Online Supplementary Materials and Methods

Online supplementary Materials and Methods Materials P2RX7 antagonists A438079 and A740003 were from TOCRIS bioscience. A438079 was given during DSS treatment by i.p. injections (2 μmoles per mouse, every 2 days, which represented 3 injections per cycle of DSS). A740003 was administrated following the same route at 1 μmole per mouse, every 2 days, 4 injections. For TGFB neutralization, C57BL/6 mice were injected by the i.p. route with 0.25 mg of anti-TGFB (clone 1D11.16.8; BioXCell) or with a control isotype (clone MOPC-21; BioXCell), at the beginning of DSS treatment and every two days with a total of 4 injections.

Animal experiments Mice were housed in SPF conditions and were provided commercial rodent food (#A03-10, Safe Laboratories) and water ad libitum. All experiments were conducted with age-matched male and female mice, as we observed no difference between the two genders. Control C57BL/6J mice were supplied by Harlan laboratories (Gannat, France). Experimental cohorts were co-housed for at least 15 days prior to the beginning of the experimental protocol to homogenize their microbiota and the co-housing was maintained during the entire experiment. We obtained similar disease scores (inflammation and tumor growth) when C57BL/6 and P2x7-/- mice were housed separately.

Induction of colitis and inflammation-driven tumor formation Experimental colitis was induced by intraperitoneal injection of azoxymethane (AOM, 8 mg/kg body weight from Wako chemicals, Neuss, DE) followed by addition of dextran sodium sulfate (DSS of 35,000-50,000 kDa from TdB consultancy Cat# DB001) to the drinking water at a concentration of 5% (w/v) for 5 days followed by regular tap water for an additional 9 days. Of note, intraperitoneal (i.p.) injection of azoxymethane (AOM) prior to DSS treatment did not change the nature of the inflammatory response. Mice were euthanized at the end of each protocol, or earlier if the animals became moribund, for tumor assessment.

Clinical score A semi-quantitative clinical score was generated by scoring body weight, presence of rectal bleeding and stool consistency (adapted from23). Body weigh loss was scored as follows: (0) no loss, (1) loss < 5%, (2) loss < 10%, (3) loss < 20% and (4) loss > 20%. The scores for stool consistency were measured as (0) normal, (1) loose stools, (2) watery diarrhea or (3) severe watery diarrhea. Rectal bleeding was scored as (0) no blood, (1) presence of petechia, (2) stools with a trace of blood or (3) bleeding. Mice were sacrificed at days 4 or 9 after DSS treatment and the colon of each animal was removed (caecum to rectum), flushed with ice cold PBS, the clinical score evaluated, cut longitudinally and processed for histological and biological analyses. To allow comparable analysis of mice, equivalent colonic regions were analyzed for each assay. For this, the last third portion of the colon (where inflammation takes place) was removed, the upper 0.5 cm distal section was fixed in buffered formalin (used for transversal sections), the lower 1 cm section was snap frozen for biological analyses (RT-qPCR and protein) and the remaining tissue was fixed in formalin for scoring of inflammation (longitudinal scoring). A double-blind evaluation of inflammatory scores was performed by trained pathologists, as previously described.50 Briefly, the severity of inflammation (none, mild, moderate, severe), extent of inflammation (none, mucosa, mucosa and submucosa, transmural), crypt damage (none, basal to 1/3, basal to 2/3, crypt loss, crypts and epithelium loss), and percentage of tissue affected by inflammation (0, 25, 50, 75 and 100%) were scored.

