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Methods Reagents, cell lines and cell culture
HhAntag was synthesized as described in Supplementary Fig.13. Cycoplamine was purchased from Toronto Research Chemicals, Inc.. The mouse anti-Shh monoclonal antibody, 5E129, was protein A-purified and confirmed for lack of aggregation and endotoxin, as well as the ability to block ligand-activated Hh signaling, following gel filtration.
HEPM, human embryonic palatal mesenchyme cells30, were stably transfected with a luciferase reporter gene driven by 6 continuous repeats of the Gli DNA binding element. The human colon fibroblast line, CCD-18Co, was purchased from ATCC (CRL-1459). MEFs from CAGGCre-ER;SmoC/C and control mice were isolated according to published methods and treated with tamoxifen (1μM) in vitro for 5 days before testing for Hh responsiveness in vitro and/or mixing with HT29-luc cell for implantation into CD-1 nude mice.
Cell viability was measured at 72h using the Celltiter-Glo Luminescent Cell Viability Assay kit (Promega), and the concentration of HhAntag resulting in 50% inhibition of cell viability was determined from a minimum of 2 experiments. Co-culture experiments were carried out by culturing tumor cells at a 4:1 ratio with C3H10T1/2 S12 fibroblast GLI-reporter cells11. Luciferase activity was measured using the SteadyLite HTS kit (Promega) after 24h. Assessment of Hh pathway target gene expression was performed by culturing cells in triplicate in 24-well plates for 24 hours with 300ng/ml rSHH and/or Hh antagonists in 0.5% serum containing medium. RNA was isolated using Qiagen RNeasy Mini Kit. Reporter gene assays Twenty-four hours after plating, cells were transfected for 18h with either Gli-luciferase or NFkB-luciferase reporter plasmids in combination with an HSV-TK plasmid utilizing the Fugene6 transfection reagent (Roche) then re-plated into 96 well culture plates. Six hours later, Hh antagonist, and/or 1µg/ml rSHH were added to cells in quadruplicate in 0.5% serum containing medium. Cultures were re-fed 24 hours later, and plates were assessed for firefly and renilla luciferase activity after an additional 24 hours utilizing the Promega Dual-Glo luciferase kit. Immunofluorescence
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doi: 10.1038/nature07275
To evaluate Hh pathway activation in vivo, Ptch1-lacZ; RAG2-/- mice were implanted with pancreatic tumor cells lines expressing differential levels of Hh ligands. Fourteen days following implantation, xenografts were excised and fixed in 4% paraformaldehyde prior to embedding in OCT. Sections were incubated with anti-βgal (1:10000, Cappel) and FITC conjugated anti-ESA (epithelial specific antigen) (1:100, Biomeda) overnight, followed by secondary Cy3-anti-rabbit (1:400, Jackson Immunoresearch) incubation for one hour. Nuclei were visualized with DAPI (blue) in Vectamount (Vector Laboratory). Xenograft models Primary tumor samples were provided by the National Disease Research Interchange (NDRI) and the Cooperative Human Tissue Network which is funded by the National Cancer Institute. Other investigators may have received samples from these same tissues. Primary human xenografts were established by direct implantation of surgical material into female CD1 nu/nu mice of 6-8 weeks of age (Charles River Laboratories, Inc., Wilmington, MA). All mice were housed and maintained according to the animal use guidelines of Genentech, Inc, conforming to California State legal and ethical practices. Tissue specimens were shipped in RPMI containing antibiotics on wet ice and implanted within 24 hours after rinsing in DMEM containing 0.11 mg/ml sodium pyruvate, 1.125 ug/ml Amphotericin B, and 1 mg/ml Kanamycin. Tissue was minced with scalpels to a size of < 