Wnt pathway inhibition via the targeting of Frizzledreceptors results in decreased growth andtumorigenicity of human tumorsAustin Gurney1, Fumiko Axelrod, Christopher J. Bond, Jennifer Cain, Cecile Chartier, Lucas Donigan, Marcus Fischer,Aurélie Chaudhari, May Ji, Ann M. Kapoun, Andrew Lam, Sasha Lazetic, Shirley Ma, Satyajit Mitra, In-Kyung Park,Kellie Pickell, Aaron Sato, Sanjeev Satyal, Michelle Stroud, Hoang Tran, Wan-Ching Yen, John Lewicki,and Timothy Hoey

OncoMed Pharmaceuticals, Redwood City, CA 94063

Edited by Roeland Nusse, Stanford University School of Medicine, Stanford, CA, and approved June 6, 2012 (received for review December 6, 2011)

TheWnt/β-catenin pathway,which signals through the Frizzled (Fzd)receptor family and several coreceptors, has long been implicated incancer. Here we demonstrate a therapeutic approach to targetingthe Wnt pathway with a monoclonal antibody, OMP-18R5. This an-tibody, initially identified by binding to Frizzled 7, interacts with fiveFzd receptors through a conserved epitope within the extracellulardomain and blocks canonical Wnt signaling induced bymultipleWntfamily members. In xenograft studies with minimally passaged hu-man tumors, this antibody inhibits the growth of a range of tumortypes, reduces tumor-initiating cell frequency, and exhibits synergis-tic activity with standard-of-care chemotherapeutic agents.

differentiation | cancer stem cell | pancreatic | breast | lung

Investigation into the role of the Wnt pathway in human tumorshas been hampered by a lack of therapeutic agents able to

inhibit Wnt signaling. The Wnt pathway is complex, with a largenumber of known ligands, receptors, coreceptors, and regulatorycomponents. Members of the Wnt family have been shown toinduce several distinct signaling events, including activation ofthe β-catenin signaling (termed the “canonical pathway”) as wellas other signaling cascades, including the planar cell-polaritypathway and the Ca+2 pathway (1–5). Lack of a detailed un-derstanding of the receptor-ligand specificities of the variouspathway components and their relationship to the Wnt-mediatedsignaling pathways has also hindered development of therapeuticagents. However, mutations in the Wnt signaling pathway occurin most cases of human colon cancer, and active signaling hasbeen noted in multiple major tumor types, highlighting thispathway as a potentially promising target for the development ofnew anticancer agents (6, 7). In recent years, several smallmolecules that impact the Wnt pathway have been identified.The nonsteroidal anti-inflammatory compound Sulindac and theantibiotic salinomycin have been reported to possess ability todown-regulate β-catenin (8, 9). Inhibitors of the poly-ADPribosylating enzymes tankyrase 1 and tankyrase 2 also have beenfound to be inhibitors of Wnt signaling, by a mechanism thatresults in stabilization of axin and the β-catenin destructioncomplex (10). However, these agents also impact other signalingevents beyond the Wnt pathway. To date, highly potent smallmolecule agents that specifically inhibit Wnt signaling have notbeen reported.An alternative approach to developing agents that block Wnt

signaling is to focus on the extracellular receptor–ligand inter-actions. There are 19 human Wnts and 10 Frizzled (Fzd) re-ceptors. In addition, Wnt signaling through Fzds involves twocoreceptors, LRP5 and LRP6, as well as other coreceptors, suchas ROR and Ryk. Although this substantial complexity andfunctional redundancy makes the prospect of developing effec-tive inhibitors more challenging, it also offers the potential forthe identification of agents that block only portions of Wnt

signaling. This selectivity relative to potential intracellular sig-naling components may offer advantages in safety and thera-peutic index. Previous studies have indicated that use of solubleWnt inhibitors and decoy receptor molecules can impact tumorgrowth (11, 12). In this article we demonstrate the ability togenerate a potent Wnt inhibitory antibody that functions bybinding to select Fzd receptors, and that this antibody is activeacross a range of human tumor types.

