The microbiome-derived metabolite TMAO drives immune activation and boosts responses to immune checkpoint blockade in pancreatic cancer

Abstract

The composition of the gut microbiome can control innate and adaptive immunity and has emerged as a key regulator of tumor growth, especially in the context of immune checkpoint blockade (ICB) therapy. However, the underlying mechanisms for how the microbiome affects tumor growth remain unclear. Pancreatic ductal adenocarcinoma (PDAC) tends to be refractory to therapy, including ICB. Using a nontargeted, liquid chromatography-tandem mass spectrometry-based metabolomic screen, we identified the gut microbe-derived metabolite trimethylamine N-oxide (TMAO), which enhanced antitumor immunity to PDAC. Delivery of TMAO intraperitoneally or via a dietary choline supplement to orthotopic PDAC-bearing mice reduced tumor growth, associated with an immunostimulatory tumor-associated macrophage (TAM) phenotype, and activated effector T cell response in the tumor microenvironment. Mechanistically, TMAO potentiated the type I interferon (IFN) pathway and conferred antitumor effects in a type I IFN-dependent manner. Delivering TMAO-primed macrophages intravenously produced similar antitumor effects. Combining TMAO with ICB (anti-PD1 and/or anti-Tim3) in a mouse model of PDAC significantly reduced tumor burden and improved survival beyond TMAO or ICB alone. Last, the levels of bacteria containing CutC (an enzyme that generates trimethylamine, the TMAO precursor) correlated with long-term survival in patients with PDAC and improved response to anti-PD1 in patients with melanoma. Together, our study identifies the gut microbial metabolite TMAO as a driver of antitumor immunity and lays the groundwork for potential therapeutic strategies targeting TMAO.

Figure 1.. Administration of TMAO or TMA drives immune activation in the PDAC TME.

(A) Measurement of tumor weight in PDAC bearing C57BL/6 (B6) mice on d21 after orthotopic implant of PDAC cells and treatment with metronidazole antibiotic (1g/l) provided in drinking water. n=8 mice in control and n=5 mice in metronidazole treated group. Data are representative of three independent experiments. (B) Volcano plot comparing metabolite profiles of serum from metronidazole-treated and control mice in (A). Metabolomics analysis was performed by non-targeted LC-MS/MS. n=8 mice in control and n=5 mice in metronidazole treated group. (C) Schematic representation of the experiment (shown on left) with treatments of TMAO or TMA in PDAC bearing mice. Assessment of tumor weight in PDAC bearing mice on d21 after orthotopic implant of PDAC cells and treatment with TMAO (80mg/kg i.p., 4x per week) or TMA (40mg/kg i.p., 4x per week) starting d7 after tumor cell implants (shown on right). n=5 mice per group. Data are representative of 3–4 independent experiments. (D) Assessment of tumor weight in PDAC bearing mice d21 after orthotopic implant of PDAC cells and treatment with metronidazole antibiotic and TMAO or TMA. n=5 mice per group. (E, F, G) Flow cytometry analyses on PDAC tissues from (C) for (E) expression of MHCI, MHCII, and CD86 by TAMs shown as MFI (mean fluorescent intensity), (F)expression of Arg1 by TAMs shown as MFI, and (G) percent IFNγ⁺ TNFα⁺ of CD8⁺ and CD4⁺ T cells. Histogram shows MFI for Arg1 on TAMs and scatter plot shows percent IFNγ⁺ TNFα⁺ on CD8⁺ T cells. n=5 mice per group. (H) Kaplan–Meier Analysis showing survival of PDAC bearing mice treated with TMAO or TMA. n=10 mice per group. In (A, C-G), data are presented as mean +/- SD. In (A), p-values were determined by two-tailed Student’s t-tests. In (B), significant change defined as |FC| > 2; q-value < 0.1 (Benjamini-Hochberg FDR-adjusted p-value). In (C-G), p-values were determined by one-way ANOVA with post-hoc multiple comparisons. In (H), p-value was determined using a log-rank (Mantel–Cox) test.

Figure 2.. Dietary and gut microbiome interventions phenocopy the anti-tumor effects of TMAO.

