment with Id1-PCAOsand removal of primary tumors, metastatic growth of LLC wassubstantially delayed as was observed after genetic reduction of Id1and Id3 levels14.

The tumor vessel phenotype observed after intervention withId1-PCAOs and after genetic Id1 loss is different from that foundafter treatment with anti-VEGF agents like bevacizumab (Avastin).Whereas VEGF ablation is reported to lead to a normalization of thetumor vasculature40, Id1 loss causes increased hemorrhage andvascular permeability. That Id1 is a downstream target of VEGF/VEGF-R2 signaling19 suggests that inhibition of different arms of theVEGF-pathway may have different effects on neo-vascularization. Theincreased vascular leakage observed after Id1-PCAO treatment couldbe due to effects on endothelial progenitor cells or on non-bonemarrow–derived endothelial cells because a requirement for Id1 inboth populations has been demonstrated16,25,41. Vascular disruptingagents (VDAs) like combretastatins and 5,6-dimethylxantheonone-4-acetic acid show a similar enhancement of tumor vasculaturepermeability as Id1-PCAO. Treatment with VDAs has been shownto lead to Id1-dependent mobilization of EPCs, which partially rescuesthe tumor from the therapeutic effect41. Combining VDAs and geneticId1 loss led to a drastically enhanced antitumor effect, which can nowbe further tested with a combination of Id1-PCAO and VDAs.

Because we observed increased hemorrhage and vascular perme-ability in treated tumors, which is in general associated with a higherrate of tumor cell embolization, it is probable that Id1-PCAO inter-feres with metastasis by blocking angiogenesis in the new distal bedrather than by inhibiting escape of cells into the circulation from theprimary tumor. Indeed metastatic cells were observed in the lungs ofId1-PCAO–treated animals but these cells failed to colonize as long astreatment was applied. This is in accordance with earlier findingsthat genetic Id1 loss prevents the establishment of metastasis in thelung after intravenous injection of LLC cells14. Also, as shownpreviously, metastatic LLC cells that start to colonize the lungs staydormant and are unable to induce angiogenesis as long as the primarytumors are not removed32. After elimination of the inhibitoryeffect emanating from the primary tumor, the dormant micrometa-stases still fail to stimulate angiogenesis as long as the PCAO treatmentis continued.

The exact mechanism by which Id1-PCAOs and 17-AAG cooperateis not clear. Most plausible is a model by which Id1-PCAOs lead toincreased tumor hypoxia (Fig. 4e,f) because of vascular leakage andtherefore enhanced dependence on Hif1a, a protein that is destabilizedby 17-AAG42. However, we also observed a decrease in tumorvascularization after treatment with 17-AAG alone. This is likelycaused by the inhibition of tumor-derived VEGF as both HER2/neuand Hif1a are upstream effectors of VEGF-A expression43. Therefore,a simple additive effect on tumor endothelial cell viability by combin-ing Id1-PCAOs with 17-AAG is also possible.

As shown in the preclinical models presented, PCAOs have anumber of attractive characteristics. First, they are remarkably stablein plasma over prolonged periods thereby bypassing a major impedi-ment that has plagued the development of antisense therapeutics inthe past. In addition, the specificity imposed by F3 toward tumorendothelium makes toxicity unlikely and indeed no adverse effects ontreated mice have been observed to date over a wide range of drugconcentrations. Although clearance of the PCAOs by the kidneys mostlikely reduced their efficacy, the PCAOs nonetheless were activeenough to reduce Id1 protein levels dramatically in the treated animalswith no obvious kidney toxicity. Conversely, systemic delivery ofnonconjugated Id1-AO did not yield any therapeutic effect or

Tumor growth

0 7 14 22 Days

Tumorimplantation

Pumpimplantation

Removalprim. tumors

End work-periodof pumps

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rcld1-PCAO (n = 5)ld1-AO + F3 (n = 5)

3432302826242220181614121086420Days post tumor removal

0

20

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Tum

or fr

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%)

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rcld1-PCAOF3 + ld1-AOa c

d P = 0.0026

P < 0.0001P = 0.00064

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are

a(%

)

Saline

Id1-

PCAO

rcld1

-PCAO

F3 +

ld1AO

b

Drug deliveryMetastatic spread

Figure 6 Id1-PCAO inhibits primary tumor growth and metastatic spread of Lewis lung carcinoma allografts.