ResultsIdentification of an Antagonistic Wnt Pathway Antibody That TargetsMultiple Fzd Receptors. Antibodies to the Fzd receptors wereidentified using phage display and were subsequently screenedfor the ability to inhibit Wnt3A signaling in a cell-based assay.Through this approach, an antibody that was initially isolated byability to bind to FZD7, OMP-18R5, was found to block mostβ-catenin signaling in response to Wnt3A (Fig. 1A). In sub-sequent experiments it was also found to inhibit β-catenin sig-naling in response to each of the Wnt family members that wereactive in these reporter studies (Fig. 1B). OMP-18R5 also re-duced both Wnt3A-induced accumulation of β-catenin andphosphorylation of LRP6 (Fig. 1C). OMP-18R5 did not inhibitβ-catenin signaling in response to intracellular pathway activa-tion with the GSK3 inhibitor compound BIO (6-bromoindirubin-3′-oxime) (Fig. S1A) and also did not inhibit activation ofa Notch pathway reporter in response to the Notch ligand DLL4(Fig. S1B). The OMP-18R5 anti-Fzd antibody was also screenedby flow cytometry with cells overexpressing each individual Fzd.OMP-18R5 was found to bind 5 of the 10 Fzd receptors: FZD1,FZD2, FZD5, FZD7, and FZD8 (Fig. 2A and Fig. S2). In-terestingly, each of these receptors has been previously impli-cated in canonical Wnt signaling (12–17). The epitope on the Fzdproteins bound by OMP-18R5 was mapped through a survey ofintroduced amino acid substitutions within FZD8, several ofwhich eliminated OMP-18R5 binding (Fig. 2B and Fig. S3).OMP-18R5 binds to a discontinuous epitope that spans a “cleft”region that is apparent in the reported crystal structure (18) of

Author contributions: A.G., F.A., C.C., A.M.K., S.L., I.-K.P., A.S., W.-C.Y., J.L., and T.H.designed research; A.G., F.A., C.J.B., J.C., C.C., L.D., M.F., A.C., M.J., A.L., S.L., S. Ma, S. Mitra,I.-K.P., K.P., A.S., S.S., M.S., H.T., and W.-C.Y. performed research; A.G., F.A., C.J.B., J.C., C.C.,L.D., M.F., A.M.K., S.L., S. Ma, S. Mitra, I.-K.P., A.S., S.S., H.T., W.-C.Y., J.L., and T.H. analyzeddata; and A.G. and T.H. wrote the paper.

Conflict of interest statement: All of the authors are employees of OncoMed Pharma-ceuticals, which provided research funding. R.N. is a member of the OncoMed ScientificAdvisory Board and holds stock in the company.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: austin.gurney@oncomed.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1120068109/-/DCSupplemental.

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mouse Fzd8 (Fig. 2C). Interestingly, the residues lining the baseof this cleft are highly conserved across the Fzd family, suggestingthat this site is functionally important. To determine whether

OMP-18R5 directly blocked the ability of Wnt to interact withFzd, binding studies were conducted. OMP-18R5 blockedWnt3A binding to Fzd5 ECD, suggesting that a mechanism by

Fig. 1. Anti-Fzd antibody OMP-18R5 inhibits Wnt signaling. (A) OMP-18R5 antibody inhibits recombinant Wnt3A signaling as assessed by β-catenin responsiveTOP-FLASH luciferase reporter and a FOP-FLASH control reporter. (B) OMP-18R5 antibody (50 nM) inhibits the ability of several Wnts to induce canonicalsignaling. Shown is relative luciferase signal normalized to control Renilla luciferase and relative to signal in the absence of Wnt (no Wnt). Bars represent themean ± SD. (C) OMP-18R5 blocks phosphorylation of LRP6 and the accumulation of active β-catenin. HEK-293 cells were treated with purifiedWnt3A and OMP-18R5, as indicated. Cells were lysed in the presence of phosphatase inhibitor and Western blot analysis was performed. OMP-18R5 inhibited the induction ofphosphorylated (Ser1490) LRP6 by Wnt3A and attenuated the Wnt induced accumulation of β-catenin and unphosphorylated (Ser37, Thr41) β-catenin.

