Dll4 blockade inhibits tumor growth by promoting nonproductive angiogenesis

doi: 1.138/nature5355 Dll4 blockade inhibits tumor growth by promoting nonproductive angiogenesis Irene Noguera-Troise, Christopher Daly, Nicholas J Papadopoulos, Sandra Coetzee, Patricia oland, Nicholas W. Gale, Hsin Chieh Lin, George D. Yancopoulos, and Gavin Thurston Regeneron Research Laboratories, 777 Old Saw Mill River Road, Tarrytown, NY, 1591 ddress correspondence to: Gavin Thurston, Ph.D., Regeneron Pharmaceuticals, Inc, 777 Old Saw Mill River Road, Tarrytown, NY, 1591, Phone: 914-345-7575, Fax: 914-347545, Email: Gavin.Thurston@Regeneron.com Lack of effects of on Normal Tissue Vasculature: Systemic treatment of tumor-bearing mice with protein for up to 25 days produced conspicuous effects on tumor growth and macroscopic appearance, as well as on tumor vasculature as revealed in thick tumor sections that were immunostained with antibodies to Pecam/CD31. In contrast, we did not observe significant effects on body weight Supplementary Figure 1 or macroscopic organ appearance, nor did we find detectable effects in the normal Noguera-Troise al. organs examined to date using similar histological approaches -- skin, skeletalet muscle, subcutaneous fat, and intestinal wall. We note that is likely to block multiple Notch receptors, thus more extensive study of the effects on normal vasculature of specific Dll4 blockade requires specificdll4-blocking reagents. Dll4 expression in tumor vessels -- confirmation using Dll4-beta-gal reporter mouse and by Dll4 in situ hybridization PECM b beta-gal b D C PECM b Figure S 1. dditional evidence of Dll4 expression in tumor vessels. (, ) Lewis lung tumor grown subcutaneously in heterozygous Dll4 gene targeted mouse in which the Dll4 gene was replaced with a β-galactosidase expression cassette, to reveal sites of endogenous Dll4 expression. ) Section from Lewis lung tumor showing immunoreactivity for CD31/PECM is equally strong in tumor vessels and adjacent skin vessels. ) djacent section from Lewis lung tumor showing immunoreactivity for β-galactosidase (as marker for Dll4) is strong in tumor Dll4 in situ vessels and weaker in vessels in adjacent skin. Weak staining for β-gal (Dll4) is also seen in many large tumor vessels (arrow). C, D) Histologic sections of C6 tumor, showing blood vessel immunoreactivity for CD31 / PECM (black) (C), and adjacent section with in situ hybridization showing strong Dll4 expression in tumor tissue, and weak Dll4 expression in nearby normal skin tissue (D). Dll4 expression associated with tumor vessels. 1

doi: 1.138/nature5355 Retroviral approach to transduce C6 tumor cells 6 5 5 LTR GFP IRES Number mdll4(-tm) hfc 3 LTR C6-Parental C6-GFP 4 3 C6-2 mdll4(-tm) 1 1 C 2 1 1 GFP 1 1 GFP fluorescence 4 3 1 Concentration of hfc (ng/ml) D GFP fluorescence throughout a retrovirally transduced C6 tumor 2 15 1 5 C6-GFP C6- #1 C6- #2 C6-Flt1-Fc cell type E F Dll4 in situ in a control C6 tumor Figure S2. Retroviral approach was used to over-express or Dll4 in C6 glioma cells. ) Schematic diagram showing retrovirus vector used to generate over-expression in combination with GFP expression. Control tumor cells were transduced with vector containing IRES-GFP elements. ) FCS analysis of C6 glioma cells expressing. Levels of GFP were used to sort cells from control C6 tumor cells (C6-GFP) and overexpressing cells (C6-). Pools of cells were generated and introduced into mice, without cloning or in vitro selection. C) Section of C6 tumor retrovirally expressing GFP shows GFP fluorescence (green) throughout the tumor, but not in adjacent skin. Dll4 in situ in a C6-Dll4 tumor D) Concentration of hfc tagged proteins of interest in conditioned media of retrovirally transduced rat glioma C6 tumor cells. Media from control C6 cells show no detectable hfc in the media, whereas media from C6 cells transduced with (or control Flt1-Fc) show levels of 1-2 µg/ml. E, F) In situ hybridization for Dll4 performed on section from control C6 tumor (E) shows punctate vessel-specific expression of Dll4 (white grains), whereas section from C6-Dll4 tumor expressing retrovirally transduced Dll4 (F), in situ labeling (white grains) is seen throughout tumor. Thus tumor cells maintain their expression of the transduced gene, even after growing into a large tumor in vivo. 2