(a) Time-line of the experiment. Male C57/B6 were engrafted with GFP/fluc expressing Lewis lung carcinoma cells.

After tumor establishment osmotic pumps were implanted to deliver Id1-PCAO or control substances. (b) Tumor

growth was followed for 14 d post implantation. Treatment started on day 8 after the pumps implanted on day 7

started working (gray field: working period of pumps during primary tumor growth). (c) Kaplan-Meier plot of tumor-

free survival after primary tumors were surgically removed 14 d after injection. Metastatic growth was monitored

by intravital luminescence imaging (gray field, residual working period of pumps after removal of primary tumors).

(d) Increased hemorrhage in treated tumors was evaluated by imaging of whole tumor sections stained with H&E.

Error bars, ± s.e.m.

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reduction in Id1 levels up to a dosage where hepatic and renal toxicityfor phosphorothioated oligonucleotides has been reported44. Thus,although optimization of a number of parameters might improvePCAO activity, the results presented here indicate that they are likelyto be active and of low toxicity in a clinical setting.

The PCAO technology should allow rapid validation of othertherapeutic targets in the tumor endothelium in a preclinical settingfor what has been previously considered undruggable proteins. Selec-tion for an effective antisense molecule can be done rapidly in vitro. Inaddition, phage display panning methods have already yielded amultitude of peptides with in vivo homing activities to other cellulartargets like lymphatics45, tumor cells46, adipose tissue47, urothelium48,synovium49 and hematopoietic cells in the bone marrow50. Whetherthese peptides will allow efficient delivery of antisense oligonucleotidesremains to be determined. If so, the potential already exists to inhibittargets for a variety of diseases in a tissue-specific way. The high localconcentrations achievable with PCAOs allows for substantially lowertherapeutic doses, thereby decreasing side effects. Thus, directeddelivery of antisense molecules or other biologically active moleculesusing a peptide conjugate may prove to be an important avenue oftherapeutic treatment of human cancers and other diseases.

METHODSGeneral. Chemicals were purchased from Sigma unless otherwise indicated.

GMBS was purchased from Fluka. Oligonucleotides were obtained from

Operon Biotechnologies. Peptides were synthesized in the microchemistry

core-facility at Memorial Sloan–Kettering Cancer Center. 17-AAG and EPL

were obtained from the National Cancer Institute. Antibodies for western blot

(WB) and immunohistochemistry (IHC) were obtained from Santa Cruz

Biotechnology (Id1 (WBs), SC-488; Nucleolin, SC-9893, CD31), Biocheck

(Id1 (IHC)), BD Pharmingen (p16ink4a, CD31, CD34), Sigma (b-actin),

Chemicon (Hif1a). Mass spectrometry was performed by M-Scan Inc.

Cell lines. Human umbilical vein endothelial cells (HUVEC-2) were obtained

from BD Biosciences, maintained inEGM-2 media (Cambrex), and were used

at passages four through six in all experiments. Normal human dermal

fibroblasts (NHDF) were purchased from Cambrex and maintained in FGM-

2 growth medium (Cambrex). Murine embryonic fibroblasts (MEFs) were

obtained from animals with a mixed C57B6/129SV background, as described

previously16. HeLa, MDA-MB-435S, LLC and EOMA cells were purchased from

ATCC and maintained in DME high-glucose medium supplemented with 10%

FBS and glutamine. KYSE-520 were obtained from the DSMZ (Deutsche

Sammlung von Mikroorganismen und Zellkulturen) and maintained in RPMI

1640 media with 10% FBS.