Fig. 2. Anti-Fzd antibody OMP-18R5 binds to 5 of the 10 human Fzd receptors and inhibits Wnt binding. (A) Binding curves of OMP-18R5 to cells thatoverexpress the indicated Fzd. Binding was assessed by flow cytometry analysis with cells transfected with cDNA encoding the indicated FZD protein. (B)Epitope mapping of OMP-18R5 to FZD8. Expression vectors comprising N-terminal FLAG-tagged FZD8 variants encoding the indicated amino acid substitutionwere transiently transfected along with GFP and OMP-18R5 binding was assessed by flow cytometry. (C) Surface rendering of the cysteine-rich domain ofFZD8 (18), highlighting important residues involved in OMP-18R5 binding in green and highly conserved residues in pink. (D) Binding of OMP-18R5 to FZDinhibits the interaction of Wnt with FZD. Wnt3A, FZD5-Fc and OMP-18R5 were coincubated as indicated, and then OMP-18R5 and FZD5-Fc were removed byprotein A immunoprecipitation. Supernatant was then assayed for Wnt activity in an 8xTCF luciferase reporter assay.

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which OMP-18R5 inhibits Wnt signaling may involve the directinhibition of Wnt binding (Fig. 2D and Fig. S4).

Wnt Pathway Blockade Inhibits the Growth of Human Tumor Xenografts.The impact of inhibitingWnt/β-catenin signaling on tumor growthwas assessed using human tumor xenografts in mice. These modelswere conducted with minimally passaged human tumors (19, 20)(Table S1). OMP-18R5 treatment resulted in inhibition of growthin several types of human tumors, including breast, pancreatic,colon [wild-type adenomatous polyposis coli (APC) and β-catenin]and lung tumors (Fig. 3). Striking synergy was observed whenOMP-18R5 was combined with several standard-of-care chemo-therapeutic agents, including taxol in nonsmall-cell lung cancerand breast cancer models, irinotecan in colon cancer models, andgemcitabine in pancreatic cancer models (Fig. 3 B,C, E, andG–I).OMP-18R5 had activity in 6 of 11 pancreatic tumors, 3 of 6 breasttumors, and 7 of 8 nonsmall-cell lung tumors tested. OMP-18R5did not display activity in colon tumors harboring APC or β-cat-enin mutations, but was highly active in a colon tumor with wild-type APC and β-catenin. OMP-18R5 also inhibited the growth ofthe established cell line PA-1 (derived from a human teratocar-cinoma), which had previously shown to be inhibited by FZD8-fcprotein (12) (Fig. 3F). In tumor recurrence studies (Fig. 3 G andH), OMP-18R5 treatment induced an extended delay in theregrowth of tumors following a treatment with high-dose

chemotherapy, whereas tumors treated with control antibodydemonstrated rapid regrowth.Several tumors showed synergistic response to OMP-18R5 and

