Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) Model

Supplemental Materials and Methods

Animals

Establishment of mouse lines deficient in each PG receptor, Ptger1 (EP1), Ptger2 (EP2), Ptger3 (EP3), Ptgir (IP), Ptgdr (DP), or Tbxa2r (TP) was previously reported (1). C57BL/6CrSlc mice and Ptges (mPGES-1)-/- mice were purchased (Japan SLC, Hamamatsu, Japan, and Jackson Laboratory, Bar Harbor, ME, respectively). All mice have a free access to chow and water and are maintained on a 12-h/12-h light/dark cycle under the specific pathogen-free condition.

Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) model

AOM/DSS model was performed according to the previous report with some modification (2). Briefly, 8-12-week-old female mice received intraperitoneal injection of 10 mg/kg AOM (Sigma, Saint Lom3, Mo) on Day 0, and received three cycles of DSS treatment, in which DSS (MP Biomedical, Santa Ana, CA. Molecular weight; 36,000-50,000 Da) was added in drinking water at 2% (Figure 1A). Finally, mice were sacrificed at Day 80 or Day 200 by the intraperitoneal injection of the lethal dose of pentobarbiturate, and whole colon were harvested for analysis.

Bone marrow transplantation

Bone marrow cells were isolated from femurs and tibia of 8-week old female wild type mice (expressing CD45.1 leukocyte antigen) or Ptger2-/- mice (expressing CD45.2 leukocyte antigen). Isolated cells (1x107/body) were intravenously injected to g-ray (9.5Gy/body) irradiated recipient mice. Transplanted mice were treated with neomycin for 4 weeks before subjecting to AOM/DSS model. At the day of sacrifice, the percentage of CD45.1+ or CD45.2+ cells in peripheral blood was measured by FACS to verify the efficiency of bone marrow reconstitution.

Immunohistochemistry

Colon tissues were collected from mice subjected to AOM/DSS model and sectioned at 10-μm thickness. Mouse primary neutrophils were prepared from bone marrow cells by discontinuous density gradient centrifuge. After blocking, the sections or cells were incubated with primary antibodies followed by incubation with secondary antibodies conjugated with fluorescent dye (Jackson ImmunoResearch). Finally, immunofluorescence images were acquired on a confocal fluorescence microscope system (CTR6500, Leica Microsystems, Tokyo, Japan or Lsm710, Carl Zeiss Microscopy GmBH, Gottingen, Germany).

Primary antibodies used in timmunohistochemistry were as follows: mouse monoclonal anti-Ki67 antibody (Leica Biosystems Inc., Wetzlar, Germany), rabbit polyclonal anti-PTGER2 (EP2) antibody (LifeSpan BioSciences, Inc., Seattle, WA), rabbit monoclonal anti-Gr-1 antibody (Abcam Inc., Cambridge, UK), goat polyclonal anti-GROa (CXCL1) antibody (Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit polyclonal anti-TNF-a antibody (Abcam), mouse monoclonal anti-a-SMA antibody (Thermo Fisher Scientific, Inc., Waltham, MA), rabbit polyclonal anti-IL-6 antibody (Abcam), goat polyclonal anti-COX-2 antibody (Santa Cruz), mouse monoclonal anti-myeloperoxidase antibody (Abcam), rabbit polyclonal anti-phosphorylated form of NF-kB p65 subunit (Ser 276) antibody (Cell Signaling Technology Inc., Beverly, MA), rabbit polyclonal anti-phosphorylated form of NF-kB p65 subunit (Ser 468) antibody (Cell Signaling Technology Inc.), rabbit monoclonal anti-phosphorylated form of NF-kB p65 subunit (Ser 536) antibody (Cell Signaling Technology Inc.), rabbit polyclonal anti-BDNF antibody (Santa Cruz), goat polyclonal anti-osteopontin antibody (R&D Systems, Inc., McKinley Place, NE), rabbit polyclonal anti-b-catenin antibody (Abcam).