Animals. Female nude NCR mice were obtained from Taconic at 4-5 weeks

of age. Genetic tumor models used (MMTV-HER2/neu (YD) and PTEN+/�)

were described previously15,16. Generation of Id1-deficient mice (Id1�/� and

Id1+/�) was described previously14. These animals were bred back into a pure

C57BL/6 background and used with wild-type littermates as controls in the

experiments. All experiments involving animals were approved by MSKCC’s

Institutional Animal Care and Use Committee (IACUC).

Coupling of antisense-oligonucleotides to cysteine-modified peptides. Cou-

pling was done according to Harrison and Balasubramanian51 with some

modifications as described below. Fully modified oligonucleotides were

obtained directly from Operon Biotechnologies with a C6-amino linker

attached to the 5¢-end of a gap-mer with the sequence GCACCagctccttgaggc

GUGAG (upper case: 2¢O-methyl RNA bases; lower-case phosphorothioate-

linked DNA bases). The reverse complimentary sequence with the same

modification was used as a control oligonucleotide (rcId1-AO and in

conjugated form as rcId1-PCAO). For uptake and homing studies, oligos were

obtained from the supplier with a 3¢-end fluorescein or Rhodamine red label.

The cysteine modified F3-peptide sequence is 5¢-CKDEPQRRSARLSAK

PAPPKPEPKPKKAPAKK-3¢.

The oligos were dissolved in 200 mM TrisHCl pH 8.4 to a final concentra-

tion of 1 mM and stored at –20 1C. 2.8 mg GMBS (4-maleimidobutyric acid

N-hydroxysuccinimide ester, 10 mmol, 100 eq.) in 40 ml acetonitrile were added

to 100 ml (100 nmol) of the oligo solution. The reaction vessel was wrapped in

aluminum foil and incubated with shaking at 25 1C for 90 min. The

oligonucleotide was precipitated with 1 ml acetonitrile and remaining GMBS

was removed by vigorous washing with acetonitrile (9 � 1 ml). After

drying in vacuo the activated oligo was dissolved in 50 ml buffer (100 mM

Na-phosphate, 400 mM NaCl at pH 7.0) to which 400 nmol F3-N-Cys (4 eq.)

in 50 ml buffer (40 mM sodium-phosphate, 20 mM EDTA, pH 7.0) were slowly

added. The reaction mixture was incubated with shaking for 24 h at 25 1C. The

coupling-product was purified by reversed-phase high-performance liquid

chromatography (Akta Purifier System, GE Healthcare; Column: OligoDNA

RP 150 � 7.8 mm, Tosoh Bioscience). To first eluate the nonconjugated peptide

a 0.05% (vol/vol) transcription factor A in water/acetonitrile gradient (5–20%

(vol/vol) acetonitrile) was used. To separate the nonconjugated antisense

oligonucleotide from the PCAO a second step was done using an ammonium

acetate (20 mM, pH 6.8)/acetonitrile gradient (5–25% (vol/vol) acetonitrile).

Fractions containing the PCAO were combined, concentrated in a vacuum

concentrator system (Eppendorf), and precipitated with ethanol. Yields in a

multitude of experiments (410, scales up to 600 nmol) varied between 40 and

78% as calculated from absorbance measurements at 260 nm. Identity of the

conjugate was verified by mass spectrometry. Negative ion electrospray ioniza-

tion mass spectronomy was performed on a Sciex Q-star/Pulsar instrument

(MDS Sciex). Id1-PCAO: calculated mass: 11441.6 Da, found: 11441.8 Da.

Transfection of endothelial cells with antisense oligonucleotides. Endothelial

cells (HUVEC-2 or MS-1) were seeded 16 h before transfection at 105 cells/well

in six-well multi-well dishes (MWD) in standard growth medium without

antibiotics. Antisense oligonucleotides were reprecipitated with ethanol from

sodium acetate (10 mM, ph 4.8) buffer before use. Cells were transfected in

1 ml OptiMEM I medium (Invitrogen) with Lipofectin (Invitrogen) or

Cytofectin (Gene Therapy Systems) according to the manufacturers’ recom-

mendations. The transfection was repeated at 24 and 48 h.

