Chemotherapy-induced ileal crypt apoptosis and the ileal microbiome shape immunosurveillance and prognosis of proximal colon cancer

Abstract

The prognosis of colon cancer (CC) is dictated by tumor-infiltrating lymphocytes, including follicular helper T (TFH) cells and the efficacy of chemotherapy-induced immune responses. It remains unclear whether gut microbes contribute to the elicitation of TFH cell-driven responses. Here, we show that the ileal microbiota dictates tolerogenic versus immunogenic cell death of ileal intestinal epithelial cells (IECs) and the accumulation of TFH cells in patients with CC and mice. Suppression of IEC apoptosis led to compromised chemotherapy-induced immunosurveillance against CC in mice. Protective immune responses against CC were associated with residence of Bacteroides fragilis and Erysipelotrichaceae in the ileum. In the presence of these commensals, apoptotic ileal IECs elicited PD-1+ TFH cells in an interleukin-1R1- and interleukin-12-dependent manner. The ileal microbiome governed the efficacy of chemotherapy and PD-1 blockade in CC independently of microsatellite instability. These findings demonstrate that immunogenic ileal apoptosis contributes to the prognosis of chemotherapy-treated CC.

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Fig. 1: Ileal apoptosis and microbiome dictate prognosis in stage IV pCC.
Fig. 2: Impact of the microbiome in the efficacy of OXA in MC38 tumor bearers.
Fig. 3: Protective role of intestinal caspase-3 and caspase-7 in the cell death of ileal IECs against CC.
Fig. 4: Mandatory role of TFH cells in the immunogenicity of dying ileal IECs.
Fig. 5: Role of Batf3 DC, IL-1R and IL-12 in the elicitation of TFH cells during ileal cell death.
Fig. 6: Immunogenic ileal commensals boost the anticancer effects of immunotherapies against CC.

Data availability

All raw sequencing data and de-aggregated and de-identified patient and mouse metadata can be found at the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject or https://www.ncbi.nlm.nih.gov/sra/) under accession number PRJNA478491 (Ileal Apoptosis and Microbiome Shape Immunosurveillance and Prognosis of Proximal Colon Cancer).

All BioSample (PATIENTS_Metadata.csv, MICE_Metadata.csv) and Run Metadata files (PATIENTS_Run.csv, MICE_Run.csv) are also provided in Supplementary Dataset 1 to ease metadata availability.

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Acknowledgements

We thank the animal facility team at Gustave Roussy. We thank technicians and pathologists from Centre GF Leclerc: L. Guyard, L. Arnould and S. Ladoire. We thank S. Brutin, A. Paci, M. Vétizou, T. Yamazaki, J.-E. Fahrner and A. G. Goubet for technical assistance. We thank D. Goere, R. Bonnet, P. Sauvanet, D. Pezet, J. Gagnière, F. Pagani and A. Martinetti for helping with human samples collection. We thank M. Merad, for scientific advice. We thank R. Förster (Institut für Immunologie) and J. A. Harker (Imperial College) for providing knockout models. L.Z. and G.K. were supported by the Ligue Contre le Cancer (Equipe Labelisée); Agence Nationale de la Recherche (ANR) Francogermanique ANR-19-CE15-0029, ANR Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la Recherche sur le Cancer; BMS Foundation, Cancéropôle Ile-de-France; Chancelerie des Universités de Paris (Legs Poix), Fondation pour la Recherche Médicale; a donation by Elior; the European Commission (ArtForce); the European Research Council; Fondation Carrefour; Institut National du Cancer; Inserm; Institut Universitaire de France; LeDucq Foundation; the LabEx Immuno-Oncology; the RHU Torino Lumière (ANR-16-RHUS-0008); H2020 ONCOBIOME, the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination; the SIRIC Cancer Research and Personalized Medicine (CARPEM); FHU CARE, Dassault and Badinter Philantropia and the Paris Alliance of Cancer Research Institutes. D.D. was supported by the German Research Foundation (CRC1181-A7). B. Ryffel was supported by Centre National de la Recherche Scientifique, the University of Orleans, the Conseil Général du Loiret and European Regional Development Fund (FEDER No. 2016-00110366 and EX005756). M.C. was supported by ITMO Cancer AVIESAN (Alliance Nationale pour les Sciences de la Vie et de la Santé, National Alliance for Life Sciences & Health) within the framework of the Cancer Plan (HTE201601).