(A) Schematic representation of the experiment (shown on left) with supplementation of 1% choline or control diet in PDAC bearing mice. Volcano plot showing differences in serum metabolites in PDAC bearing mice on d21 after orthotopic implant of PDAC cells and supplement of 1% wt/wt choline diet or control diet (shown on right). Metabolomics was performed by non-targeted LC-MS/MS. n=5 mice in control diet and n=5 mice in 1% choline diet group. (B) Assessment of tumor weight in PDAC bearing mice on d21 after orthotopic implant of PDAC cells and supplement of 1% wt/wt choline diet or control diet. n=7–8 mice per group. Data are representative of 2–3 independent experiments. (C, D) Flow cytometry analyses on PDAC tissues from (B) for (C) expression of MHCI and MHCII by TAM shown as MFI and (D) percent IFNγ⁺ TNFα⁺ of CD8⁺ and CD4⁺ T cells. n=7–8 mice per group. (E) Assessment of tumor weight in PDAC bearing mice on d21 after orthotopic implant of PDAC cells and provided with normal chow, 1% wt/wt choline diet, or 1% wt/wt choline diet plus metronidazole. n=5 mice per group. (F) Quantification of TMAO levels by LC-MS/MS in sera from PDAC bearing mice on d21 after orthotopic implant of PDAC cells and receiving fluoromethylcholine (FMC) or metronidazole in drinking water. n=5 mice per group. (G) Measurement of tumor weights in orthotopic PDAC bearing mice on d21 after orthotopic implant of PDAC cells and treatment with fluoromethylcholine (FMC). n=8 mice in control and n=5 mice in FMC treated group. Data are representative of two independent experiments. (H, I) Flow cytometry analysis on PDAC tissues from (G) for (H) expression of Arg1 and MHCII by TAM shown as MFI and (I) percent IFNγ⁺ TNFα⁺ of CD8⁺ and CD4⁺ T cells. n=8 mice in control and n=5 mice in FMC treated group. In (A), significant change defined as |FC| > 2; q-value < 0.1 (Benjamini-Hochberg FDR-adjusted p-value). In (B-I), data are presented as mean +/- SD. In (B-D, G-I), p-values were determined by two-tailed Student’s t-tests. In (E, F), p-values were determined by one-way ANOVA with post-hoc multiple comparisons.

Figure 3.. TMAO drives immune activation in the PDAC TME.

(A) Heat map showing differential gene expression of indicated RNA transcripts of genes of interest (p-value < 0.05) in FACS-sorted TAMs from tumors of d21 orthotopic PDAC bearing mice treated with TMAO (80mg/kg i.p., 4x per week starting d7 after tumor cell implants). n=4 mice per group. (B) Ingenuity Pathway Analysis (IPA) of activated (red bars) and inhibited (blue bars) regulators in TAMs from (A). (C) Uniform Manifold Approximation and Projection (UMAP) plot of scRNA-seq on tumor infiltrating CD45⁺ cells sorted from pooled tumors of d21 orthotopic PDAC bearing mice treated with TMAO. Mice received TMAO treatments as in (A). Tumors from n=7 mice were pooled together per group. (D) Violin plot of scRNA-seq data in (C) showing number of cells in each immune cell cluster from control and TMAO treated groups. (E) IPA for regulators of scRNA-seq data in (C) showing activated (red bars) and inhibited (blue bars) regulators in indicated immune cell clusters. Differentially expressed genes passing FDR<0.05 were analyzed. (F) IPA for functions of scRNA-seq data in (C) showing activated (red circles) and inhibited (blue circles) functions in T cell cluster. Differentially expressed genes passing FDR<0.05 were analyzed. (G) Measurement of tumor weights in PDAC bearing mice on d21 after orthotopic tumor cell implants and receiving treatments with TMAO or anti-CSF1 plus clodronate or combination. n=5 mice per group. (H) Assessment of tumor size in PDAC bearing mice on d21 after s/q tumor cell implants and receiving treatments with TMAO or CD8⁺ T cell depleting antibody or combination. n=6–8 mice per group. In (G, H), data are presented as mean +/- SD. p-values were determined by two-way ANOVA with post-hoc multiple comparisons. ns= not significant.

Figure 4.. TMAO signals by promoting activation of the type-I IFN response.

(A) IPA of RNAseq for activated (red bars) and inhibited (blue bars) regulators in BMDM treated with 300µM TMAO for 8hr. Differentially expressed genes passing FDR<0.05, and |FC|>=1.5 were analyzed. n=3. (B) IPA of RNAseq for activated (red bars) and inhibited (blue bars) regulators in BMDM pre-treated with TMAO (300µM) for 1hr or left untreated and then stimulated with 20% PDAC tumor conditioned media (TCM) for 8hr. Differentially expressed genes passing FDR<0.05, and |FC|>=1.5 were analyzed. n=3. (C) RT-PCR showing relative expression of indicated genes in BMDM pre-treated with TMAO (300µM) for 1hr or left untreated and then stimulated with ISD (interferon stimulatory DNA) or poly(dG:dC). The mRNA relative expression is shown compared to β-actin. n=4. (D) Flow cytometry analyses of indicated activation markers on BMDM pre-treated with TMAO (300µM) for 1hr or left untreated and then stimulated with ISD (interferon stimulatory DNA). Histograms show MFI for CD86 on BMDM. n=3–4. (E) Schematic representation of the experiment (shown on left) with treatments with anti-IFNAR1 and TMAO in PDAC bearing mice. Assessment of tumor size in PDAC bearing mice on d21 after s/q PDAC cell implants and receiving treatments with TMAO or IFNAR1 blocking antibody or combination (shown on right). n=7 mice per group. (F) Kaplan–Meier Analysis showing survival of PDAC bearing mice from (E). In (C-E), data are presented as mean +/- SD. p-values were determined by two-way ANOVA with post-hoc multiple comparisons. In (F), p-value was determined using a log-rank (Mantel–Cox) test. ns= not significant.

Figure 5.. TMAO shapes macrophage responses to promote effector T cell activity and reduce PDAC growth.