Plasma stability assay. Female BALB/c mice were bled by submandibular

punctuation using a 4.5 mm lancet (Medipoint). Plasma was separated from

heparinized whole blood by centrifugation (18,000g, 5 min at 4 1C). Antisense

oligonucleotides and PCAOs were dissolved in plasma at 25 mM and 10 ml

aliquots in microfuge tubes and were incubated at 37 1C for the indicated time.

After incubation, 5-ml quencher solution (1.6 M NaCl, 100 mM EDTA pH 8.0)

were added and the samples were stored at –80 1C. For analysis samples

were diluted to 60 ml with agarose sample buffer, incubated 5 min at 95 1C

and separated in a 1.5% (wt/vol) low melting point agarose gel.

Antisense oligonucleotides/PCAOs were visualized with ethidium bromide

under UV-light.

Transfection of cells with Id1-PCAOs. HUVEC were seeded 24 h before

transfection at 105 cells/well in six-well MWDs in standard growth medium

(EGM-2) without antibiotics. At the day of transfection the antisense oligo-

nucleotides–conjugates and control oligos were diluted in EGM-2 to the

indicated concentration. Medium was replaced with the supplemented EGM-

2. The procedure was repeated every 24 h for two more days, samples were

drawn at the indicated time points and analyzed by western blot analysis. Other

cell lines were treated similarly, with the exception that Id1-PCAO was added to

standard growth medium, DME or RPMI 1640, according to cell type.

Uptake studies in different cell lines with fluorescence labeled Id1-PCAO

were performed in both the standard growth medium (DME, RPMI 1640

or EGM-2) or in OptiMEM I (supplemented with 2% FBS) to control for

media effects.

For uptake studies monitoring dependence on VEGF stimulation, HUVEC

were starved for 36 h in OptiMEM I. Serum-free medium was exchanged with

different amounts (0, 2, 5, 20 ng/ml) of VEGF-A165 (Peprotech) and cells were

incubated for 10 h. Id1-PCAO-FAM was added at 200 nM and cells were

incubated for an additional 2 h, fixed and counterstained.

Scratch assay. HUVEC were plated in fibronectin-coated two-well chamber

slides (BD Bioscience) at 2.5 � 104 cells/well. Growth medium was

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supplemented with 200 nM Id1-PCAO (or Id1-AO) and renewed every 24 h.

Seventy-two hours after plating, a scratch was applied using a 20 ml pipette tip.

Chambers were washed with medium and supplemented medium was added.

Eighteen hours after the scratch was applied, cells were fixed and imaged.

Transduction of LLC cells with an eGFP/Fluc dual-modality reporter.

Ecotropic retrovirus based on the SFG vector34, expressing an Aequorea

Victoria eGFP/firefly luciferase (eGFP/FLuc) fusion protein was produced

in PhoenixE cells and was used with at least 1 � 106 infectious particles/ml

against NIH3T3cells. The in vitro transduction of early passage LLC cells

with the retroviral vector was accomplished by exposing the cell monolayer

to a filtered (0.45 mm) culture medium obtained from the vector producer

cells for 8 h in the presence of 8 mg/ml polybrene (Sigma). Stably trans-

duced cells were enriched by FACS using eGFP-expression as an marker for

successful transduction.

Tube formation assay. HUVEC were grown for 72 h in T25 dishes in media

containing Id1-PCAO (or rcId1-PCAO or Id1-AO plus F3) at 200 nM. Medium

was renewed every 24 h. Treated cells were detached with trypsin/EDTA and

seeded at 2.5 � 104 cells on top of a matrigel layer in 24-well multiwell dishes in

the treatment medium. After incubation for 18 h, cells were counterstained

with Calcein AM (Invitrogen) and imaged under an inverted fluorescence

microscope (Zeiss Axiostar 200). Tube length was evaluated with the Meta-

Morph software.

