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1 DOI: /ncb3206 Supplementary Figure 1 Autophagy-related gene expression in murine and human intestinal tumors. (a) Representative LC3 immunostaining in colonic adenoma and adjacent non tumoral tissue from Apc +/- mice. (b) Transcript levels of Atg7, Atg5, Beclin1 and Lamp2 mrna were analyzed by qpcr in samples of small intestinal dysplasia (VilCreER T2 Apc flox/flox mice 5 days post tamoxifen induction, Apc -/- mice) and adenomas (VilCreER T2 Apc flox/+ 135 days post induction, Apc +/- ). The abundance was assessed relative to control tissue (tamoxifen injected Apc flox/flox mice). Data were normalized to the abundance of 18s rrna (mean ± SD, n: Controls=3 extracts from 3 control mice, n: Dysplasias=3 extracts from 3 Apc -/- mice, n: Adenomas=3 adenomas from 3 Apc +/- mice, significant differences, *1P=0.0074, *2 P=0.0108, *3P=0.0009, *4P=0.0023, *5P=0.0028, *6P=0.0039, *7P=0.0089, *8P=0.0436, two-tailed unpaired t-test). (c) The expression of Atg7, Atg5, Beclin1 and Lamp2 mrna were analyzed by qpcr in human untreated intestinal tumors and in normal matched tissue. Twenty-six colonic adenomas, 31 non-metastatic colon carcinomas, 39 metastatic colon carcinomas, and 24 liver metastasis were analyzed. The percentage indicate the tumors that showed a higher level of Atg gene expression than normal matched-tissue (>1.5 fold) for at least 3 out of 4 tested genes. Gene expression levels were normalized to the abundance of 18s rrna for each sample. 1

2 Supplementary Figure 2 Immune cell infiltration following IEC-Atg7 deficiency. (a) Transcriptomic analysis of normal duodenum of Apc +/- Atg7 -/- mice compared to Apc +/ mice revealed that among 129 genes that were significantly deregulated following Atg7 deletion, 32 are related to type I IFN signaling response (one way ANOVA, P<0.05). (b) qpcr analyses confirmed that the IFN-mediated response was deregulated in the duodenum and colon from Apc +/- and Apc +/- Atg7 -/- mice. (mean±sd., n: Apc +/- =9 extracts from 9 mice, Apc +/- Atg7 -/- =10 extracts from 10 mice; mice were pooled from 2 independent experiments, significant differences, *1P=1.97x10-05, *2P=0.0002, *3P=0.0007, *4P=0.0005, *5P=0.0005, *6P=0.0376, *7P=0.0003, *8P=0.0005, *9P=0.0008, *10P=0.0091, two-tailed unpaired t-test). (c) Representative immunostainings for CD45 and CD3 showing immune cell infiltration in non tumoral colonic mucosa of Apc +/- Atg7 -/- and Apc +/- mice. (d) Representative CD11c immunostaining showing an increase in CD11c + cells in the lamina propria of the small intestine of Apc +/- Atg7 -/- compared to Apc +/- mice. (e) Percentages of DC subsets gated on CD11c high MHCII high by flow cytometry analyses in the MLN from Apc +/- Atg7 -/- and Apc +/- mice (mean±sd, n: Apc +/- =3 and Apc +/- Atg7 -/- =4 mice, significant differences, *1P=0.0155, *2P=0.0483, *3P=0.0422, two-tailed unpaired t-test). Representative dot plots of DC subsets based on CD103, CD11b expression in MLN from Apc +/- Atg7 -/- and Apc +/- mice. (f) Secretion of IL- 12p70 produced following PMA/Ionomycin stimulation of immune cells extracted from the small intestine lamina propria of Apc +/- Atg7 -/- and Apc +/ mice (mean±sd, n: Apc +/- =3 and Apc +/- Atg7 -/- =3 mice, significant differences, *1P=0.0335, two-tailed unpaired t-test). 2

3 Supplementary Figure 3 Immune gene expression following IEC-Atg7 deletion. (a,b) The expression of genes associated with cytotoxic CD8 + T cells (CD2, CD3g, CD8a, CD8b, IL15, ZAP70, GZMA, GZMb, GZMk, PRF1), T H 1 cells (Stat1, IRF1, IFNg, TBX21, IL12Rb1), T reg cells (Foxp3, CTLA4, TGFb1), and T H 17 cells (Il17Rb, IL23a, ROR C, IRF4, CCL20, CCR6, STAT3) assessed by qpcr in normal colon (a) and duodenum (b) from Apc +/- Atg7 -/- compared to Apc +/- mice. (c) Transcript levels of of CX3CL1, CXCL9, CXCL10 mrna was analyzed by qpcr in the colon and duodenum from Apc +/- mice or Apc +/- Atg7 -/- mice. Gene expression levels were normalized to the abundance of 18s rrna for each sample (mean±sd, (a-c) n: Apc +/- =9 extracts from 9 mice and Apc +/- Atg7 -/- =10 extracts from 10 mice, mice were pooled from three independent experiments, significant differences, (a) *1P=0.0108, *2P=0.0257, *3P=0.0185, *4P=0.0150, *5P=0.0180, *6P=0.0123, *7P=0.0109, *8=0.0200, *9P=0.0006, *10P=5.25x10-08, *11P=2.51x10-07, *12P=0.0006, *13P=0.0134, *14P=0.0009, *15P=0.0154, *16P=0.0195; (b) *1P=0.0366, *2P=0.0472, *3P=0.0413, *4P=0.0313, *5P=0.0006, *6P=0.0156, *7P=0.0055, * 8P=0.0473, *9P=0.0187, *10P=0.0294, *11P=0.0142, *12P=0.0356, *13P=0.0002, *14P=0.0070, *15P=0.0038, *16P=0.0006, *17P=0.0305, *18P= (c) *1P=0.0005, *2P=0.0278, *3P=0.0136, *4P=0.0281, *5P=0.0338, *6P=0.0021, two-tailed unpaired t-test). 3