chemotherapy. Gene-expression analysis of the response ofbreast tumor PE-13 to OMP-18R5 alone or in combination withtaxol indicated that OMP-18R5 and taxol induced opposingchanges in the expression of numerous genes involved in stressresponses, as well as ABC family transporters, suggesting Wntpathway inhibition may alter cellular responses to taxol treat-ment (Fig. S5). mRNA for several genes previously associatedwith breast tumor tumorigenicity (20–22), including CD44,ALDH1A1, SOX1, and SOX2, were decreased by OMP-18R5.Similarly, OMP-18R5 also displayed synergy with gemcitabine inpancreatic tumors. OMP-18R5 was noted to induce changes inthe histopathology of the pancreatic tumors. In PN4, a pancre-atic tumor with a moderately differentiated ductal phenotype,the ducts were greatly enlarged and the cells lining the ductswere larger following OMP-18R5 treatment. Staining withAlcian blue, to reveal mucin expression, revealed large increasesin mucin content (Fig. 4 A and B). Immunohistochemical (IHC)staining also revealed reduced nuclear β-catenin levels followingOMP-18R5 treatment. (Fig. 4 C and D). Proliferation, as mea-sured by Ki-67 staining, was also reduced by OMP-18R5 treat-ment (Fig. 4 E and F). Quantitative PCR of known targets ofWnt/β-catenin signaling Wnt target genes and genes associated

Fig. 3. Inhibition of canonical Wnt signaling inhibits tumor growth as monotherapy and in combination with chemotherapy. Results of nine tumor models:colon tumor C28 (A), breast tumor T3 (B), lung NSCLC Lu24 (C), pancreatic tumor PN8 (D), breast tumor PE13 (E), teratocarcinoma line PA1 (F), pancreatictumor PN4 (G), breast tumor PE13 (H), and colon tumor C28 (I). Mean tumor volumes with SEs are presented (n = 10). (G and H) Duration of chemotherapy andantibody treatment is indicated by brackets. Asterisks denote statistical significance P < 0.02 (* vs. control Ab; ** vs. chemotherapy alone).

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with pancreatic lineages was performed (Fig. 5). Down-regula-tion of AXIN2 and osteopontin (SPP1) was observed in tumorstreated with OMP-18R5, consistent with inhibition of Wnt sig-naling (23, 24). In addition, in these pancreatic xenografts,multiple genes within the mucin family and certain cytokeratinswere induced by OMP-18R5. Gemcitabine treatment resulted inelevated mRNA for several genes associated with epithelieal-mesenchymal transition, including fibronectin, vimentin, slug,and snail. These effects were blocked by combined treatmentwith OMP-18R5.

Targeting Fzd Receptors Reduces Tumorigenicity. Several studieshave suggested that Wnt signaling plays a key role in mediatingself-renewal essential for tumor initiating cells (25–27). To assessthe impact of OMP-18R5 on tumor-initiating cell frequency,limiting dilution studies were conducted wherein defined num-bers of human cells from treated or control tumors were assessedfor their ability to initiate tumor growth after secondary trans-plant into a new cohort of mice in the absence of further treat-ment. OMP-18R5 treatment of the pancreatic tumor PN4 (Fig.6A) and the breast tumor PE13 (Fig. 6B) reduced the tumor-initiating cell frequency of the tumor cells by threefold. Neithergemcitabine treatment of the pancreatic tumor nor taxol treat-ment of the breast tumor reduced tumorigenic cell frequency (infact, taxol treatment increased tumorigenicity, P < 0.05). Incontrast, the combination of OMP-18R5 and chemotherapeuticagents reduced tumorigenicity by 10-fold in both pancreas andbreast tumors.

Intracellular Blockade of the Canonical Wnt Pathway Inhibits TumorGrowth. The OMP-18R5 antibody blocks Wnt signaling by bindingto multiple Fzd receptors, suggesting a requirement for activationof the canonical Wnt pathway during tumor growth. AlthoughOMP-18R5 is a human IgG2 isotype—an isotype that mediateslittle effector function, such as complement mediated cytotoxicityor antibody dependent cell killing—and further that these studieswere performed in immunocompromised mice, it is conceivablethat immune function contributes to OMP-18R5 efficacy. Toprobe Wnt pathway involvement further, we tested the impact ofan alternative approach to Wnt pathway inhibition. Overexpres-sion of the intracellular Wnt pathway component Axin leads todestabilization of β-catenin and inhibition of canonical signaling(28). We find that introduction of Axin into tumors by lentivirus-mediated transduction substantially abrogated tumor growth (Fig.S6), suggesting importance of canonical Wnt pathway signalingwithin the tumor cells.