doi: 1.138/nature5355 C6 tumor over-expression of or full length Dll4 affects Notch target genes in host mouse tissue in the tumor relative RN level 5 4 3 2 1 C6 tumors * Downstream gene/ type Figure S3. Manipulation of Notch signaling in C6 tumors in vivo. C6 tumor cells were engineered to over-express of full length Dll4 and implanted subcutaneously in mice. RT-PCR (Taqman) analysis of downstream host mouse Notch genes in retrovirally transduced C6 tumors. PCR probes and primers were designed to distinguish mouse cdn from tumor-derived rat cdn. nalysis of tumor tissue shows that mouse Notch downstream genes are downregulated in C6 tumors over-expressing compared to control C6 tumors, including HES1, HEY2, and NRRP. Reciprocally, the same mouse downstream genes are upregulated in C6 tumors over-expressing full-length Dll4 compared to control C6 tumors. Values significantly different from control group (* P<.5; P<.1). Co-culture experiments in vitro - manipulation of Notch target genes in HUVEC by addition of C6-Dll4 or C6- cells huhes1/β actin Relative Expression 2. 1.5 1..5. Control Dll4 Relative Expression 2. 1.5 1..5. huhey2/β actin Control Dll4 Relative Expression 2. 1.5 1..5. NRRP/β actin Control Dll4 Figure S4. Manipulation of Notch signaling pathway in cultured HUVEC by coculture with C6 glioma cells engineered to overexpress, full length Dll4, or egfp (controls). Co-culture with C6- cells resulted in a significant decrease of human HES1 (), HEY2 (), and NRRP (C) as assessed by Taqman primers and probes specific for human expression. 3

doi: 1.138/nature5355 Treatment of cultured HUVEC with protein rapidly reduces Notch target genes relative RN level 1. HES1.75.5.25. Fc 2 hrs HUVEC 8 hrs relative RN level 1..75.5.25. Fc Hey2 2 hrs HUVEC 8 hrs C relative RN level 1..75.5.25. Fc Nrarp 2 hrs HUVEC 8 hrs Figure S5. Treatment of cultured endothelial cells (HUVEC) with results in reduced expression of Notch downstream genes, including HES1 (), HEY2 (), and NRRP (C). Confluent HUVEC monolayers were treated with protein (1 µg/ml) for 2 hr or 8 hr, or hfc protein as control. Gene expression was assessed by quantitative PCR (Taqman) for amount of RN in HUVEC treated with relative to HUVEC treated with hfc. The actions of soluble Delta and Notch molecules have been somewhat varied in different experimental systems. In some systems, soluble versions of Dll4 have been reported to activate the Notch pathway, inducing classic Notch target genes such as HES1 and HEY1 ( 1 ), whereas in other systems they can inhibit the pathway ( 2 ). Part of the apparent discrepancy may be due to differences in the oligomerization state of the soluble Notch ligands used in the particular experiments ( 3 ). In our experiments, cultured HUVEC treated with purified dimeric protein showed clear down-regulation of several consensus Notch target genes, strongly suggesting that acts an as inhibitor in this system. Furthermore, co-culture of HUVEC with tumor cells overexpressing produced similar decreases in target gene expression, whereas coculture with tumor cells overexpressing full length, membrane bound Dll4 produced increased expression in these target genes. Consistent with these findings in cultured endothelial cells, in vivo production of soluble by tumor cells, or systemic delivery of by either adenovirus or protein injection, decreased the same Notch downstream genes, and thus appears to inhibit the Notch pathway. Reciprocally, production of full