Proliferation assay. Cells were plated at 2 � 104 cells in the wells of 24 well

MWDs. After 18 h medium was exchanged with standard growth medium

(EGM-2) supplemented with Id1-PCAO or Id1-AO plus F3 (200 nM or

1 mM). Supplemented medium was renewed every 24 h. Media was removed

from sample plates at different time points, and the plates were stored at

–80 1C. All samples were analyzed in parallel using the CyQuant system

(Invitrogen) according to the manufacture’s instructions.

Nucleolin-antibody blocking studies. HUVEC were plated in EGM-2 medium

at 2.5 � 104 cells in 4-well chamber slides (BD Bioscience). After 24 h

antinucleolin antibodies were added to the medium (tested antibodies: MS-3

(SantaCruz Biotechnologies), H-250 (SantaCruz Biotechnologies), ZN004

(MBL) and 3G4B2 (Millipore). After 2 h cells were fixed with 4% (wt/vol)

PFA, probed with fluorescein-labeled secondary antibodies and counterstained

with Hoechst 33342. Ability to recognize extracellular epitopes of cell surface

nucleolin was assessed by confocal laser microscopy.

To study blocking of PCAO-uptake, Id1-PCAO-FAM was added at 200 nM

after the 2 h incubation step with the antibody. Cells were incubated an

additional 2h at 37 1C, fixed with 4% (wt/vol) PFA and counterstained.

To study blocking of PCAO-mediated Id1-downregulation, HUVEC were

seeded at 4 � 105 cells in 6-well MWDs, and incubated for 16 h. Antibody

(ZN004, 10 mg/ml) or IgG-control (mouse-IgG2b, BD Pharmingen) were

added for 2 h before Id1-PCAO or rcId1-PCAO (200 nM) was supplemented.

After 24 h and 48 h medium was exchanged, containing the same concentration

of AB and Id1-PCAO. Seventy-two hours after the first treatment, cells were

lysed and lysates were probed by western blot analysis.

Delivery of Id1-PCAOs in vivo. 12 nmol fluorescence-labeled Id1-PCAOs

(B6.8 mg/kg body weight (BW)) or Id1-AOs were dissolved in TBS and

injected into the tail vein or subcutaneously of tumor-bearing mice. Mice were

killed, organs and tumors were dissected, fixed overnight in 4% (wt/vol) PFA

and finally immersed in 20% (wt/vol) sucrose for 24 h. After embedding in

OCT (Miles Inc.) and sectioning, samples were probed for CD31 using a

biotinylated secondary antibody and a streptavidin-Alexa488 conjugate as a

tertiary agent.

Allograft model of Her2-overexpressing breast cancer. Female nude NCR

mice (Taconic) were engrafted with 5 � 106 MMTV-HER2/neu (YD) Id1�/�

tumor cells in the left flank. The animals were randomly divided into three

cohorts of four animals and treatment was started 96 h later when tumors

became palpable. The first cohort received 10 nmol Id1-PCAO conjugate in

200 ml TBS. The other two cohorts served as negative controls and received

either TBS or 10 nmol F3-peptide plus 10 nmol Id1-AO in TBS (B5.7 mg/kg

BW). Application of the conjugate and control solutions was performed by

intravenous injection into the tail vein and was repeated every 24 h for

7 consecutive days. 24 h after the last injection animals were killed. Tumors,

kidneys, livers and femurs were collected, fixed with paraformaldehyde and

embedded in paraffin.

Alternatively, animals were implanted s.c. with osmotic pumps (Durect

Corp.) that delivered 7 nmol/d Id1-PCAO (3.5 mg/kg BW) in TBS over a 14-d

period. Controls received 20 nmol/d F3-peptide plus 20 nmol/d Id1-AO (in

TBS) or TBS. 2 � 106 MMTV-HER2/neu (YD) Id1�/� tumor cells were

injected into the left flank 24 h after implantation of the pumps. After the 14-d

treatment period, pumps were replaced using a model with a work period of

7 d. Treatment with 17-AAG (75 mg/kg, i.p. on three consecutive days/week)

was started when tumors reached a size of 20 mm3. 17-AAG was dissolved at

50 mg/kg in DMSO and diluted with EPL 1:1 before injection. Control animals

received DMSO:EPL 1:1 i.p. at the same schedule. Tumour size was measured

using a calliper. Volume was calculated as V ¼ (p/6 � longest diameter �perpendicular diameter2).