Macroscopic polyp analysis and histopathology Colons (caecum to rectum) were removed from animals, the length measured, flushed with ice cold PBS, and cut longitudinally. A picture of the colon was taken with a Digimax L60 Digital Camera (Samsung) and macroscopic polyps were analyzed under a dissecting microscope (10X magnification). The number and the size of the polyps were measured (taking into account their maximal dimensions) prior to their collection for histological and biochemical analyses. For histopathology, paraffin embedded samples were sectioned (5 μm thick slices), stained with hematoxylin and eosin and evaluated by a trained pathologist. In brief, sections were scored blindly for inflammation, epithelial defects, crypts atrophy, hyperplasia, dysplasia/neoplasia, and areas affected by dysplasia. Hyperplasia, dysplasia and carcinoma were scored based on the severity of the cellular changes induced by the disease. Dysplasia was defined as abnormal crypts displaying misalignment of epithelial nuclei and loss of mucosal secretion. Colon tissue sections with at least two dysplasic lesions were scored 1 in contrast to 0 where no lesion was observed.

Production of organ cultures Organ cultures from control, DSS, and AOM/DSS challenged mice were generated by incubating PBS washed colon sections (1 cm from the distal part of the last 1/3 colon section) with DMEM media containing penicillin/streptomycin in a humid 5% CO 2 incubator at 37°C for 16 hrs. Cell-free supernatants were harvested and assayed by an ELISA for IL1B (IL-1 β), TGFB1 (TGF-β1), (R&D systems), CXCL1 and CXCL2 (PeproTech) according to the manufacturer’s protocol.

Quantitative real time PCR and immunoblotting Total RNA was isolated from colonic tissue using TRI Reagent (Sigma) and 1 µg was reversed transcribed using the High capacity cDNA RT kit (Applied Biosystems). RT-qPCR analysis was performed on a StepOne Real-Time PCR system using SYBR Green PCR Master Mix (Applied Biosystems; Life Technology,), as previously described.51 Relative changes in gene expression were reported as fold changes compared to the untreated colon tissue of each animal model. Protein was purified from phenol-ethanol supernatants obtained after precipitation of RNA, as described by the manufacturer (Sigma Aldrich). Protein extracts were resolved by SDS- PAGE and transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore). After saturation, membranes were incubated overnight with the indicated antibody, washed in Tris-NaCl buffer (TN) supplemented with 0.1% Triton X-100 (TNT) three times and finally with TN buffer. Membranes were then incubated with the secondary anti-rabbit or anti- mouse horseradish peroxidase conjugated antibody. Bound antibodies were revealed using an ECL system (Pierce) and signal quantified with a Pxi Camera (Ozyme, FR). Phospho-STAT3 (#9131), STAT3 (#9139) and BCL2 (Bcl2, #50E3) antibodies were from Cell Signaling, anti- BCL2L1 (Bcl-XS/L , sc-634) and anti-BAX (Bax, sc-526) were from Santa Cruz and the anti- ACTB (Actin, clone AC40) antibody was from Sigma Aldrich.

Immunohistochemical analyses of mouse colon tissues Resected mouse colon tissues were fixed in 10% formalin, paraffin embedded, and further sliced into 3 μm sections. Immunohistological staining for proliferating cell nuclear antigen (PCNA), CD3, FOXP3, EMR1 (F4/80) and LY6G was performed using sections from the mid and distal colon. Antigen retrieval was performed by boiling sections for 10 min in citrate buffer (pH 6.0) and cooling at RT°, followed by blocking of endogenous peroxidase activity with 0.3% H2O2 for 30 min. The sections were blocked with 2.5% horse serum in TBS solution for 30 min in a humid chamber prior to incubation with optimal dilutions of anti-PCNA (Epitomics), anti-cleaved-caspase-3 (Imgenex), anti-CD3 (Abcam), anti-Foxp3-biotin (ebioscience), anti-F4/80 (Abcam), and anti-Ly6G (Abcam) overnight at 4°C. Positive cells were detected using an ImmPRESS HRP anti-rabbit detection Kit or directly with an anti- Streptavidin-Alexa-594 antibody. The immune complexes were visualized using a Peroxidase Substrate DAB kit (Vector) according to the manufacturer’s protocol and slides were counterstained with hematoxylin. For PCNA and cleaved caspase 3 quantification, on average, 200 intestinal epithelial cells from well-formed crypts were scored per mouse (a well-formed crypt was defined as a crypt with mucosal secretion and displaying well-aligned nuclei) and positive cells were expressed as the percentage of total cells. For immune cell quantification at least 400 cells per sample were counted, from 4 randomly selected visual fields per section at 400X magnification using ImageJ software, and positive cells were expressed as the percentage of total cells.

