Journal: bioRxiv

Article Title: Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

doi: 10.1101/2020.01.25.919795

Figure Lengend Snippet: Characterization of IL-11 + Cells in CAC Using Il11-Egfp Reporter Mice. a Protocol for induction of AOM/DSS-induced CAC in mice. Il11-Egfp reporter mice were intraperitoneally injected with AOM on day 0, followed by repeated DSS administration. Colorectal cancer gradually develops on ∼30 days after AOM injection. Unless otherwise indicated, the following experiments used tumor and nontumor tissues collected on day 98–105 after AOM/DSS treatment. b Representative image of tumor and adjacent nontumor tissues in the colon of mice on day 77 after AOM injection. Colonic sections were stained with hematoxylin & eosin (H&E). Scale bar, 200 μm. c Prepared mRNA samples from tumor and nontumor tissues were analyzed by qPCR to determine expression of the indicated genes. Results are mean ± SE (n = 6–10 mice). d, e From colonic tumor tissues of wild-type or Il11-Egfp reporter mice, single-cell suspensions were prepared, and percentages of IL-11 + cells were determined. Representative flow cytometry images are shown ( d ). Results are mean ± SE (n = 7 mice). Cells were stained with the indicated antibodies, and expression on EGFP + cells was analyzed by flow cytometry ( e ). Results are representative of four independent experiments. f Nontumor and tumor tissue sections were stained with anti-GFP antibody. Scale bars, 100 μm. g, h Tumor sections were stained with the indicated antibodies (red) and with anti-GFP antibody (green) (n = 3 mice). Right panels show enlarged images of white boxes from left panels. White arrowheads indicate merged cells. Scale bars, 100 μm, unless otherwise indicated. Statistical significance was determined by two-tailed unpaired Student’s t -test ( c, d ). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Total RNAs were extracted from the indicated tissues of mice by using TRI Reagent (Molecular Research Center) or Sepasol II Super (Nacalai Tesque), and cDNAs were synthesized with the RevertraAce qPCR RT Kit (Toyobo).

Techniques: Injection, Staining, Expressing, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

doi: 10.1101/2020.01.25.919795

Figure Lengend Snippet: IL-11 + Cells Appear in Tumor Tissues in the Absence of Inflammation. a Elevated Il11 and Egfp expression in colonic tumors from Apc min/+ ;Il11-Egfp reporter mice. Experiments were performed using tumor and nontumor tissues from the colon of 20- to 24-week-old Apc min/+ ;Il11-Egfp reporter mice. Il11 and Egfp mRNA expression was determined by qPCR. Results are mean ± SE (n = 5 mice). b Colonic tissue sections from Apc min/+ ; Il11-Egfp reporter mice were stained with H&E (upper panels) or anti-GFP antibody (lower panels). Middle and right panels, respectively, show enlargements of the black and red boxes from the left panels. Red arrowheads indicate tumor cells. Scale bar, 100 μm. c, d IL-11 + cells express stromal cell markers and, to a lesser extent, hematopoietic or epithelial cell markers. Colonic cells were prepared from tumors of Apc min/+ ; Il11-Egfp reporter mice, stained with the indicated antibodies, and analyzed by flow cytometry. Percentages of IL-11 + cells were determined ( c ). Results are mean ± SE (n = 3 mice). Representative histograms show expressions of the indicated markers on EGFP + cells ( d ). Results are representative of three independent experiments. e–g Characterization of IL-11 + cells by IHC. Colonic tumor sections from Apc min/+ ; Il11-Egfp reporter mice were stained with the indicated antibodies (red) and anti-GFP antibody (green). White arrowheads indicate merged cells. Scale bar, 100 μm, unless otherwise indicated. Statistical significance was determined by two-tailed unpaired Student’s t -test ( a, c ). **p < 0.01; ***p < 0.001.

Article Snippet: Total RNAs were extracted from the indicated tissues of mice by using TRI Reagent (Molecular Research Center) or Sepasol II Super (Nacalai Tesque), and cDNAs were synthesized with the RevertraAce qPCR RT Kit (Toyobo).