length Dll4 by tumor cells increased expression of the Notch downstream genes, and thus appears to locally activate the Notch pathway. The tumor cells that were engineered to overexpress or Dll4 did not have different growth characteristics in vitro than the control tumor cells. Thus we believe that the effects that we observe on tumor vascularity and growth with Dll4 and treatments are the result of activation / inhibition of Notch signaling in the vasculature, respectively. lthough we were limited in our approaches to activate the Dll4/Notch pathway in tumor blood vessels (i.e., local tumor overexpression of full-length Dll4 in C6 tumors), we were able to employ several approaches to inhibit the pathway in multiple tumor models. Reassuringly, we observed reciprocal effects of activation / blockade by several endpoints. Moreover, the various approaches to block the pathway - regardless of delivery approach or tumor type - yielded largely similar results. Further support for our conclusions comes from contemporary studies from several groups exploring the effects of Dll4 / Notch blockade during postnatal retinal vessel development [I. Lobov et al, submitted; M. Hellstrom et al, submitted;. Eichmann, personal communication] and from previous studies on the vascular development in Dll4 gene targeted mice 4, 5. It must be noted that in all of the biological systems examined to date (developing embryo, developing retina, and growing tumor), the effect of inhibiting Dll4 / Notch may have secondary effects, such as increasing tissue hypoxia and thus upregulating VEGF, that may complicate the interpretation of the results. However, all of these studies came to similar conclusions, most notably that Dll4/Notch normally acts to restrain angiogenic sprouting and branching activity, and that blockade of this pathway results in excessive levels of angiogenic activity. Our findings extend this role of the Dll4 / Notch pathway to the setting of tumor angiogenesis. 4

doi: 1.138/nature5355 4 C6- tumors grown for 17 days are smaller than corresponding C6 control tumors Volume (mm3) 3 2 1 C6 control C6- Type Figure S6. Growth of larger C6 tumors is inhibited by tumor over-expression of. C6 tumors over-expressing either GFP (control) or (an independent cell pool from that shown in Figure 2) were implanted into SCID mice and grown for 19 days. gain, with this independent pool and with somewhat larger tumors, the C6- tumors were consistently smaller than the C6-GFP tumors. iacore analysis of Dll4 blocking antibodies 14 12 1 8 6 4 2 DLL4-Fc (1µg/ml) + b (1µg/ml) + b (2µg/ml) + b (4µg/ml) DLL4-Fc + Control b (4µg/ml) Figure S7. iacore competition binding assay of mouse to rat Notch1-hFc, and blocking by increasing amounts of polyclonal antibody to Dll4. The specific blocking of the binding of mouse Dll4-hFc to rat Notch1-hFc using anti-human Fc depleted rabbit polyclonal against mouse was assessed using iacore (iacore, Piscataway, New Jersey, US). Rat Notch1-hFc was amine-coupled (45 resonance units [RU]) onto a CM5 chip. Varying concentrations of specific mouse Dll4 polyclonal antibody, as well as irrelevant control polyclonal antibody (Fc depleted anti-mouse LYVE-1), were incubated with a fixed amount mouse (1µg/ml) prior to flowing the solution across the Notch1 chip. Nearly complete inhibition of Dll4 / Notch1 binding is seen at 4-fold excess of polyclonal antibody. 5