FACS analysis

Colon tissues were treated with EDTA and collagenase D solution to disperse cells. Inflammatory cells were purified by discontinuous density gradient centrifugation in Percoll PLUS reagent (GE Healthcare, Piscataway, NJ). After counting the number of purified cells, cells were incubated with fluorescence-conjugated antibodies and analyzed by FACS analysis (FACS Calibur, Becton, Dickinson and Company, San Jose, CA).

Antibodies used in FACS analysis were American hamster monoclonal anti-CD3e PE antibody (eBioscience Inc., San Diego, CA), rat monoclonal anti-CD4 PerCP-Cy5.5 antibody (BioLegend Inc., San Diego, CA), rat monoclonal anti-CD8a FITC antibody (eBioscience), mouse monoclonal anti-CD45.1 FITC antibody (BioLegend), mouse monoclonal anti-CD45.2 APC antibody (BioLegend), American hamster monoclonal anti-CD11c PE antibody (eBioscience), rat monoclonal anti-CD11b FITC antibody (eBioscience), rat monoclonal anti-Ly6G PerCP-Cy5.5 antibody (BioLegend), rat monoclonal anti-F4/80 PE antibody (BioLegend), rat monoclonal anti-Ly6C APC antibody (BioLegend), rat monoclonal anti-CD115 PE antibody (eBioscience).

Quantitative real time-PCR (Quantitative RT-PCR)

Total RNA was prepared from colon tissue using a RNeasy Fibrous Tissue Mini Kit (QIAGEN, Hilden, Germany) or from cells using a RNeasy Mini Kit (QIAGEN), and transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Life Technologies Corporation). Quantitative RT-PCR was, then, performed with SYBR Premix Ex Taq II (Takara, Shiga, Japan) and Real Time PCR Detection System CFX96 (Bio-rad, CA). mRNA expression of glyceraldehyde-3-phosphte dehydrogenase (Gapdh) or actin a1 skeletal muscle (ACTN1) was used as an internal control. For quantification, the second derivative maximum method was used for crossing point determination.