Techniques: Expressing, Staining, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

doi: 10.1101/2020.01.25.919795

Figure Lengend Snippet: IL-11 + Cells Appear in the Colon of DSS-Treated Mice and Express Stromal Cell Marker. a Il11-Egfp reporter mice were treated with 1.5% DSS in drinking water for 5 days, followed by a change to regular water. On day 7 after DSS treatment, Il11 and Egfp mRNA expression in the colon was determined by qPCR. Results are mean ± SE (n = 9 mice). b, c Appearance of IL-11 + cells in submucosal tissues of the colon of Il11-Egfp reporter mice on post-DSS treatment day 5 (b) or day 1 ( c ). Colonic tissue sections from untreated or DSS-treated Il11-Egfp reporter mice were H&E stained or immunostained with anti-GFP antibody. Right panels show enlargements of the boxes (b). Scale bar, 100 μm. d, e Characterization of cell surface markers on IL-11 + cells. Colonic cells were prepared from the colon of Il11-Egfp reporter mice as in ( b ). We determined the percentages of EGFP + (IL-11 + ) cells from the colon before and after DSS treatment ( d ). Cells were stained with the indicated antibodies, and marker expressions were analyzed on GFP-positive cells ( e ). Results are representative of three independent experiments. f Representative immunostaining of IL-11 + cells. Colonic tissue sections were prepared from Il11-Egfp reporter mice as in ( b ), and immunostained with the indicated antibodies and anti-GFP antibody. Results are merged images. Right panels are enlarged images from the boxes (n = 3–4 mice). White arrowheads indicate merged cells. g IL-11 + cells do not proliferate in situ . Il11-Egfp reporter mice were treated with DSS as in ( a ), and intraperitoneally administered BrdU (40 mg/kg) on day 6. On day 7, colonic sections were prepared and stained with anti-GFP and anti-BrdU antibodies. Results are representative images from three independent experiments. Scale bars, 100 μm, unless otherwise indicated. Statistical significance was determined by two-tailed unpaired Student’s t -test ( a ). *p < 0.05.

Article Snippet: Total RNAs were extracted from the indicated tissues of mice by using TRI Reagent (Molecular Research Center) or Sepasol II Super (Nacalai Tesque), and cDNAs were synthesized with the RevertraAce qPCR RT Kit (Toyobo).

Techniques: Marker, Expressing, Staining, Immunostaining, In Situ, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

doi: 10.1101/2020.01.25.919795

Figure Lengend Snippet: IL-11 + Cells Express Genes Associated with Cell Proliferation and Tissue Repair. On day 7 after DSS treatment, cells were isolated from the colon of Il11-Egfp reporter mice, and IL-11 + cells (EGFP + ) were sorted by flow cytometry. We isolated mRNA from IL-11 − and IL-11 + cells, and analyzed the gene expression by microarray analysis (n = 3). a Heat map of microarray gene expression of IL-11 − and IL-11 + cells. Legend on right shows gene-expression color normalized by Z-score transformation. b Volcano plot of whole genes. Horizontal line indicates genes differentially regulated in IL-11 + cells compared to IL-11 − cells, shown in log2. Vertical line indicates p values of statistical significance, shown in −log10. Significantly upregulated and downregulated genes are indicated by red and blue dots, respectively. Several upregulated genes are plotted. c Gene Ontology (GO) terms that were significantly enriched in IL-11 + cells compared to IL-11 − cells. d, e Gene expressions were analyzed by qPCR. Results are mean ± SE (n = 4 mice). Statistical significance was determined by two-tailed unpaired Student’s t -test ( c, d ). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Article Snippet: Total RNAs were extracted from the indicated tissues of mice by using TRI Reagent (Molecular Research Center) or Sepasol II Super (Nacalai Tesque), and cDNAs were synthesized with the RevertraAce qPCR RT Kit (Toyobo).

Techniques: Isolation, Flow Cytometry, Gene Expression, Microarray, Transformation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