DiscussionThe role of the Wnt-Fzd pathway in cancer has been the subjectof investigation for nearly three decades following the initialobservation that mouse mammary tumor virus retroviral in-tegration events resulting in inappropriate Wnt1 expressioncould result in murine mammary gland tumors (29). Recognitionof the importance of APC and β-catenin mutations in coloncancer has provided strong evidence that the pathway playsa major role in the genesis of at least one major type of humancancer (30, 31). Although not as frequently mutated in othertumor types, increased Wnt pathway activation and epigeneticsilencing of Wnt antagonists has been noted in several tumortypes (7, 32–37).In the present study, we provide evidence that it is possible to

generate potent inhibitors of canonical Wnt signaling by simul-taneous targeting of several Fzd receptors. The antibody de-scribed in this study binds to five distinct Fzd receptors througha conserved epitope. Interestingly, this epitope is distinct fromthe region of FZD8, previously implicated in Wnt binding byalanine scanning mutation analysis (18). The structure of Wnthas not yet been reported, but both Wnt and the antibody aresubstantially larger proteins than the relatively small cysteine-richdomain of Fzd receptors, so it is possible that steric hindranceprevents simultaneous binding of Wnt and antibody. Our dataindicate that blockade of Wnt signaling can inhibit growth ina variety of human tumor types. Inhibition of tumor growth wasnoted in lung, breast, colon, and pancreatic tumors. Interestingly,there was strong synergy observed with several chemotherapyagents, particularly taxol. Gene-expression analysis suggests thatrather than “enhancing” the magnitude of taxol-induced effects,the combination with OMP-18R5 actually inhibits some taxol-induced gene changes and may therefore be altering cellular

Fig. 4. Inhibition of Wnt signaling in pancreas tumor induces mucininousdifferentiation and decreased nuclear β-catenin and cellular proliferation.Shown is staining of sections of pancreatic tumor PN4 with Alcian blue tohighlight mucin (A and B), and with anti–β-catenin (C and D) and with anti-Ki-67 to indicate proliferating cells (E and F). Sections are from tumors fol-lowing treatment (41 d) with control antibody (A, C, and E) or OMP-18R5 (B,D, and F).

Fig. 5. Expression changes induced by Wnt pathway inhibition. Shown isthe relative mRNA expression (normalized to control antibody and to humancontrol mRNA GAPDH) for the indicated human genes from pancreatic PN4tumors treated with OMP-18R5, gemcitabine, or the combination. Datashown is average from four tumors in each treatment group analyzed in-dividually. Shown are genes with statistically significant (Student’s t test P <0.05) expression difference in response to OMP-18R5 or combination treat-ment relative to either control or gemcitabine treatment arms) with theexception of CDH1 which is included for completeness.

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response to taxol treatment. The robust ability of overexpressedaxin within tumors to also inhibit tumor growth is evidence thatthe tumor cells themselves are dependent upon this pathway.Generally, single-agent activity was observed to be significant,

but modest relative to the efficacy observed in combination withchemotherapy. This finding may reflect the fact that OMP-18R5does not target all Wnt pathway receptors. However, it haspreviously been demonstrated that robust Wnt inhibition byadenovirus-mediated delivery of the pan-Wnt pathway inhibitordickkopf 1 (DKK1) results in profound inhibition of gastroin-testinal tract homeostasis with disruption of the crypt-villus ep-ithelial cell layer (38). Because of the high degree of evolutionaryconservation in this pathway, the OMP-18R5 binds to both hu-man and mouse Fzd receptors. In fact, within the cysteine-richdomain FZD1, -2, -7, and -8 are identical between human andmouse, and FZD5 has only two amino acid changes that do notimpact OMP-18R5 binding (Fig. S2). There was no apparentimpact of OMP-18R5 on the gastrointestinal tract at the dosesused in these studies. At very large doses (100 mg/kg) we didobserve an intestinal colitis that resembled, although was lesssevere, the results reported with DKK1. OMP-18R5 did howeversubstantially inhibit the expression of several known Wnt targetgenes in the liver (Fig. S7) (39).OMP-18R5 reduces tumor cell proliferation and tumor-initi-