Primer sets used in quantitative RT-PCR were

forward 5’- CCGAAGTCATAGCCACACTCAA-3’,

reverse 5’- GCAGTCTGTCTTCTTTCTCCGTTAC-3’ for Cxcl1,

forward 5’- AGTGAACTGCGCTGTCAATG -3’,

reverse 5’- GCCCTTGAGAGTGGCTATGA -3’ for Cxcl2,

forward 5’- CAAGCAGGTAAAGACACATCCA -3’,

reverse 5’- CACCCAGACAGAAGTCATAGCC -3’ for Cxcl3,

forward 5’- AGTGGCGTCCTGCCTTGAT -3’,

reverse 5’- GTCCCGAAGAAAGCGATGG -3’ for Pf4 (Cxcl4),

forward 5’- GCGTGAACAGCAACAGAAATG -3’,

reverse 5’- TCCACACCTCCTCCAGCATA -3’ for Cxcl5,

forward 5’- TGCCTATGTCTCAGCCTCTTC -3’,

reverse 5’- GAGGCCATTTGGGAACTTCT -3’ for Tnf,,

forward 5’- TCCATCCAGTTGCCTTCTTG -3’,

reverse 5’- GGTCTGTTGGGAGTGGTATC -3’ for Il6,

forward 5’- TCGCAGGAAGGGGATGTTGT -3’,

reverse 5’- CTGAAGCCCACCCCAAACAC -3’ for Ptgs2,

forward 5’- GATGAAGCAACCAGAGCAGAC -3’,

reverse 5’- GTGCATCCTTTCGTTACCAGA -3’ for Ptger2,

forward 5’- CAAGGACGACAGCGAAGATG -3’,

reverse 5’- CTGCTACGGGTTCACACTGC -3’ for Cxcr2,

forward 5’- TTGCTGATGTCTCCTGTGAAGC -3’,

reverse 5’- GATTGTTTTGTGCCGTTTACCA -3’ for Bdnf,

forward 5’- AAACCAGCAAGCACCCTTCA -3’,

reverse 5’- GAGGTATGATGTGAGGCAGGAG -3’ for Mmp12,

forward 5’- CCCGGTGAAAGTGACTGATT -3’,

reverse 5’- TTCTTCAGAGGACACAGCATTC -3’ for Spp1,

forward 5’- CTCTAGCGTCCACGAACTCC -3’,

reverse 5’- CAAATAGGCAGCCGAGAGAC -3’ for Wnt5a,

forward 5’-TGAACGGGAAGCTCACTGG- 3’,

reverse 5’-TCCACCACCCTGTTGCTGTA- 3’ for Gapdh.

forward 5’- CATACTCCTGCTTGCTGATCC -3’,

reverse 5’- GATGCAGAAGGAGATCACTGC -3’ for ACTN1,

forward 5’- AGGTCCCATTTTTCCTTTCG -3’,

reverse 5’- CTTCTCATTGTCTCGGTGCTC -3’ for PTGER2,

forward 5’- CTACCAGAAGGGCAGGATACAG -3’,

reverse 5’- GAGCAGGCAGATGAAATACCAG -3’ for PTGS2,

forward 5’- CCTCTTTGCTGCTTTCACACAT -3’,

reverse 5’- CTTCGGTCCAGTTGCCTTCT -3’ for IL6,

forward 5’- CGCCGTTACCCACTCACTAATAC -3’,

reverse 5’- GCCTCCTCTTCTCTTTCTGCTG -3’ for BDNF,

forward 5’- TATTTCCCACGGTAGTGACAGC -3’,

reverse 5’- ATCAACACATTTCGCCTCTCTG -3’ for MMP12,

forward 5’- CCTTTCCTTTCCTTTCCTTTGC -3’,

reverse 5’- GGAGTTGTATTTGCCATCACCA -3’ for WNT2,

forward 5’- TAAGCCTCTTCTCCTTGGCATC -3’,

reverse 5’- GGGTAGTCCACGCTATTACTCG -3’ for WNT2B,

forward 5’- GGCATCTCTCTTTCACCATTCC -3’,

reverse 5’- TAGCAGCATCAGTCCACAAACA -3’ for WNT5A.

Cells

Mouse primary neutrophils were prepared according to the previous report with some modifications (3). In brief, femurs and tibiae were dissected from 8-12-week-old female C57BL/6 mice or Ptger2-/- mice, and neutrophils in bone marrow cells were isolated by discontinuous density gradient centrifuge in Percoll PLUS reagent (GE Healthcare, Piscataway, NJ). Purity of primary neutrophils was determined as CD11b and Ly-6G double-positive cells in FACS analysis. Purified neutrophils were stimulated with PGE2 (10 mM, Nacalai Tesque, Inc., Kyoto, Japan), the EP2 agonist, ONO-AE1-259 (0.5 mM), dibutyryl cAMP (100 mM, Sigma), TNF-a (1 ng/ml, R&D) as indicated in Results or Figure Legends in the presence of indomethacin (Sigma).

Human tumor-associated fibroblasts cell line (CCD-18Co) established from colon (4) was purchased (#CRL-1459, lot number; 61619649, American Type Culture Collection, Manassas, VA). Cultured cells were stimulated with ONO-AE1-259 (0.5 mM) alone or in combination with TNF-a (10 ng/ml) as indicated in Results or Figure Legends in the presence of indomethacin.

PCR array

Primary culture of neutrophils was stimulated with TNF-a alone (1 ng/ml) or in combination with ONO-AE1-259 (0.5 mM) for 15 min, 1 h or 6 h, and total RNA was prepared from stimulated cells as described above. PCR array analysis was done by RT2 Profiler PCR array system from QIAGEN (RT² Profiler™ PCR Array, Mouse NF-kB Signaling Pathway, #PAMM-025Z) using cDNA transcribed from purified total RNA by RT² First Strand Kit (QIAGEN) as a sample. The induction of each gene was calculated as a ratio of gene expressions in vehicle-treated group.