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Contributions

The authors responsible for conceptualization were M.P.R. and L.Z. The authors responsible for performing experiments and data collection were M.P.R., S.Y., C.P.M.D., M.P., G.F., M.T.A., C. Rauber, C.H.K.L., B. Routy, S. Becharef, P. Ly, E.P., C.F., R.D., A.F., A.V., S.K., P.O., F.M. and P.D. The authors responsible for formal data analysis were C. Richard, L.D., C.K., P. Lepage and V.I. The author responsible for data curation was V.I. The authors responsible for reagents, animal models and clinical sample resources were D.D., I.V.S., M.C., S. Benoist, J.-Y.S., A.H., D.M., F. Pietrantonio, F. Pagès, I.G.B., A.E., D.R., F.G., B. Ryffel and P.V. The author responsible for methodology and creation of models was T.V.B. The authors responsible for writing of the original draft were M.P.R. and L.Z. The authors responsible for writing (review and editing) were M.P.R., G.K. and L.Z. The author responsible for visualization was M.P.R. The authors responsible for supervision were G.K. and L.Z. The authors responsible for funding acquisition were G.K. and L.Z.

Corresponding author

Correspondence to Laurence Zitvogel.

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Competing interests

L.Z. and G.K. are cofounders of EverImmune, a biotech company devoted to the use of commensal bacteria for the treatment of cancers.

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Peer review information Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Description of sample origin and pCC cohorts.

a, Schematic view of the anatomic distribution of the specimen utilized in the clinical study. Ileal and colonic materials and sampling from patients that have been utilized for various analyses are described. b-c, Enumeration of samples for each type of experiments or analyses in both cohorts.

Extended Data Fig. 2 Ileal apoptosis and microbiome dictate prognosis in stage IV proximal colon cancer.

a-b, Automated quantification of cleaved in immunohistochemical staining of paired ilea (a) and colons (b) in different tissue compartments (lamina propria, villus, crypts) of pCC patients from the discovery cohort (Supplementary Table 1), with preoperative chemotherapy or untreated prior to surgery. Non-previous chemotherapy ileum n = 35, colon n = 30; previous chemotherapy ileum n = 12, colon n = 12.Each dot represents one pCC patient, box plot center lines correspond to the median value; lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles); and lower and upper whiskers extend from the box to 5 and 95 percentiles, respectively. Two-tailed Mann Whitney U test p-value is shown. c, Quantification of T lymphocytes in tumor beds (according to the immunoscore methodology, CD3 and CD8, in IM (invasive margin) and CT (core of tumor)) in the discovery cohort of pCC patients, with preoperative chemotherapy or untreated prior to surgery. Non-previous chemotherapy: CD3IM n = 59, CD3-CT n = 59, CD8-IM = 60, CD8-CT = 60; previous chemotherapy CD3-IM n = 16, CD3-CT n = 15, CD8-IM = 16, CD8-CT = 16. Each dot represents one pCC patient, Box plot center lines correspond to the median value; lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles); and lower and upper whiskers extend from the box to 5 and 95 percentiles, respectively. Two-tailed Mann Whitney U test p-value is shown. d, Quantification of TFH in the lamina propria of pCC patients with available data from discovery cohort. Non-previous chemotherapy n = 35; previous chemotherapy n = 12. Each dot represents one pCC patient, boxes and whiskers depict medians, first and third quartiles and ± 595 percentiles, respectively. Two-tailed Mann Whitney U test p-value is shown. e, Heatmap of correlations between immunoscore components shown in (c) and TFH (CD4+Bcl6+) for n = 40 patients with available data in the discovery cohort. Spearman’s rank coefficients of correlation (rs) and two-tailed p-values<0.05 (*) are depicted. f, Correlation between TFH densities in ileum and tumor paired samples from pCC patients of the discovery cohort. Each dot represents one pCC patient, n = 44. The continuous and dotted lines show the regression line and 95% of confidence intervals, respectively. Spearman’s rank coefficient of correlation (rs) and two-tailed p-value are shown. g, Correlation between the number of ileum crypt cells positive for cleaved caspase 3 stainings and TFH cell density in tumors from stage IV patients from the discovery and validation cohort, n = 31. The continuous and dotted lines show the regression line and 95% of confidence intervals, respectively. Spearman’s rank coefficient of correlation (rs) and two-tailed p-value are shown.