ating cell frequency. The reduction in tumor-initiating cell fre-quency was quantified using limiting-dilution tumorigenicityassays. This functional assay measures in vivo tumorigenicity andmakes no assumption about the frequency, FACS marker profile,or heterogeneity of the tumor-initiating cell population. A strengthof this approach is that it can be applied to any tumor. In contrast,cell-surface markers may vary in different tumors and their ex-pression may be influenced by environment, which changes in re-sponse to drug treatment. OMP-18R5 reduced tumor-initiatingcell frequency both as a single agent and in combination withchemotherapy. In contrast neither gemcitabine nor taxol reducedtumorigenicity as single agents. Taxol treatment actually resultedin an increase in tumor-initiating cell frequency, consistent withprevious reports that tumor-initiating cells are selectively resistantto various chemotherapeutics and radiation (40, 41).In conclusion, we have developed a unique antibody that

antagonizes the Wnt signaling pathway. Targeting the Wntpathway through by blockade of selected members of the Fzdreceptor family may provide an efficacious approach for thetreatment of a broad range of tumors.

Materials and MethodsAll animal work was performed according to OncoMed Pharmaceutical’sInstitutional Animal Care and Use Committee guidelines.

Antibody Generation and Reporter Studies. OMP-18R5 was generated bypanning the HuCALGOLD phage-display library (MorphoSys) with recombinantFZD7 protein (42). DNA fragments encoding the fAb generated from thephage display library were subcloned into a full-length human IgG2 expressionvector. The antibody was expressed in CHO cells and purified. The ability ofWnt to activate T-cell factor (TCF)-dependent transcription was assessed bytransient transfection of HEK293 responder cells with the TOP-FLASH/FOP-FLASH luciferase reporter assay and a Renilla luciferase transfection controlreporter, followed by treatment with purified Wnt3A (R&D Systems) or 24-hcoculture (1:1) exposure to HEK293 cells transiently transfected with expres-sion vectors encoding human Wnt proteins, and then assayed using the Dual-Glo luciferase assay reporter system (Promega). The TOP-FLASH reporter wassynthesized and includes eight copies of a TCF binding site (AGATCAAAGG)upstream of a minimal promoter and firefly luciferase. BIO was obtained fromSigma-Aldrich. The ORF cDNA encoding human Fzd, Wnt, Smoothened, andNotch2 proteins were isolated by PCR or synthesized (DNA2.0). The Notch lu-ciferase reporter assay was performed essentially as previously described (19).Briefly, PC3 cells were transfected with an expression vector encoding full-length human Notch2 and an 8xCBF1-luciferase reporter vector and a Renillaluciferase vector as a transfection control. Cells were plated in wells coatedwith 100 ng of human DLL4 (R&D Systems) and exposed to the indicatedantibodies at 10 μg/mL and then assayed 18 h later using the Dual-Glo lucif-erase system. Fzd-Fc fusion proteins were produced in baculovirus and con-tained the N-terminal extracellular domain of human Fzd receptors fused tothe CH2-CH3 domains of human IgG1. Lentiviral vectors containing CMV-mouse AXIN-IRES-GFP or CMV-IRES-GFP cassettes were generated by trans-complementation in HEK 293 cells transiently cotransfected with the plasmidcontaining the vector genome along with the gag-pol, rev, and VSV-G envpackaging constructs. For lentiviral transduction studies, tumor cells wereisolated as single cells and cultured (43) in the presence of lentivirus for 24 h.Transduced cells were then isolated by flow cytometry gating on positiveGFP expression.