Human samples

Human samples are dissected during surgery to resect colorectal cancer lesions or on biopsy for diagnosis. The use of resected samples to the present study was approved by the local ethical committee at Kyoto University Graduate School of Medicine (Approved number; E1975).

Pathological diagnosis of human specimen used in his study according to the International Union Against Cancer (UICC) colorectal cancer classification and modified scoring system for histological assessment of ulcerative colitis (5) are as follows.

Specimen #1; H05-9943 (1)Tubular adenocarcinoma, pT1N0M0, pStage I (2) Ulcerative colitis, Grade 4,

Specimen #2; H06-1126 (1) High grade dysplasia, (2) Ulcerative colitis, Grade 3,

Specimen #3; H11-11283 (1)Tubular adenocarcinoma, pT4bN0, pStage IIC(2) Ulcerative colitis, Grade 3,

Specimen #4; H12-12291 (1) High grade dysplasia, (2) Ulcerative colitis, Grade 3,

Specimen #5; H13-7354 (1) Tubular adenocarcinoma, pT2N0M0, pStage I (2) Ulcerative colitis, Grade 4,

Specimen #6; H13-10226 (1) High grade dysplasia, (2) Ulcerative colitis, Grade 4,

Specimen #7; H14-7294 (1)Carcinoma in situ, pTisN, pStage 0 (2) Ulcerative colitis, Grade 2.

For immunostaining, paraffin-embedded slices were deparaffinized and used after antigen retrieval. The procedures of immune-staining were the same as those in animal specimen and cultured cells. Antibodies used were mouse monoclonal anti-neutrophil elastase antibody (DAKO, Agilent Technologies, Santa Clara, CA), mouse monoclonal anti-a-smooth muscle actin antibody (DAKO) and rabbit polyclonal anti-EP2 antibody (Santa Cruz).

EP2 antagonist

PF-04418948, 1-(4-fluorobenzoyl)-3-[(6-methoxy-2-naphthyl)oxy]methyl azetidine- 3-carboxylic acid, a selective EP2 antagonist with the Ki value of 16 nM for human EP2 (6, 7) was synthesized at KNC Laboratories Co.,Ltd. (Hyogo, Japan). The purity of synthesized compound examined by HPLC was 99.1%.

Statistical Analysis

Statistical comparison between two groups was made using Mann-Whitney U test. Statistical comparisons among more than 2-groups were conducted using Kruskal-Wallis test followed by post hoc Dunn’s test.

Supplemental References

1. Kabashima K, Saji T, Murata T, Nagamachi M, Matsuoka T, Segi E, et al. The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut. J Clin Invest. 2002;109:883-93.

2. Tanaka T, Kohno H, Suzuki R, Yamada Y, Sugie S, Mori H. A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate. Cancer Sci. 2003;94:965-73.

3. Boxio R, Bossenmeyer-Pourie C, Steinckwich N, Dournon C, Nusse O. Mouse bone marrow contains large numbers of functionally competent neutrophils. J Leukoc Biol. 2004;75:604-11.

4. Sugarman BJ, Aggarwal BB, Hass PE, Figari IS, Palladino MA, Jr., Shepard HM. Recombinant human tumor necrosis factor-a: effects on proliferation of normal and transformed cells in vitro. Science. 1985;230:943-5.

5. Geboes K, Riddell R, Ost A, Jensfelt B, Persson T, Lofberg R. A reproducible grading scale for histological assessment of inflammation in ulcerative colitis. Gut. 2000;47:404-9.

6. af Forselles KJ, Root J, Clarke T, Davey D, Aughton K, Dack K, et al. In vitro and in vivo characterization of PF-04418948, a novel, potent and selective prostaglandin EP2 receptor antagonist. Br J Pharmacol. 2011;164:1847-56.

7. Birrell MA, Nials AT. At last, a truly selective EP2 receptor antagonist. Br J Pharmacol. 2011;164:1845-6.