Extended Data Fig. 3 Impact of the microbiome in the efficacy of OXA in MC38 tumor bearers.

a, Experimental setting (a) for Fig. 2a and S2b. ATB were administered for 2 weeks prior to s.c. injection of MC38. OXA was administered on day 7 without ATB interruption. b, Lipocalin-2 (LCN2) levels in a kinetics study monitored by ELISA in stools of tumor bearing mice treated with OXA (or PBS) i.p. ± ATB treatment. Blue dotted lines indicate day of tumor s.c. injection and orange dotted lines indicate day of OXA/PBS treatments. n = 5 mice/group. c, Representative micrographs of immunohistochemical staining for Ki67 in ileal and colonic mucosae 3 days after i.p. OXA (or PBS) treatment in WT mice. One experiment. d, Quantification of Ki67+ cells in ileal (PBS n = 5, OXA n = 5) and colonic (PBS n = 14, OXA n = 15) crypts and according to cellular compartment. Each dot represents one sample, mean ± SEM is depicted. e, Experimental setting for Fig. 2d. FMT was performed in germ-free mice 2 weeks prior to s.c. injection of MC38. OXA (or PBS) is administered i.p. on day 7. Mice are housed in isolators throughout the experiment. f, The PLS-DA / VIP method was used to compare abundances of all bacterial species between aNR (n = 35) and aR (n = 72) subgroups. Species with differential abundance between both groups were used as input for the Partial Least Square Discriminant Analysis to calculate the variable importance (VIP score > 1). Two-stages Benjamini-Hochberg False Detection Rate (FDR) at 10% was applied on normalized and standardized relative abundances before PLS-DA / VIP analysis. g, Representative flow cytometry plots of TFH cells in tdLN 21 days post PBS or OXA treatment in tumor bearers in aR versus aNR. Staining for PD-1, and CXCR5 in CD4+ T cells gated on the viable CD3+CD45+ population. CXCR5hiPD-1hi cells within live CD4+ T cells from one representative aNR and one aR mouse treated with OXA are shown. In total, 4 aNR and 4 aR groups were evaluated; concatenated data of all individual samples is shown in Fig. 2g. h, FACS determination of CCR6+CXCR3-/CD4+ T cells gated in the viable CD3+CD45+ population in tumor tdLN in aR and aNR mice with or without OXA treatment on day 21 at sacrifice. Concatenated data from 12 FMT patients. Each dot represents one mouse, mean ± SEM is depicted. aNR PBS n = 19, aNR OXA n = 20; aR PBS n = 22, aR OXA n = 30.Refers to Fig. 2f. Statistics: Mann-Whitney U test used to compare two independent groups (after Kruskal-Wallis was implemented for multiple groups) (d, f, h). Significant two-tailed p-values are shown in the figures.

Extended Data Fig. 4 Protective role of intestinal caspases -3 and -7 in the cell death of ileal IEC against colon cancer.