(A) Flow cytometry analysis of Cell Trace Violet (CTV) labelled OT-I T cell proliferation after co-culture with TCM stimulated BMDM (+/- TMAO) in the presence of DCs and OVA_257–264 peptide for 3 days. n=3. (B) Flow cytometry analysis of Cell Trace Violet (CTV) labelled CD8⁺ T cell proliferation after co-culture with TCM stimulated BMDM (+/- TMAO) in the presence of anti-CD3 and anti-CD28 for 3 days. n=3. (C) Measurement of tumor weights in PDAC bearing mice on d21 after orthotopic tumor cell implants and receiving i.v. injections of BMDM (either primed with TMAO for 24hr or control, 3x per week, 10⁶ per mouse). n=5 mice per group. Data are representative of two independent experiments. (D, E) Flow cytometry analysis on PDAC tissues from (C) for expression of IFNγ, Ki67, CD103, and CD44 on CD8⁺ T cells shown as MFI. Histogram shows MFI for indicated markers on CD8⁺ T cells. (F) RT-PCR showing relative expression of indicated genes in human monocyte derived macrophages (HMDM) pre-treated with TMAO for 1hr and then stimulated with 20% PDAC conditioned media (TCM) for 8hr. TCM was generated using PANC-1 human PDAC cell line. The mRNA relative expression is shown compared to β-actin. n=4. Data are presented as mean +/- SD. In (A, B), p-values were determined for comparing division 5 to 6 between the groups by one-way ANOVA with post-hoc multiple comparisons. In (C, E), p-values were determined by one-way ANOVA with post-hoc multiple comparisons. In (F), p-values were determined by two-tailed Student’s t-tests. ns= not significant.

Figure 6.. Administration of TMAO or TMA renders PDAC responsive to ICB.

(A, B) Flow cytometry analysis of percent PD1⁺Tim3⁺ CD8⁺ T cells assessed from tumor tissues of PDAC bearing mice on d21 after orthotopic implant of PDAC cells and receiving (A) treatments with TMAO or TMA and (B) supplementation of 1% choline diet or control diet. Scatter plot shows percent PD1⁺Tim3⁺ on CD8 T⁺ cells. (A) n=5 mice per group and (B) n=7–8 mice per group. (C) Measurement of tumor weights in PDAC bearing mice on d21 after orthotopic PDAC cell implants and receiving treatments with TMAO or anti-PD1 or combination. n=5 mice per group. Data are representative of 2–3 independent experiments. (D, E) Flow cytometry analysis on PDAC tissues from (C) for (D) the expression of MHCI by TAMs, CD103⁺ DCs, and MDSCs shown as MFI, and (E) percent CD44⁺ Ki67⁺ or IFNγ⁺ TNFα⁺ of CD8⁺ T cells. (F) Measurement of tumor weights in PDAC bearing mice on d21 after orthotopic PDAC cell implants and receiving treatments with TMAO or anti-Tim3 or combination. n=5 mice per group. (G) Assessment of tumor size in PDAC bearing mice on d21 after s/q PDAC cell implants and receiving treatments with TMAO, TMA, anti-PD1, anti-Tim3, or indicated combinations. n=7–8 mice per group. (H) Kaplan–Meier Analysis showing survival of PDAC bearing mice from (G). In (A-G), data are presented as mean +/- SD. In (A, C-G), p-values were determined by one-way ANOVA with post-hoc multiple comparisons. In (B), p-values were determined by two-tailed Student’s t-tests. In (H), p-value was determined using a log-rank (Mantel–Cox) test. ns= not significant.

Figure 7.. Abundance of CutC containing bacteria and elevated CutC gene expression correlate with improved survival and response to anti-PD1 in cancer patients.

(A, B) Box-and-whisker plot showing relative abundance of CutC containing bacterial counts of (A) Genus Bacillus (top) and Genus Paenibacillus (bottom), within the tumor microbiome of short-term survivors (STS) and long-term survivors (LTS) of PDAC and (B) Genus Bacillus, within the gut microbiome of melanoma patients who were responders (R) or non-responders (NR) to anti-PD1 checkpoint immunotherapy after FMT. (C) Heat map showing normalized CutC level in CutC expressing bacterial strains between responders (R) vs non-responders (NR) to anti-PD1. (D) Box-and-whisker plot indicating relative abundance of CutC containing bacterial families Clostridiaceae and Enterococcaceae in responders (R) vs non-responders (NR) to anti-PD1. (E) Box-and-whisker plot of CutC gene expression shown as ppm across responders (R) vs non-responders (NR) to anti-PD1. (F) Overall survival of patients stratified by expression of CutC. In (A, B), Mann-Whitney non-parametrical test was used for comparison of relative abundance values between LTS vs STS and R vs NR groups with Benjamini-Hochberg used for correction for multiple testing. Significant results that passed FDR<0.05 were shown. In (C, D), CutC presence was associated with response through Fisher exact test. Bacterial CutC with Fisher exact p < 0.01 and Mann-Whitney p < 0.03 are presented. In (E), p-value was determined by the Wilcoxon rank-sum test, and (F) p-value was determined by the log-rank test.