doi: 1.138/nature5355 size (mm 3) Treatment of MMT tumors with decreases tumor growth and increases tumor vascularity MMT tumors 8 6 4 2 Control MMT tumor control C Supplementary Figure 9 Noguera-Troise et al. MMT tumor DII4-Fc Treatment Figure S8. Systemic delivery of to mice bearing MTT tumors results in smaller tumors and dramatic changes in tumor vessels. delivered using adenoviral system from the time of implantation. ) Size of MMT tumors treated with from time of implantation. caused a reduction in tumor size. The graphs show data from individual tumors, as well as mean ± SD for each group., C) also caused increase in tumor vessel density and dramatic changes in vessel morphology in MMT tumors. sections stained for CD31 (PECM, black). Vessel sprouting / branching Well-organized vessel network VEGF Induction of Dll4 Suppression of unnecessary branches / sprouts Vessel sprouting / branching Excessive sprouting / branching VEGF Induction of Dll4 Suppression of unnecessary branches / sprouts Poorly functional vascular network Dll4 / Notch blockade Figure S9. Proposed model of Dll4 involvement in tumor angiogenesis. Increased VEGF expression in tumor tissue induces vascular sprouting and branching. VEGF also induces Dll4 expression in a subset of tumor endothelial cells. Dll4 in the tumor vasculature modulates the actions of VEGF on endothelial cells by decreasing sprouting and branching (), thus helping form a well-organized and functional vascular network. lockade of Dll4 allows unrestricted actions of VEGF on endothelial cells, resulting in excessive vascular sprouting and branching (), and a poorly functional vascular network. 6

doi: 1.138/nature5355 METHODS C6 tumors retrovirally expressing or Dll4 cdn was engineered to the extracellular portion of murine Dll4, ending in PSFPW, joined to human Fc from IgG1. The fragment was cloned into a retroviral vector containing 3 and 5 LTR repeats from Moloney murine leukemia virus, a CMV promoter, and a multi-cloning site to insert the gene of interest in front of an IRES- GFP cassette (Figure S2). Full length murine Dll4 coding sequence was cloned into the same retrovirus vector. C6 rat glioma tumor cells (merican Type Tissue collection) were infected with retrovirus to over-express GFP, GFP plus, or GFP plus full length Dll4; cells infected with GFP alone were used as controls. Infected cell populations were fluorescent activated cell sorted (FCS) twice on a DakoCytomation MoFlo high-speed cell sorter (Fort Collins, CO). Positive GFP fluorescent cells comprised more than 95% of the cell population. These cells were expanded and implanted subcutaneously in SCID mice. (FCS sorting of transduced C6 rat glioma tumor cells based on egfp expression rapidly yielded over-expressing pools of transductants (representing 1-2% of the initially infected cells) without the need for antibiotic selection or subcloning. fter culturing the initial sorted population, re-analysis by FCS typically revealed >95% positivity for egfp (Figure S2), as well as substantial production of the introduced protein product (e.g., 15 ng/ml of soluble, Figure S2). In addition, use of such transduced pools also avoids phenotypic artifacts that might result from examination of a limited number of tumor subclones. Expression of the retrovirally-introduced GFP and the transgene was maintained when the cells were grown as tumors ( Figure S2). fter tumors became palpable, size was measured every three days (volume= (length x width 2 )/2). fter 12-16 days, mice were sacrificed, and tumor size was measured ex vivo, using the formula length x width x height to calculate tumor volume. s were harvested and processed for histology or gene expression analysis. For histology, tumors were cut into 8 µm sections, stained with antibodies to CD31/Pecam-1 followed by D-peroxidase reaction, and counterstained with pyronin Y. Two independent pools of C6 cells over-expressing either Dll4- Fc or full length Dll4 were generated; the separate pools from each Dll4 construct gave consistent effects on the size and vessel morphology of the resultant tumors. denovirus construction and delivery Recombinant E1-, E3-deleted adenovirus vectors expressing hfc or Dll4-hFc under control of the cytomegalovirus (CMV) promoter were constructed using the d Max Cre-lox based system (Microbix iosystems Inc). riefly, the transgene was cloned into a shuttle plasmid, which was then