4 Supplementary Figure 4 Anti-CD8 and anti-cd25 antibody treatments on Apc +/- Atg7 -/- and Apc +/- mice. (a) Protocol of CD8 and CD25 depletion. Apc +/- Atg7 -/- and Apc +/- mice were injected with tamoxifen and treated with anti-cd8, anti-cd25 antibodies or IgG isotype and their diet was supplemented with tamoxifen for 5 days per month. Mice were injected with antibodies or with control IgG twice a week for 90 days. (b) Successful CD8 + T and T reg -cell depletion in Apc +/- Atg7 -/- and Apc +/- mice was monitored by flow cytometry of splenocytes. Percentage of CD8 + IFNg T cells or Foxp3 CD4 + T cells within the CD45 + cell population from mice of the indicated genotype and treatment. (mean±sd, for CD8 + T cells: n: Apc +/- Isotype IgG=3, Apc +/- dcd8= 3, Apc +/- Atg7 -/- Isotype IgG=3, Apc +/- Atg7 -/- dcd8=4 mice; for CD4 + T cells: n: Apc +/- Isotype IgG=3, Apc +/- dcd8=3, Apc +/- Atg7 -/- Isotype IgG=5, Apc +/- Atg7 -/- dcd8=5 mice, significant differences, *1P=0.0144, *2P=0.0066, *3P=0.0261, *4P=0.0076, two-tailed unpaired t-test). 4

5 Supplementary Figure 5 IEC-autophagy deficiency induces microbial dysbiosis. (a) Transcriptomic analysis of normal duodenum of Apc +/- Atg7 -/- mice compared to Apc +/ mice revealed that among the 54 genes that were significantly downregulated following Atg7 deletion, 20 genes encode Paneth cell markers (one way ANOVA, P<0.05). qpcr analyses confirmed that Paneth cell markers are less abundant in the distal small intestine of Apc +/- Atg7 -/-. Gene expression levels were normalized to the abundance of 18s rrna for each sample (mean±sd, n: Apc +/- =9, Apc +/- Atg7 -/- =10 mice, significant differences, *1P=4.15x10-05, *2P=7.61x10-04, *3P=1.39x10-05, two-tailed unpaired t-test). (b) Heatmap of differentially represented bacterial species in feces between Apc +/- and Apc +/- Atg7 -/- mice. Log10-transformation was applied on the relative abundance data matrix. Phyla assignment of the different bacterial species is indicated by a cap letter at the beginning of the species name. F: Firmicutes, B: Bacteroidetes, P: Proteobacteria, A: Actinobacteria. (n=8 extracts of feces from 8 mice per condition, mice were pooled from 2 independent experiments, P values are listed in Supplementary Table 2 (two-tailed unpaired t-test). (c) Diversity of the gut microbiota in Apc +/- and Apc +/- Atg7 -/- mice. Simpson diversity index was calculated to estimate bacterial diversity in both fecal and ileal mucosa samples of Apc +/- and Apc +/- Atg7 -/- mice (mean±sd, for feces, n: Apc +/- =8 extracts from 8 mice, n: Apc +/- Atg7 -/- =8 extracts from 8 mice, for ileum n: Apc +/- =7, n: Apc +/- Atg7 -/- =7, mice were pooled from 2 independent experiments, P=0.015, two-tailed unpaired t-test). (d) Main bacterial genera repartition in both ileal and feces mucosa of Apc +/- and Apc +/- Atg7 -/- mice (n: Apc +/- =8 extracts from 8 mice, n: Apc +/- Atg7 -/- =8 extracts from 8 mice, for ileum n: Apc +/- =7, n: Apc +/- Atg7 -/- =7, mice were pooled from 2 independent experiments, P values are listed in Supplementary Table 3 (twotailed unpaired t-test). (e) Aerobic culture of fecal and colonic microbiota (mean±sd, n: (Gram - ) Apc +/- =3, Apc +/- Atg7 -/- = 4 mice; (Gram + ) Apc +/- =4, Apc +/- Atg7 -/- =4 mice, significant differences, *1P=0.0428, *2P=0.0029, *3P=0.0276, *4P=0.0008, two-tailed unpaired t-test). (f) Protocol of short and long term antibiotic treatments (ATB). 5