1mm3, and approximately 100 mg of tissue was implanted in the subcutaneous space of the hind flank using blunt dissection and a 10G trocar. Tumor lines were serially passaged into larger cohorts of mice for efficacy testing. Mice were distributed into tumor volume-matched cohorts upon tumors reaching between 200 to 350 mm3. HT-29, HT55, and DLD-1 cell lines were purchased from ATCC and were established as xenografts by injection into the hindflank of 6-8 week old female CD1 nu/nu mice with 5 x 106 (HT-29 and HT55) or 10 x 106 cells (DLD-1) resuspended in Hanks’ balanced salt solution. Tumor-bearing mice were distributed into tumor volume-matched cohorts when the tumors reached between 80 and 120 mm3. HhAntag, or a close derivative, was resuspended in 0.5% methyl-cellulose, 0.2% Tween-80 (MCT) and administered orally twice daily at either 100 or 75 mg/kg from a 10 mg/ml suspension as indicated. 5E1 or an isotypic control (IgG1) were dissolved in PBS and administered at 60 mg/kg on day 1 and then at 30 mg/kg weekly via intraperitoneal delivery. Tumor volume and animal weights were monitored twice weekly and tumor volume calculated as (L x W x W)/2. Expression studies
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For the analysis of SHH and IHH mRNA expression in multiple human tissue specimens, data were obtained from Gene Logic, Inc. (Gaithersburg, MD). Microarray gene expression analysis of RNA extracted from primary xenograft tissue was carried out on two separate platforms, Human Genome U133 Plus 2.0 & Mouse Genome 430 2.0 arrays (Affymetrix). Preparation of complementary RNA, array hybridizations, and subsequent data analysis were carried out using the manufacturers’ protocols, with signal intensities being determined by the MAS5.0 algorithm. Hh pathway genes were quantitatively assessed by Taqman and transcript levels were normalized to the housekeeping genes β-glucuronidase (GUSB) or ribosomal protein L19 (RPL19). Results are expressed as normalized expression values (=2-ΔCt) or normalized expression relative to a cell line pool representative of multiple tissue types (=2-ΔΔCt), unless otherwise stated. For xenograft model profiling, gene expression in each compartment was normalized to the same species-specific housekeeping gene to ensure that observed differences weren’t due to differences in the degree of stromal infiltrate. Correlations in gene expression were evaluated by Spearman rank tests and p-values reported. Sequences of primer/probes are shown in Supplementary Table 3 and species-specificity of human/mouse Taqman assays is confirmed in Supplementary Table 2. FACS Cells were stained with either mouse anti-Hh antibody 5E1 or isotype control antibody and then followed by biotin-conjugated anti-mouse, streptavidin-PE, and PI. Stained live (PI-excluded) cells were analyzed on a FacsCalibur, and data were plotted using FlowJo software package. 30. Yoneda, T., & Pratt, R.M. Mesenchymal cells from the human embryonic palate are
highly responsive to epidermal growth factor. Science. 213, 563-565 (1981).
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Supplementary Table 1: Quantitative expression (qRT-PCR) of Hh pathway genes in pancreatic cell lines and relationship to in vitro HhAntagonist activity (IC50)
HhAntagIC50 (μM) Ct 2-ΔCt Ct 2-ΔCt Ct 2-ΔCt Ct 2-ΔCt