co-transfected with an adenovirus genomic plasmid into 293 cells. High efficiency site-specific recombination catalysed by Cre introduced the gene of interest into infectious adenoviral DN while simultaneously excising the recombinase gene 6. Infectious virus was plaque-purified and amplified in 293 cells. The working viral stocks were purified from infected cells and conditioned medium by 3 freeze-thaw cycles followed by two centrifugation steps in cesium chloride gradients by standard techniques 7. Viral particles were titered using TCID 5 (Tissue Culture Infectious Dose) method 8. Previous experience with such adenoviruses in mice has shown them to be taken up and expressed in hepatocytes and to provide sustained transgene expression for 2 wk 9. C6 glioma cells (1x1 6 ), mouse mammary tumor cells (MMT, 5x1 5, TCC), or HT18 cells (1x1 6, TCC) were implanted subcutaneously into the shaved right flank of male SCID/C17 mice (8-1 wk of age). fter 24 hr, 1 x 1 9 pfu of adeno-hfc or adeno- was injected into the jugular vein. Other control mice received tumors but no injection of adenovirus. fter 8 to 13 days of growth, tumors were processed for histological analysis as described above. Production of Dll4 antibodies and characterization Polyclonal antibodies were generated by immunizing rabbits against murine Dll4- hfc protein. Serum was depleted for anti-human Fc antibodies, and then used for immunostaining tumor sections at 1:1 dilution. The specific neutralization of the binding of mouse to rat Notch1-hFc by the rabbit polyclonal antibodies was assessed using iacore (iacore, Piscataway, New Jersey, US). Rat Notch1-hFc was amine-coupled (45 resonance units [RU]) onto a CM5 chip. Varying concentrations of specific mouse Dll4 polyclonal as well as irrelevant control polyclonal antibody (Fc depleted anti-mouse LYVE-1) were incubated with a fixed amount mouse (1µg/ml) prior to flowing the solution across the Notch1 chip. studies in Dll4 Lac/Z reporter mice The entire coding region of the Dll4 gene was replaced with a β-galactosidase reporter gene 5 using Velocigene technology 1. Lewis lung carcinoma cells (LL/2, TCC) (5 x 1 5 ) were implanted s.c. in the right hind flank of mice and allowed to grow to tumors. s were harvested, cut into 8 µm sections, stained with antibodies to CD31/Pecam-1 (D Pharmingen) and/or β-gal (Molecular Probes) (or X- gal reaction) followed by D-peroxidase reaction (Sigma), and counterstained with pyronin Y. Quantification of media and serum levels of Cell culture supernatant and serum samples obtained from mice bearing tumors expressing GFP or were analyzed by ELIS. The ELIS was performed by coating plates (Immuno-Maxisorp) with goat anti-human IgG (gamma chain specific) (Sigma) at 5µg/ml. Protein standard () was diluted from 1 ng/ml to.137 ng/ml. C6 cells expressing, Flt1-Fc, or egfp (as control) were plated at a density of 1 6 cells into a T75 flask and grown for three days in Ham s F1 media (Irvine Scientific) containing fetal bovine serum (Irvine Scientific). Cells were rinsed twice with PS containing Ca 2+ and Mg 2+ and incubated overnight in 6 ml of media without serum. Supernatants were collected and tested over a range of concentrations. Serum from mice treated with adeno-dll4-fc was analyzed with a similar protocol. In situ hybridization, Northern blotting and Real Time-PCR Total RN was extracted from C6 glioma tumors expressing GFP, or Dll4 using Trizol reagent (Life Technologies, NY). For Northern blot analysis, RN (1 µg) was separated on 1.2% agarose gels, transferred to nylon membrane and immobilized by UV crosslinking. fter prehybridization, 32P-labeled Dll4, GFP or glyceraldehyde-3-phosphate dehydrogenase (GPDH)-specific probes were added, and the filters were hybridized at 42 C overnight. Stringent washes were performed by standard protocols, and an autoradiograph was obtained after 48 h exposure to x-ray film with