Flow cytometry analysis Splenocytes, peripheral blood mononuclear cells and tumor cells were studied by FACS analysis as described in (16). The following fluorescent labeled antibodies were purchased from BD biosciences: CD45-PE, LY6G-FITC, ITGAM-APC (CD11b-APC) and isotype controls. All flow cytometry was done using a FACS ARIA flow cytometer and FlowJo software.

Supplementary figure legends Figure S1: Characterization of heterozygous P2rx7 +/- mice Cohorts of 5 WT or heterozygous P2rx7 +/- mice were given a colitis regimen as described in the legend to Figure 1 A. Body weight loss was analyzed as described in the supplementary Materials and Methods section. Data are presented as means  SEM; Results were not statistically different. B. Sections were stained with hematoxilin and eosin and analyzed microscopically (scale bar = 100μm). Histological scoring of mid colonic sections was calculated as indicated in the materials and methods section. Data are presented as means  SEM; Results were not statistically different. C. Epithelial cell proliferation was evaluated by PCNA-staining as indicated for Figure 3. Data are presented as means  SEM. The difference between WT and P2rx7 +/- mice was not statistically different.

Figure S2: The A740003 pharmacological inhibitor of P2RX7 dampens colitis A. Cohorts of 5 untreated WT mice or WT mice treated with the selective P2RX7 antagonist A740003 received a colitis regimen (5% DSS, 5 days). The A740003 antagonist was given i.p. during DSS treatment (1 μmole per injection, 1 injection every two days starting with DSS, 4 injections). The experiment was stopped 9 days after the beginning of the treatment. A Kaplan-Meier analysis was performed to analyze mice survival (p≤0.01, Log-rack (Mantel- Cox) test). To assay body weight scoring, mice received a ringer lactate solution (i.p., 100 l per day) from day 5 to 9 to favor recovery of mice. Data are presented as means  SEM; * p≤ 0.05. B. To favor mice recovery, mice received a ringer lactate solution (i.p., 100 l per day) from day 5 to 9. At day 9, mice were euthanized and colons removed for histological analysis (bar = 100 m). The inflammatory index was scored as indicated in the Materials and Methods section.

Figure S3: P2rx7-dependent colonic epithelial cell proliferation A. PCNA staining of colonic sections from non-treated WT and P2rx7-/- mice showed no difference in the proliferative index. B. Expression of P2RX7 in colonic sections of non-treated WT mice. P2RX7 (Chemicon, ref AB5246) staining was performed on colonic sections of WT mice. P2RX7 was expressed by enterocytes (arrow), stromal cells (arrow head) and the muscularis mucosa. C. P2RX7-dependent proliferation of human colon cancer cell lines (T84, ATCC, CCL-248). 1x104 cells were seeded on a 96-wells plate pre-treated or not with oATP (500 M) or A438079 (5 M) for 2 hrs and then stimulated with ATP (3 mM). XTT was added to each well and its incorporation was followed for 4 hrs. ATP alone slightly increases XTT incorporation in T84 cells. In contrast, in the presence of either oATP or the specific P2RX7 antagonist, XTT incorporation was increased by 3- to 5-fold demonstrating that P2RX7 inhibition favored epithelial cell proliferation. D. Cohorts of 5 untreated WT mice or WT mice treated with the selective P2RX7 antagonist A438079 received a colitis regimen (5% DSS, 5 days). The A438079 antagonist was given i.p. during DSS treatment (2 mole per injection, 3 injections per cycle). Nine days after the beginning of the treatment, mice were sacrificed and the colons removed. Quantification of the number of PCNA-positive cells was performed. On average 500 epithelial cells from inflammatory lesions were scored per mouse (Scale bar = 50 μm). Data are presented as means  SEM; ** p ≤ 0.01. We observed that A438079 administration reproduced the effect of genetic deletion of P2rx7 since the proliferation index increased 6-fold. E. Cleaved caspase-3 staining of A438079-treated colonic biopsies from DSS-treated mice reproduced the effect of genetic deletion of P2rx7 since the apoptotic index decreased 2.5- fold.