AsPC-1 ≥30 23.3 0.9140 31.4 0.0033 26.1 0.1236 26.3 0.1138BxPC-3 5.4 26.9 0.0823 38.7 0.0000 26.3 0.1329 35.6 0.0002Capan-1 20.0 23.1 0.9252 28.3 0.0241 26.6 0.0768 32.2 0.0016Capan-2 nd 26.5 0.0647 35.2 0.0002 25.8 0.1029 31.2 0.0025CFPAC 5.8 26.1 0.0864 27.8 0.0272 28.2 0.0213 34.8 0.0002HPAC 2.7 28.8 0.0246 >40 27.7 0.0509 33.3 0.0010HPAF-II 6.2 25.7 0.1307 >40 26.7 0.0665 30.9 0.0036Hs 766T nd 26.2 0.2199 29.9 0.0172 26.2 0.2330 29.1 0.0297HuP-T3 ≥30 33.4 0.0009 27.1 0.0709 26.7 0.0908 33.5 0.0008KP4 10.3 27.0 0.1012 24.0 0.8241 22.9 1.7584 23.2 1.4079MIA PaCa-2 ≥30 25.9 0.0799 24.1 0.2763 26.8 0.0421 30.6 0.0030Pan C 02.03 6.0 32.0 0.0036 31.7 0.0044 26.3 0.1821 33.2 0.0015Pan C 04.03 9.4 32.2 0.0019 34.3 0.0005 25.6 0.1763 32.7 0.0013Pan C 05.04 10.8 27.9 0.0569 25.3 0.3479 26.0 0.2106 33.0 0.0017Pan C 08.13 8.9 26.5 0.1163 38.8 0.0000 26.0 0.1656 32.2 0.0023Panc 03.27 3.5 25.5 0.1677 26.8 0.0693 26.1 0.1115 33.3 0.0008Panc 10.05 ≥30 32.0 0.0019 36.0 0.0001 26.3 0.0987 33.8 0.0005PANC-1 ≥30 23.9 0.7840 28.4 0.0337 26.9 0.0972 27.7 0.0570PA-TU-8902 2.9 27.0 0.1647 39.2 0.0000 27.8 0.0907 33.9 0.0013PA-TU-8988T ≥30 32.9 0.0012 24.6 0.3932 25.7 0.1768 31.7 0.0027PL45 ≥30 30.4 0.0057 34.6 0.0003 27.0 0.0605 34.6 0.0003PSN1 5.8 23.6 1.4144 26.1 0.2502 27.2 0.1166 31.1 0.0081SU.86.86 2.7 28.8 0.0297 25.3 0.3389 26.0 0.2023 32.6 0.0022SW 1990 ≥30 26.1 0.0767 27.2 0.0378 26.2 0.0745 28.6 0.0143nd: not done
SHH SMO PTCH1 GLI1
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Supplementary Table 2: Confirmation of the species-specificity of human and murine primer/probe sets for detection of Hh pathway genes using universal human or murine reference RNA sources. Cycling threshold (Ct) values are shown.
Primer/probe setUniversal
human reference RNA
Universal mouse
reference RNA
no template control
Human_GUSB 22.6 nd ndHuman_GLI1 26.9 nd ndHuman_PTCH1 24.0 nd ndHuman_PTCH2 28.4 nd ndHuman_SMO 21.8 nd ndMurine_Gusb nd 22.1 ndMurine_Gli1 nd 27.6 ndMurine_Ptch1 nd 22.6 ndMurin_Ptch2 nd 30.9 ndMurine_Smo nd 22.9 ndnd, not detected (Ct above 40)
Cycling Threshold (100ng input RNA)
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Supplementary Table 3: qRT-PCR primer/probes
Gene species SequenceGUSB human forward primer TGGTTGGAGAGCTCATTTGGA
reverse primer GCACTCTCGTCGGTGACTGTTprobe TTTGCCGATTTCATGACT
GLI1 human forward primer GTTCACATGCGCAGACACACTreverse primer TTCGAGGCGTGAGTATGACTTCprobe CACACAAGTGCACGTTT
PTCH1 human forward primer CGGCAGCCGCGATAAGreverse primer TTAATGATGCCATCTGCATCCAprobe AGCCAGTTGACTAAACAG
PTCH2 human forward primer GCTTTGCCAGAGTGACTACCT reverse primer AGGGGCTGGATGGATGTA probe CATGACCGTGGCCATCCACC
SHH human forward primer CGGCTTCGACTGGGTGTACTreverse primer GCAGCCTCCCGATTTGGprobe CGAGTTCTCTGCTTTCA
IHH human forward primer CTCACAAAGCATGGGACACT reverse primer GAGTCTCAGGGGCCAGAA probe CCGTGGCTGACCACCACCTG
SMO human forward primer GTGCTGGCCCCAATCGreverse primer GCAGCATGGTCTCGTTGATCTprobe CTCATCCGAGGAGTCAT
Gusb mouse forward primer CTGCCACGGCGATGGAreverse primer ACTGCATAATAATGGGCACTGTTGprobe CCTCAACACCACTCTCATGTCGGTATCTTG
Gli1 mouse forward primer GCAGTGGGTAACATGAGTGTCT reverse primer AGGCACTAGAGTTGAGGAATTGT probe CTCTCCAGGCAGAGACCCCAGC
Ptch1 mouse forward primer GCTACGACTATGTCTCTCACATCAACTreverse primer GGCGACACTTTGATGAACCAprobe ATGGCGGCTGCCCTGTCTTCATT
Smo mouse Applied Biosystems Cat. #: Mm01162702_g1www.nature.com/nature 6
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Supplementary Figure 1: No correlation of PTCH1 or SMO mRNA levels (Affymetrix-derived) with HhAntag activity in cell lines
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Supplementary Figure 2: No correlation of GLI1 mRNA levels (qRT-PCR) with cyclopamine activity in cell lines
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Supplementary Figure 3: rSHH does notinduce GLI luciferase reporter activity in tumor cell lines. The mesenchymal cellline, 10T1/2, serves as a positive control
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Supplementary Figure 4: Normalized activity of transiently transfected pathway reporter constructsin cell lines treated with Hh antagonists (solid bars, cyclopamine; open bars, HhAntag). Note thatHh antagonists inhibit multiple pathway reporters at the concentrations required to inhibit growthin these cells