doi: 10.1101/2020.01.25.919795

Figure Lengend Snippet: The MEK/ERK Pathway is Involved in Il11 Upregulation in Tumor Tissues. a, b Wild-type mice were treated with DSS with or without NAC ( a ) or Abx ( b ). Colonic cells were prepared and stained with CellRox-green, and ROS accumulation was analyzed by flow cytometry. Left panels show representative histograms of ROS levels in colonic cells. Right panels show percentages of CellRox-green-positive cells of an individual mouse. Results are mean ± SE (n = 3–4 mice). c Wild-type mice were treated as in A. Colonic expressions of 16S rRNA, Hmox1, and Il11 mRNA were determined by qPCR. Results are mean ± SE (n = 11). d Abx blocked Il11 mRNA upregulation in the colon of DSS-treated mice. Wild-type mice were untreated or treated with DSS in the absence or presence of Abx. On day 7 after DSS treatment, qPCR was performed to determine the expression of bacterial 16S rRNA and Il11 mRNA in the colon. Results are mean ± SE (n = 10). e, f Trametinib inhibits ERK phosphorylation and Il11 mRNA expression in the colon of DSS-treated mice. Wild-type mice were treated with DSS in the absence or presence of trametinib, and then colonic sections were prepared and stained with anti-pERK antibody ( e ). Scale bars, 100 μm. Il11 mRNA expression was determined by qPCR ( f ). Results are mean ± SE (n = 13–14). g NAC inhibits ERK phosphorylation in the colon of DSS-treated mice. Wild-type mice were treated with DSS in the absence or presence of NAC in the drinking water for 5 days. Colonic sections were stained with anti-pERK antibody. Results are representative of three independent experiments. Scale bar, 100 μm. h Tgfbs expression in the colon of DSS-treated wild-type mice. Wild-type mice were treated with DSS as in . On day 7 after DSS treatment, qPCR was performed to determine Tgfb1, Tgfb2 , and Tgfb3 mRNA expression in the colon. Results are mean ± SE (n = 8–10 mice). i Treatment with anti-TGFβ antibody does not downregulate Il11 mRNA expression in the colon of DSS-treated mice. Mice were intraperitoneally administered 100 μg anti-TGFβ antibody on days 2 and 4 after DSS administration, and sacrificed on day 5. Il11 mRNA expression was determined by qPCR. Results are mean ± SE (n = 11–16 mice). j Schema of administration of various inhibitors in AOM/DSS-treated mice. Mice were treated with Abx for 8 weeks, NAC for 4 weeks, trametinib at −6 and −30 hours, or anti-TGFβ antibody on day −1, −3, −5 (just before sacrifice). k–m, o Mice were treated as in , and then treated with Abx ( k ) (n = 7–9 mice), NAC ( l ) (n = 7–11 mice), trametinib ( m ) (n = 10–12 mice), or anti-TGFβ antibody (n = 6–7 mice) ( o ) as in ( j ). On day 98–105 after AOM injection, mRNA was extracted from tumor and non-tumor tissues, and Il11 expression was determined by qPCR. Results are mean ± SE. n Mice were treated as in . Tgfb1 , Tgfb2, and Tgfb3 expressions in tumor and nontumor tissues were determined by qPCR. Results are mean ± SE (n = 7 mice). Statistical significance was determined using the unpaired two-tailed Student’s t -test ( c, h ), Mann-Whitney U test ( f, i ), two-way ANOVA with Bonferroni’s test ( d, k, l, m, o ), or one-way ANOVA with Tukey’s post-hoc test ( a, b ). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Article Snippet: Total RNAs were extracted from the indicated tissues of mice by using TRI Reagent (Molecular Research Center) or Sepasol II Super (Nacalai Tesque), and cDNAs were synthesized with the RevertraAce qPCR RT Kit (Toyobo).

Techniques: Staining, Flow Cytometry, Expressing, Phospho-proteomics, Injection, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

doi: 10.1101/2020.01.25.919795

Figure Lengend Snippet: IL-11 Preferentially Induces Signals to Fibroblasts. a Relative expressions of Il11ra1 , Il22ra1 , and Il6st in colonic epithelial organoids and colonic fibroblasts. Colonic epithelial organoids and fibroblasts were established from wild-type mice as described in the methods. Expressions of the indicated genes were determined by qPCR. Results are mean ± sd of triplicate samples and representative of two independent experiments. b Colonic epithelial organoids and fibroblasts were unstimulated or stimulated for 30 min with IL-11 (10 or 100 ng/mL) or IL-22 (10 or 100 ng/mL). Total STAT3 and phosphorylated STAT3 (pSTAT3) were analyzed by Western blotting. STAT3 and pSTAT3 signaling intensities were calculated by Fiji, and the relative ratio of pSTAT3/STAT3 is shown. Results are representative of two independent experiments. c Administration of IL-11R agonist induces expression of the genes expressed in IL-11 + cells. We injected 8-week-old wild-type mice with 10 μg IL-11R agonist. At 3 hours after injection, mRNA was isolated from the colon, and gene expressions were analyzed by microarray analysis (n = 2 for untreated samples; n = 3 for injected samples). Heat map of microarray gene expression in the colon of untreated and treated mice ( c ). Legend on the right shows gene-expression color normalized by Z-score transformation. d Volcano plot of whole genes. Horizontal line indicates differentially regulated genes in the colon after IL-11R agonist injection compared to before injection, shown in log2. Vertical line indicates p values of statistical significance, shown in −log10. Significantly upregulated genes are indicated by red dots. Several upregulated genes are plotted. e Venn diagram of genes elevated in IL-11 + IAFs compared to IL-11 − cells, and genes elevated in the colon with IL-11R agonist treatment compared to untreated colon. Overlapping area includes 18 genes. Statistical significance was determined by two-tailed unpaired Student’s t -test ( a ). *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Total RNAs were extracted from the indicated tissues of mice by using TRI Reagent (Molecular Research Center) or Sepasol II Super (Nacalai Tesque), and cDNAs were synthesized with the RevertraAce qPCR RT Kit (Toyobo).

Techniques: Western Blot, Expressing, Injection, Isolation, Microarray, Gene Expression, Transformation Assay, Two Tailed Test