Allograft model of metastatic LLC. 7.5�105 Dual reporter labeled LLC cells

were implanted in the right dorsal flank of male C57J/B6 mice (Jackson

Laboratories). After 7 d, animals were implanted with osmotic pumps (100 ml

volume, work period 14 d). The pumps were filled with saline solution of either

Id1-PCAO (3.5 mM), rcId1-PCAO (3.5 mM) or F3-peptide plus Id1-AO

(12.5 mM each). Concentration and release rate of the pumps resulted in a

delivery rate of 229 mg/d (Id1-PCAO and rcId1PCAO) or 265 mg/d and 580 mg

(F3 and Id1-AO). Fourteen days after tumor implantation animals were

anaesthetised and primary tumors were surgically removed. Complete removal

of the tumor tissue was checked 3 d post operation by in vivo luciferase imaging

and re-growing primary tumors were removed. For in vivo luciferase imaging,

100 ml of D-luciferin (Gold Bio Technology, 15 mg/ml potassium-salt in PBS)

were injected retro-orbitally to animals anaesthetised by isofluorane inhalation.

Photographic and luminescence images were acquired using an IVIS

100 system (Xenogen). Animals were sacrificed when distressed. Tumor

burden and metastasis data acquired by in vivo luminescence was confirmed

by histology.

Image acquisition and analysis. Epifluorescence, bright field and phase

contrast images were acquired using Zeiss Axiostar 200 microscopes. Leica

laser confocal microscopes were used for co-localisation studies. For quantifi-

cation, large fields of the tissue sections were acquired using an automated

image acquisition and montaging system (Zeiss Axiostar 200M microscope

with MetaMorph Software, Molecular Devices). For evaluation of single cell

staining (Id1, CD31, Hif1a) an average of 30 adjacent, single images were

acquired from the center of the section using a 20� objective. Images were

montaged to yield one large image covering an average area of 0.82 mm2. Three

or 4 large field images were used to quantify each section. Stained areas were

quantified using MetaMorph or ImageJ software (http://rsb.info.nih.gov/ij/).

Images were threshholded and stained area (CD31) was calculated or particles

per field were counted (Id1 or Hif1a positive cells). To quantify extent of

hemorrhage, H&E whole tumor sections were imaged with a 5x objective and

evaluated using the color threshold function of the Metamorph software.

Statistical analysis. Statistical analysis was performed using the GraphPad

Prism software (GraphPad Software). Tumour progression in different treat-

ment arms was compared using the Wilcoxon signed rank sum test. Students

t-test was used to analyze results from IHC staining and hemorrhage evaluation

experiments. The build in statistical function of the Prism software was also

used to evaluate Kaplan-Meyer survival curves. All P-values are two-tailed.

Note: Supplementary information is available on the Nature Biotechnology website.

ACKNOWLEDGMENTSThe authors thank Simona Curelariu for help with animal models and NincheAlston for help with in vivo imaging. This work was supported by the DeutscheForschungsgemeinschaft (fellowship to E.H.), the National Institutes of Health(R.B.), William H. Goodwin and Alice Goodwin and the CommonwealthCancer Foundation for Research and the Experimental Therapeutics Center ofMemorial Sloan-Kettering Cancer Center (R.B.), the Breast Cancer ResearchFoundation (R.B.) and the Mary Kay Ash Foundation (R.B.).

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AUTHOR CONTRIBUTIONSAll authors contributed significantly to the experimental design and/orexecution of the experiments described.

Published online at http://www.nature.com/naturebiotechnology/

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