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Supplementary Figure 5a: Hh pathway antagonist-mediated growth inhibition in select cell lines used in previous studies and/or colo-rectal cell lines used for in vivo studies in the accompanying manuscript
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Supplementary Figure 5b: Lack of Hh pathway activity in vitro in select cancer cell lines used in previous studies and/or colo-rectal cell lines used for in vivo studies in the accompanying manuscript; nd: not detected
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Supplementary Figure 6. Quantitative expression of SHH and IHH mRNA in a panel of tumor cell lines. Expression values (2-ΔΔCt) are relative to gene expression determined from a pool of cell lines representative of multiple tissue types. The arrows and letters indicate the pancreatic cell lines HPAF-II (a) and PL45 (b), and the colon cell lines HT55 (c), HT-29 (d) and DLD-1 (e).
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Supplementary Figure 7. Cell surface expression of Hh ligands in a panel of pancreatic and colon cancer cell lines determined by FACS analysis (mAb 5E1, black line). Grey shade represents isotype-matched control Ab staining.
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Supplementary Figure 8: Stromal activation of the Hh pathway in HPAF-II, but not PL45, xenografts in vivo. Additional sections demonstrating β-galactosidaseactivity (red) in the stromal compartment following implantation of HPAF-II cells in Ptch1-lacZ;RAG2-/- mice with serial sections taken for hematoxylin and eosin stain (right panels). Epithelial-specific antigen staining is depicted in green.
HPAF-II HPAF-II
PL45 PL45
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Supplementary Figure 9:Rapid infiltration of murine stromal cells in primary human xenograft models
B. C.
A. Primary human tumor hybridized with a mouse chromosome X painting probe fails to detect any positive cells within the tumor (arrow head) or stroma (arrow). B. Second passage xenograft tumors derived from this primary tumor reveal strong and ubiquitous hybridization within the stromal compartment (arrow) while the tumor cells (arrow head) fail to hybridize. C. Immunofluorescence of a separate section from the same xenograft was double labeled with BerEP4 (green) and vimentin (red) to highlight the tumor and stromal compartments, respectively. Hybridization was carried out as suggested by the manufacturer (Catalog 1189-XMF-02, Cambio Ltd, 1 The Irwin Centre, Scotland Road, Dry Drayton, Cambridge, CB23 8AR).
A.
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Supplementary Figure 10:Comparison of the GLI1 mRNA expression levels originating from the tumor epithelium and stromal microenvironment in human primary tumor implants. The red boxes reflect the human and mouse GLI signal respectively in a reference source representative of a pool of tissue types.
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Supplementary Figure 11: Maximal inhibition of Gli1 in vivo is observed at concentrations required to inhibit tumor growth (ie. 100mg/kg) followingHhAntag treatment
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Supplementary Figure 12: Reduction in Smo mRNA expression in tamoxifen treated MEFs derived from CAGGCreER;SmoC/C mice
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Supplementary Figure 13a: Synthesis of HhAntag (N-[4-Chloro-3-(5-dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5-dimethoxy-benzamide)
N-[4-Chloro-3-(5-dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5-dimethoxy-benzamide.
N
NH
NCl
HNO
O
O
2HCl
1). 2-chloro-N-(5-(dimethylamino)-2-nitrophenyl)-5-nitrobenzamide.