6 Supplementary Figure 6 b-catenin signaling in adenoma following IEC-Atg7 deletion. Representative hematoxylin/eosin staining of colonic adenomas from Apc +/- and Apc +/- Atg7 -/- mice. Polyps from Apc +/- Atg7 -/- mice were smaller than those from Apc +/- mice. (b,c) Gene expression levels of several b-catenin target genes (Axin2, c-myc, c-jun, Sox9) assessed by qpcr of adenomas (Ade) from the indicated genotype relative to non-tumoral colon of Apc +/- mice (NT). Gene expression levels were normalized to the abundance of 18s rrna for each sample (mean ± SD., n: NT Apc +/- =8 extracts from 8 mice ; Ade Apc +/- =8 tumors from 8 mice and Ade Apc +/- Atg7 -/- =8 tumors from 8 mice, mice were pooled from two independent, significant differences, (b) colon, *1P=0.0038, *2P=0.0042, *3P=0.0032, *4P=0.0025, *5P=0.0047, *6P=0.0485, *7P=0.0466, *8P=0.0472, (c) duodenum *1P=0.0091, *2P=0.0192, *3P=0.0086, *4P=0.0082, *5P=0.0037, *6P=0.0008, *7P=0.0384, *8 P = two-tailed unpaired t-test). 6

7 Supplementary Figure 7 Effect of metformin treatment on intestinal adenomas from Apc +/- mice. (a) Collected ultrasound measurements of colonic tumor volumes from Apc +/ mice and those from metformin-treated- Apc +/- mice. Box plots show the 5-95 percentiles which are delineated by the upper and the lower limits of the box and the median is shown by the horizontal line inside the box. n: Apc +/- =10, Apc +/- Met=32 tumors pooled from 3 mice. P(genotype)<0,0001 and P(Time)<0,0001 (two-way ANOVA test). (b) Western blotting for phosphorylated-ampk (pampk), AMPK, phosphorylated-raptor (praptor), Raptor, phosphorylated-p70s6 kinase (pp70s6k), p70s6 kinase (p70s6k), phosphorylated-s6 ribosomal protein (ps6) and S6 ribosomal protein (S6) in adenomas from Apc +/ mice and those from metformin-treated-apc +/- mice. g-tubulin served as a loading control. Each lane represents a sample from a different animal. Unprocessed original scans of blots are shown in Supplementary Fig

8 Supplementary Figure 8 Model of the differential effects of IEC-Atg7 deletion on the initiation and progression of intestinal tumorigenesis driven by Apcloss. IEC-autophagy blockade by Atg7 deletion alters Paneth cell numbers and leads to abnormal mucin accumulation in goblet cells. This host defense alteration is accompanied by an increase in intestinal permeability and contributes to a shift in the composition of the gut microbiota characterized by an outgrowth of Firmicutes and a decrease in Proteobacteria. Remodeling of the microbiota architecture and composition is associated with an increased abundance of CD103 + CD11b - within the mesenteric lymph node reported to prime Th1 and CD8 T cell through IL-1b and IL-12 secretion. Infiltration of cytotoxic CD8 + T cells in the lamina propria has a major impact on the elimination of transformed cells induced by Apc loss. However, this antitumoral response is incomplete as few cancer cells escape and persist in Apc +/- Atg7 -/- mice. At this stage, cell cycle arrest associated with p53 accumulation, AMPK signaling activation and low abundance of glycolytic enzymes lead to a drastic decrease in Atg7-deficient tumor cell growth. 8

9 Supplementary Figure 9 Unprocessed original scans of Western blots presented in the indicated Figures and Supplementary Figures. 9

10 Supplementary Table legends Supplementary Table 1 Transcriptomic analysis of normal duodenum of Apc +/- Atg7 -/- mice compared to Apc +/ mice. 129 genes were selected on the basis of passing a relative significant 1.5 fold increase or decrease cut-off (54 genes < 1.5 fold and 75 genes > 1.5 fold; one way ANOVA, P<0.05) Supplementary Table 2 Differentially represented species in feces of Apc +/- Atg7 -/ mice compared to Apc +/- mice. The gut microbial communities within feces of Apc +/- and Apc +/- Atg7 -/- mice were assessed by high throughput sequencing approach. Out of 11,273 OTUs, linear discriminant analysis effect size analysis highlighted a total of 49 bacterial species differentially represented in feces between Apc +/- Atg7 -/- and Apc +/- mice (P values are indicated, two-tailed unpaired t-test). Supplementary Table 3 Main bacterial genera repartition in both ileal and feces mucosa of Apc +/- and Apc +/- Atg7 -/- mice revealed by linear discriminant analysis effect size (LefSe) analysis (P values are indicated, two-tailed unpaired t-test). Supplementary Table 4 Primers used for qpcr assays in this study. 10