Figure S4: Scoring of pre-cancerous dysplasic lesions Cohorts of 5 to 6 WT and P2rx7-/- mice were given 3% DSS in the drinking water for 3 days followed by regular tap water for an additional 9 days. The disease activity score, inflammatory index and pre-cancerous lesions were analyzed as described in the materials and methods section. This experiment was repeated twice with similar results. Data are presented as means  SEM; * p≤ 0.05, ** p ≤ 0.01.

Figure S5: Body weight curves of WT, P2rx7-/- and A438079-treated mice on a CAC regimen A. Cohorts of 8 to 10 WT and P2rx7-/- mice were injected i.p. with AOM on day 0 followed by three cycles of DSS given in the drinking water. The weight of the mice was followed during the entire protocol. B. Untreated WT mice and WT mice treated with the selective P2RX7 antagonist A438079 and CAC was induced as described in A. The A438079 antagonist was given i.p. during DSS treatment (2 mole per injection, 3 injections per cycle). The weight of the mice was followed during the entire protocol. Error bars were removed to increase the readability of the figure.

Figure S6: Histological examination of cancerous colonic lesions of WT and P2rx7-/- mice A. Cohorts of 8 to 10 WT and P2rx7-/- mice were injected i.p. with AOM at day 0 followed by three cycles of DSS given in the drinking water. At day 80 after AOM administration, mice were euthanized, colons were resected and colon sections from the mid to distal part were fixed in formalin and stained with hematoxilin and eosin for histological analysis (scale bar = 1 mm). Quantification of the number of PCNA-positive cells was performed. On average 800 epithelial cells from cancerous lesions were scored per mouse (Scale bar = 100 μm). Data are presented as means  SEM; * p≤ 0.001. B. Cohorts of 5 WT or P2rx7 -/- C57Bl/6 mice were injected s.c. in the dorsal right flank with 1x106 Lewis Lung Carcinoma cells (LLC1, ATCC, CRL-1642). After two weeks, mice were euthanized and tumors were removed, sized and weighted. Data are presented as means  SEM; * p≤ 0.05.

Figure S7: The cancerous microenvironment of P2rx7-/- mice is enriched in neutrophils A. Increased expression of CXCL1 and CXCL2 in P2rx7-/- mice. Cohorts of 8 to 10 WT and P2rx7-/- mice were given 3 cycles of DSS and the serum concentration of CXCL-1 and CXCL-2 were determined by ELISA. Data are presented as means  SEM; ** p≤ 0.01. B. Cohorts of 8 to 10 WT and p2rx7-/- mice were injected i.p. with AOM at day 0 followed by three cycles of DSS given in the drinking water. At day 80 after AOM administration mice were euthanized, bones, spleens and colons were removed, and the presence of neutrophils (ITGAM+ (CD11b+) /LY6G+) and macrophages (ITGAM+ (CD11b+) /LY6G-) was evaluated using flow cytometry. C. Cohorts of 8 to 10 WT and P2rx7-/- mice were injected i.v. with AOM at day 0 followed by three DSS-cycles given in the drinking water. At the end of the protocol mice were euthanized, colons were resected and the mid to distal colonic sections were removed and extracted for RNA. The expression levels of mRNA transcripts for Cxcl1, Cxcl2, Ccl2, Ccl5, Arg1 (Arginase 1) and Cxcl10 were examined by RTqPCR. Data are presented as means  SEM; ** p≤ 0.01.