N
Cl
Cl
O
NO2
NO2
NH2
NO2
NH
O
N
Cl
NO2
ACN Dissolve N,N-dimethyl-4-nitrobenzene-1,3-diamine in ACN (50g/L) in a 3-neck flask equipped with overhead stirring and an addition funnel under nitrogen. Dissolve 2-chloro-5-nitrobenzoyl chloride in ACN (35g/100mL; 1.1equ.) and add drop-wise to the aniline while keeping the temperature under 25oC. After addition is complete, heat the reaction mixture to 75oC for 1 hour. (Note: LC-MS should indicate >95% product.) Cool reaction mixture to 0oC, filter, rinse with cold ACN and dry (Yields ~90%).
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Supplementary Figure 13b: Synthesis of HhAntag (N-[4-Chloro-3-(5-dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5-dimethoxy-benzamide)
Cont.
2). 2-(5-amino-2-chlorophenyl)-N,N-dimethyl-1H-benzimidazole.
NO2
NH
O
N
Cl
NO2 SnCl2
EtOH
N
NH
NCl
NH2 Dissolve 2-chloro-N-(5-(dimethylamino)-2-nitrophenyl)-5-nitrobenzamide in EtOH (50g/L) in a 3-neck flask equipped with overhead stirring and a reflux condenser and heat to 40oC. When temperature reaches 40oC, add 1st aliquot SnCl2/HCl (~7 equ. and 3 vol respectively, divided into 3 portions) and heat to 60oC. When the temperature reaches 60oC, add the 2nd aliquot of SnCl2/HCL and heat to 80oC. When the temperature reaches 80oC, add the 3rd aliquot SnCl2/HCl and heat to reflux (~87oC) for 2 hours). (Note: LC-MS should indicate >95% product.)Cool the reaction mixture to 0oC and begin NaOH addition (~30equ) while keeping the temperature under 10oC. When NaOH addition is complete, the pH is 12-13 and solution is white. Dilute the reaction mixture with EtOAc and 2.5M NaOH, stir overnight and extract. Back extract the aqueous layer with EtOAc, combine the organics and wash with NaOH followed by brine. Dry the organic layer with Na2SO4, concentrate and dry (Yields ~75% from the first crop).
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Supplementary Figure 13c: Synthesis of HhAntag (N-[4-Chloro-3-(5-dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5-dimethoxy-benzamide)
3). (N-[4-Chloro-3-(5-dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5-dimethoxy-benzamide.
N
NH
NCl
NH2
N
NH
NCl
HNO
O
O
Cl
O
O
2HCl
THF
O
Dissolve 2-(5-amino-2-chlorophenyl)-N,N-dimethyl-1H-benzimidazole in THF (25g/L) in a 3-neck flask equipped with overhead stirring and an addition funnel under nitrogen and cool to 0oC. Dissolve 3,5-dimethoxybenzoyl chloride in THF (50g/300mL; 1.2 equ.) and add dropwise while keeping the temperature under 10oC. After addition is complete, stir for an additional 30 minutes. Add additional THF to maintain consistency. (Note: LC-MS should indicate >95% product.) Dilute the reaction mixture with diethyl ether (1/2 volume of existing THF) and collect the solid by filtration. Dissolve the solid in EtOAc and extract with NaHCO3. Back extract the aqueous layer with EtOAc, combine the organics and wash with NaHCO3 followed by brine. Dry the organics with Na2SO4 and then concentrate to dryness (Yields ~60-70%; ~50% from the first crop). Dissolve the freebase in ACN (50g/1.5L) and heat to 60oC. When the solution is homogeneous, cool to 0oC and add 2.5N HCl dropwise. After HCl addition is complete, filter the product and dry in a vacuum oven (Yield from freebase ~95%). Characterization: 1H-NMR (400 MHz, CDCl3): δ 10.88 ppm (s, 1H), 8.51 ppm (d, 1H), 8.12 (m, 1H), 7.95 (bs, 1H), 7.88 (m, 1 H), 7.71 (m, 2H), 7.17 (d, 2H), 6.68 (t, 1H), 3.79 (s, 6H), 3.15 (s, 6H). MS (m/z): [M]+ calcd for C24H23ClN4O3, 450.15; found 450.2.www.nature.com/nature 22
doi: 10.1038/nature07275 SUPPLEMENTARY INFORMATION