a-b, Vaccination of naïve C57BL/6 J (a) or BALB/c (b) mice using ileal or colonic IEC which were exposed to OXA to protect against syngeneic transplantable colon cancers (such as MC38 or CT26, respectively (Fig. 3a-b)) or irrelevant syngeneic tumors (such as MCA205 (a) and 4T1 (b), respectively. Tumor growth curves showing one representative experiment out of 3 yielding similar results. MCA205 n = 5/group. 4T1 naïve n = 6, ileum-OXA n = 6, colon OXA n = 5. Mean ± SEM is depicted. c-d, Dying ileal IEC were harvested at 6 h post-OXA i.p. and separated by a FACS cell sorting based on Annexin V/7AAD expression or vaccination with IEC subjected to three freeze-thaw cycles (F/T) before being inoculated as a vaccine in naive mice as described in Fig. 3a. A representative flow cytometric analysis is shown (c) and tumor growth kinetics of MC38 in recipients vaccinated with the four cell subsets are depicted (d). Naïve n = 6, IEC annV-7AAD- n = 5, IEC annV+7AAD- n = 5, IECAnnV+7AAD + n = 6, IEC F/T n = 10. Mean ± SEM is depicted. e, Vaccination as in Fig. 3a but using an IEC cell suspension enriched in villi versus crypt cells. Naïve n = 5, Villi IEC OXA n = 10, Crypt IEC OXA n = 10. Mean ± SEM is depicted. f, Representative FACS dot plot of the IEC fraction from Lgr5EGFP-IRES-creERT2 mice used for lineage-tracing of Lgr5-expressing stem cells of the small intestine. One representative sorting data out of two performed. g, Id. as in Fig. 3a but vaccination has been performed using ileal IEC composed of either LGR5+ (purity > 95%) or LGR5-. Tumor size at day 21 in 2 pooled experiments is shown, each dot representing one mouse. Naïve n = 13, LGR5+ n = 12, LGR5- n = 11. Means ± SEM are represented. Statistics: mixed-effect modeling with a specific software (Methods) for longitudinal tumor growth analysis (a, b, d, e) and Mann-Whitney U test used to compare two independent groups (after Kruskal-Wallis was implemented for multiple groups) (g). Significant two-tailed p-values are shown in the figures.

Extended Data Fig. 5 Immunogenic cell death of ileal IEC.

a, Dying (OXA-exposed) ileal IEC were administered two weeks apart as in Fig. 3a. in WT C57BL/6 mice cotreated with CD4 or CD8 T cell depleting Abs or their isotype control Abs. CD4 and CD8 levels in peripheral blood were monitored and after their full recovery to baseline levels, mice were challenged with the MTD of MC38. Tumor sizes at sacrifice are depicted in a representative experiment. Naïve n = 5, Isotypes n = 6, αCD4 n = 6, αCD8 n = 6. b, MC38 tumor sizes at sacrifice in mice vaccinated with OXA-exposed ileal IEC harvested in mice harboring different genotypes WT (PBS n = 6, OXA n = 24) versus Cd39/Entpd1-/- (n = 16), Tlr9-/- (n = 10), Tlr2/4-/- (n = 20), and Il1a/b-/- (n = 6) and Il18-/- (n = 16). Pooled data from 3 independent experiments, mean ± SEM is depicted. c, Ileal IECs from OXA-treated mice were injected into wild type C57BL/6 J mice, along with the ATP-depleting agent dinitrophenol (DNP) and the purinergic P2Y2 receptor antagonist pyridoxalphosphate-6-azophenyl-2’,4’-disulphonic acid (PPADS) or neutralizing antibodies anti-HMGB1 or calreticulin before to challenging of recipient mice with the MTD of MC38. Tumor sizes at sacrifice are indicated. A representative experiment out of two is shown, comprising n = 6 mice/group, mean ± SEM is depicted. Statistics: Mann-Whitney U test used to compare two independent groups (after Kruskal-Wallis was implemented for multiple groups). Significant two-tailed p-values compared to vaccination controls are shown in the figures.

Extended Data Fig. 6 Role of the ileal microbiome in the adjuvanticity of ileal cell death.