Histology and IHC, and Western Blottings. Formalin-fixed and paraffin-em-bedded tumors were sectioned at 4-μm thickness. Sections were stained withAlcian blue as recommended by the manufacturer (Poly Scientific). The IHCanalysis used anti–β-catenin (mAb2081, Millipore; 1:100) and anti–Ki-67 (sp6,Vector Laboratories; 1:200) with optimal cutting temperature embeddedfrozen-tumor sections. For phospho-LRP6 and β-catenin Western blot anal-ysis, HEK-293 cells were cultured in 2% (vol/vol) FBS containing DMEM for 4h in the presence of 400 ng/mL recombinant Wnt3A (R&D Systems) and 50μg/mL OMP-18R5, as indicated. Cells were lysed in lysis buffer (Invitrogen)containing 1 tablet/10 mL PhosSTOP phosphatase inhibitor (Roche). Westernblots were probed with α-LRP6 (Cell Signaling; C47E12), α-phospho LRP6

Fig. 6. Inhibition of Wnt reduces tumorigenicity and induces the presence of nontumorigenic cells. Limiting dilution analysis of the ability of human cellsisolated from treated pancreatic PN4 tumor (A) and breast tumor PE13 (B) to initiate tumor growth in the absence of further treatment following secondarytransplant to a new cohort of mice. The primary tumor was treated with control antibody or OMP-18R5 or chemotherapy, as indicated. The tumor initiatingfrequency (TIC) refers to the average number of cells determined to be required to cause tumor growth in the recipient cohort.

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(Ser1490; Cell Signaling), α-β-catenin (BD Transduction Laboratories; 610154),α−“active”-β-catenin (unphosphorylated Ser37, Thr-41; clone 8E7; Millipore).

Xenograft Models. The establishment of minimally passaged human tumorxenograft models and determination of tumor initiating cell frequency wasperformed as previously described (19, 44). Briefly, human tumors xenograftmodels were established by subcutaneous implantation of patient-derivedsolid tissue fragments in NOD/SCID mice. Established tumors were sub-sequently disassociated and single-cell suspensions of tumor cells were fro-zen at −80C. Tumors for subsequent experiments were established by serialimplantation of frozen cell stocks. For tumorigenicity studies, single-cellsuspensions from control and treated tumors were incubated with bio-tinylated α-mouse CD45-biotin and α-mouse H2Kd on ice for 30 min followedby addition of streptavidin-labeled magnetic beads to remove murine stro-mal cells. Human tumor cells were collected, counted, and diluted, and theninjected subcutaneously in NOD/SCID mice. Tumor growth was monitoredfor up to 3 mo. Cancer stem cell frequency was determined using L-Calc

Version 1.1 software program (StemCell Technologies). OMP18R5 was dosedat 10 mg/kg twice weekly, except as indicated, and was dosed at 20 mg/kgonce weekly in Fig. 4G and 15 mg/kg twice weekly in Fig. 4C. Taxol (pacli-taxel) was dosed at 10 mg/kg once weekly except for Fig. 4H, which wasdosed at a maximum tolerated dose of 20 mg/kg twice weekly. Gemcitabinewas dosed at 50 mg/kg twice weekly. Irinotecan was dosed at 7.5 mg/kgtwice weekly. All agents were administered intraperitoneally.

Data Analysis. Data are expressed as mean ± SEM. Differences in mean valuesbetween groups were analyzed by nonparametric t test. Tumor-initiatingcell frequency was determined using Poisson distribution statistics andL-Calc software.

ACKNOWLEDGMENTS. We thank many people at OncoMed for theircontributions to this work, including JorgeMonteon, Paul Sauer, Peter Stathis,Ian Scott, Min Wang, Daniel Croom, Jim Evans, Xiaomei Song, and MichaelMulkerrin. Research funding was provided by OncoMed Pharmaceuticals.

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