intensifying screens. In situ hyridization for Dll4 was performed on 1 µm cryo-sections using a 35S-labeled RN mouse Dll4 specific probe, spanning the entire coding region. specific expression was analyzed using the Taqman (pplied iosystems, C) real-time PCR chemistry and detection system with the primers pairs and labeled probes specific for Dll4, the Notch receptors 1 and 4 and Notch downstream targets, Hes1, Hey2, and Nrarp. The number of cycles necessary to reach the threshold for amplification of the cdn (or CT value) was obtained, and normalized to a housekeeping reference (GPDH or β-actin) (=2-DCT). The results were normalized to a baseline tissue (the tumor overexpressing GFP control for the experiment), giving the relative mrn abundance change (=2DDCT) and is expressed as the mean ± s.e.m. for at least 4 separate samples run in triplicate 11. In vitro assays to determine effect of and full length Dll4 on Notch signaling in human umbilical vein endothelial cells (HUVEC) HUVEC (VEC Technologies, 1 x 1 6 ) were plated onto 6 mm dishes and grown in MDC131 complete medium to obtain 1% confluent cultures the following day. The next day, protein (1µg/ml) was added to the cell media for varying periods (2 to 8 hr). Cells were scrapped into 1 ml of Trizol reagent (Life Technologies, NY) and total RN was prepared as described. Samples were analyzed by Taqman, using probes and primers specific for human HES1, HEY2, and NRRP. In other experiments, HUVEC (4 x 1 5 ) were plated onto 6 mm dishes to obtain 5% confluent cultures the following day. The next day, 8 x 1 5 C6 glioma cells were plated on top of the HUVEC. fter 24 hr of co-culture, cells were assayed for human HES1, HEY2, and NRRP mrn levels as above. Hypoxia and lectin staining In some mice, HypoxyProbe-1 (pimonidazole hydrochloride, Chemicon) (6 mg/kg), which forms long-lived protein adducts at partial pressure of oxygen less than approximately 1 mmhg, was injected intraperitoneally 1 hr prior to sacrifice. nesthetized mice were then perfused with 4% paraformaldehyde solution in.1m NaPO 4 ph7.2. s were processed as described above for histological analysis, and tumor sections were stained using anti-hypoxyprobe antibody (Chemicon, 1:1) and biotinylated horse anti mouse IgG (Vector laboratories, 1:1), followed by the C detection system (Vector Laboratories) and D reaction. For lectin staining, anesthetized mice were injected in the left femoral vein with biotinylated Lycopersicon esculentum lectin (1 µg, Vector Laboratories). Lectin was allowed to circulate for 1 minutes. s were subsequently fixed by perfusion of paraformaldehyde solution as above. Morphometric analysis of vessel density and hypoxia in tumor sections Vessel area density was performed using the NIH Image 1.62 analysis program on images collected from 8 µm thick tumor sections stained with CD31 / PECM antibody, followed by D reaction and pyronin Y counterstaining. Images were segmented by thresholding to identify vascular structures that were immunostained for PECM. Threshold values were kept constant for all tumor measurements. The area of the tumor was demarcated manually to include the viable tumor and exclude surrounding normal tissue and necrotic regions. Vessel density was calculated as a percentage of stained PECM area compared to the total tumor area. Hypoxic area was outlined manually in tumor sections, and compared to the total vascularized tumor area. The distance to the hypoxic rim was determined by measuring the linear distance from the growing front of tumor vessels to the prominent hypoxic rim at 4-7 points for each tumor section for 5 tumors per group. Data nalysis and Statistics Statistical comparisons of treated and control groups were performed by two-tailed Student s t-test. P-value <.5 was considered significant and denoted by *, and P- value <.1 was denoted by. 7

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