a, Tumor growth kinetics of MC38 after vaccination with OXA-exposed ileal stem cell -derived enteroids to immunize WT hosts. Naïve n = 5, organoid-PBS n = 6, organoid-OXA n = 5. Mean ± SEM is depicted. b, Experimental setting aimed at reverting tolerance into immunogenicity using ileal organoids: Ileal stem cell derived organoids from WT C57BL/6 J mice were or were not exposed to one of ten pCC patients’ mucosal ileal microbiota and OXA ex vivo prior to s.c. injection to immunize naive mice against MC38. Details of each donor patient in Supplementary Table 5. Controls included organoids exposed to OXA alone, or ileal microbiota alone (without organoids. c, Percentages of tumor size reduction between immunized and naive mice at day 21 segregating responders (R) from non-responders (NR) based on statistical analyses on tumor growth kinetics. Sample sizes from left to right n = 5, 5, 5, 6, 6, 6, 6, 6, 4, 5. Mean ± SEM is depicted. d, Comparison between R (n = 6) and NR (n = 4) at family taxonomic rank level. Relative abundance of Prevotellaceae family. Box plot center lines correspond to the median value; lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). e, Linear discriminant analysis (LDA) coupled with the effect size measurements to represent species differentially present among R (n = 6) and NR (n = 4). LEfSe plots were generated with Python 2.7 and all species with LDA score ≥ 2 are shown. p-values show significant differences in pair-wise analyses between NR and R. f, Culturomics-based determination of the bacterial species from ileal mucosa microbiota. Significant differences in detection of distinct bacterial species between R and NR performed in the 10 PCC patients. Chi-square test p-values are shown. g-h, Flow cytometric analyses of migratory dendritic cells, cDC1 and cDC2 in lamina propria of ilea or colons (g, n = 6/group) or mLN (h, n = 5/group) in mice treated with OXA + /- ATB. One representative experiment out of two yielding similar results. Mean ± SEM is depicted. i, Heatmap representation of RT-PCR based- relative ratios of immune gene transcripts in mLN at 24 h post-OXA i.p. vs PBS with or without ATB. Water groups n = 11, ATB groups n = 5. j, Humoral immune responses monitored by ELISA (IgG) after 4 weeks of vaccination using OXA- exposed ileal organoids compensated or not with rIL-1β (n = 6/group, one representative experiment out of 2 yielding similar results). Two-sided Wilcoxon signed-rank test. k, Il12b gene product in WT versus Batf3 KO mice. WT groups n = 3, Batf3−/- groups n = 4. Mean ± SEM is depicted. Statistics: unless otherwise specified, mixed-effect modeling with a specific software (Methods) for longitudinal tumor growth analysis (a) and Mann-Whitney U test used to compare two independent groups (after Kruskal-Wallis was implemented for multiple groups) (d, e, g, h, k). Significant two-tailed p-values are shown in the figures; for (i): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Extended Data Fig. 7 Immunogenic ileal commensals induce DC IL-12 and IL-1β release and IgG humoral immune responses.

a, ELISA monitoring of cytokine release (IL-1β, IL-12p70) by BM-derived DC cultured in GM‑CSF + IL-4 or FLT3L conditions (which differentiate into cDC1) pulsed with various live commensals (for 2 h followed by killing with ATB for 22 h). A representative experiment out of two performed yielding similar results is shown, run in experimental triplicates. Mean ± SEM is depicted. Two-way ANOVA with Bonferroni’s correction. Significant two-tailed p-values are shown in the figure. b, IgG humoral responses evaluated by ELISA in the serum of mice, which were treated with OXA (day 0) and oral gavaged with the indicated commensals at day -1 and +1. Analysis was performed at day 7. Data pooled from two independent experiments. Germ-free groups n = 4, SPF PBS n = 8, SPF OXA n = 10, B. fragilis n = 11, E. ramosum n = 11, P. clara n = 11. Median value is depicted. Mann-Whitney U test. Significant two-tailed p-values are shown in the figure. c, IgG humoral responses evaluated by ELISA in the serum of mice, in the experimental setting described in Fig. 6f. Mean ± SEM is depicted. Mann-Whitney U test. Significant two-tailed p-values are shown in the figure. d, Quantitative PCR using dedicated and specific probe sets to monitor colonization of various commensals at day +2 post-first and second oral gavage. Data from two experiments (performed in C57BL/6 J (upper panel) or in BALB/c (lower panels)) are depicted, one dot representing one feces. Detected values by qPCR are shown, mean ± SEM is depicted. Analyzed sample sizes in C57BL/6 J groups: PBS n = 19, OXA n = 17, B. fragilis n = 16, E. ramosum n = 16, P. clara n = 21. Analyzed sample sizes in BALB/c: n = 6/group.

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Supplementary Figs. 1–6 and Supplementary Tables 1–6.

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Supplementary Data 1

PATIENTS_Metadata: De-aggregated and de-identified metadata relative to patients. PATIENTS_Run: Metadata relative to Next-Generation Sequencing (NGS) run of patients. MICE_Metadata: Metadata relative to mice. MICE_Run: Metadata relative to Next-Generation Sequencing (NGS) run of mice.

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Roberti, M.P., Yonekura, S., Duong, C.P.M. et al. Chemotherapy-induced ileal crypt apoptosis and the ileal microbiome shape immunosurveillance and prognosis of proximal colon cancer. Nat Med 26, 919–931 (2020). https://doi.org/10.1038/s41591-020-0882-8

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