Phocaeicola vulgatus induces immunotherapy resistance in hepatocellular carcinoma via reducing indoleacetic acid production

Abstract

Immunotherapy has made remarkable achievements in various cancers, but response rates in hepatocellular carcinoma (HCC) remain highly variable. Understanding mechanisms behind this heterogeneity and identifying responsive patients are urgent clinical challenges. In this study, the metagenomic analysis of 65 HCC patients reveals distinct gut microbiota profiles distinguishing responders (Rs) from non-responders (NRs). These findings are further validated through fecal microbiota transplantation (FMT) in mouse models. Notably, Phocaeicola vulgatus (P. vulgatus) is enriched in NRs and diminishes anti-PD-1 efficacy in both syngeneic and orthotopic tumor models. Mechanistically, P. vulgatus suppresses the production of indoleacetic acid (IAA), thereby weakening interferon (IFN)-γ⁺ and granzyme B (GzmB)⁺CD8⁺ T cells and impairing the antitumor immune response. Furthermore, supplementation with IAA restores CD8⁺ T cell cytotoxicity and counteracts the immune-suppressive effects of P. vulgatus. Our findings establish a causal relationship between P. vulgatus and anti-PD-1 resistance in HCC, highlighting IAA as a potential therapeutic target to enhance immunotherapy outcomes.

Keywords: Phocaeicola vulgatus; gut microbiota; immune checkpoint inhibitor; immunotherapy; indoleacetic acid; liver cancer.

Graphical abstract

Figure 1

Gut microbiota influence immunotherapy outcomes in patients with hepatocellular carcinoma (A) Overview of the patient study design. (B) Patient enrollment scheme. (C) PFS (left) and OS (right) in responders (n = 50) vs. non-responders (n = 15). (D) PCoA of gut microbiota β-diversity using Bray-Curtis dissimilarity metrics for responders (n = 50) and non-responders (n = 15). (E–H) Experimental design and results from fecal microbiota transplantation (FMT) into antibiotics (ATB)-treated pseudo-germ-free mice bearing subcutaneous RIL-175 tumors. (E) Experimental design: mice received FMT from Rs or NRs, followed by anti-PD-1 or isotype control treatment. (F) Tumor growth curves over time. (G) Representative tumor images (shown from the second of two independent experiments). (H) Final tumor weights. Each group included fecal samples from 6 Rs to 6 NRs, with 4–6 mice per patient. Data represent pooled results from two independent experiments (n = 14–17 mice/group). (I–K) FMT validation in an orthotopic HCC model via hydrodynamic tail vein injection of oncogenic plasmids. (I) Experimental design. (J) Representative liver tumor images across groups. (K) Representative bioluminescence images and quantification of total tumor flux. Each group included fecal samples from 4 Rs or 4 NRs, with 4–6 mice per patient (n = 10 mice/group). Data are shown as mean ± SEM. Statistical significance was determined by log rank test (C), PERMANOVA (D), or ANOVA (F, H, and K). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and p < 0.05. irAEs, intolerable immune-related adverse events; R_Isotype, responder FMT + isotype control; R_Anti-PD1, responder FMT + anti-PD-1; NR_Isotype, non-responder FMT + isotype control; NR_Anti-PD1, non-responder FMT + anti-PD-1.

Figure 2

Higher abundance of P. vulgatus impairs immunotherapy efficacy in hepatocellular carcinoma (A) LEfSe analysis shows differentially abundant taxa in responders (n = 50) vs. non-responders (n = 15); taxa with LDA >1.5 are shown. (B) Cladogram comparing gut microbiota in responders (n = 50) vs. non-responders (n = 15) based on metagenomic sequencing. (C) Relative abundance of P. vulgatus in feces of responders (n = 50) vs. non-responders (n = 15) detected by metagenome sequencing and qPCR. (D) PFS (left) and OS (right) in patients with low (n = 33) or high (n = 32) P. vulgatus abundance. (E) Representative MRI images of a responder (low P. vulgatus, top) and a non-responder (high P. vulgatus, bottom) before and after immunotherapy. (F–H) In vivo evaluation of P. vulgatus effects in an RIL-175 subcutaneous tumor model (n = 8 mice/group). (F) Experimental design: mice were orally gavaged with P. vulgatus, followed by tumor implantation and anti-PD-1 or isotype treatment. (G) Tumor growth curves. (H) Representative tumor images (left, two tumors in the PBS_Anti-PD1 group had completely regressed and were not visible at necropsy) and corresponding tumor weights (right). (I–K) Validation in an orthotopic in situ tumor model induced by hydrodynamic tail vein injection of oncogenic plasmids (n = 4–5 mice/group). (I) Experimental design. (J) Representative tumor images. (K) Representative bioluminescence images (left) and quantification of tumor burden by total bioluminescent flux (right). (L) Relative abundance of P. vulgatus in feces (left) and tumors (right) of PBS_Anti-PD1 and Pv_Anti-PD1 mice one week after gavage, detected by qPCR (n = 5–6). Data are shown as mean ± SEM. Statistical significance was determined by t test (C and L), log rank test (D), and ANOVA (G, H, and K). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05; ns, not significant. Pv, P. vulgatus.

Figure 3

Gut microbiota and P. vulgatus modulate immunotherapy response by modulating tumoral CD8⁺ T cell function (A) Schematic of immune profiling study design. (B) Frequencies of tumoral IFN-γ⁺ and GzmB⁺CD8⁺ T cells in the FMT model. (C) Frequencies of tumoral IFN-γ⁺ and GzmB⁺CD8⁺ T cells in the P. vulgatus transplantation model. Data are shown as mean ± SEM. Statistical significance was determined by ANOVA (B and C). ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05.

Figure 4

P. vulgatus inhibits CD8⁺ T cell effector function in the tumor microenvironment (A) Volcano plot of differentially expressed genes in tumors of PBS_Anti-PD1 and Pv_Anti-PD1 mice. (B) Bubble plot of top five GO terms enriched in tumors from PBS_Anti-PD1 and Pv_Anti-PD1 mice. (C) GSEA of leukocyte-mediated cytotoxicity in tumors from PBS_Anti-PD1 and Pv_Anti-PD1 mice. (D and E) Representative immunofluorescence images (D) and quantification (E) of GzmB⁺CD8⁺ T cells in tumors (n = 3 mice/group), Scale bars, 20 μm. (F and G) Representative immunofluorescence images (F) and quantification (G) of IFN-γ⁺CD8⁺ T cells in tumors (n = 3 mice/group), Scale bars, 20 μm. (H) IFN-γ levels in tumors of mice treated with P. vulgatus in the RIL-175 subcutaneous model (n = 4–6 mice/group). Data are shown as mean ± SEM. Statistical significance was determined by ANOVA (E, G, and H). ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05.

Figure 5

P. vulgatus impairs immunotherapy response through reducing indoleacetic acid production (A) Frequencies of (left) GzmB⁺ and (right) IFN-γ⁺ CD8⁺ T cells co-cultured with 10% culture medium of P. vulgatus (n = 3). (B) Heatmap of fecal metabolomic profiles (left) and concentrations of IAA and ILA (right) in feces from PBS and Pv-treated mice (n = 6 mice/group). (C) Heatmap of plasma metabolomic profiles (left) and concentrations of IAA and ILA (right) in plasma of PBS or Pv-treated mice (n = 11–12 mice/group). (D) Venn diagram of overlapping metabolites decreased in both plasma and feces of Pv-treated mice. (E) Concentrations of IAA and ILA in plasma of responders (n = 38) and non-responders (n = 10). (F) Correlation between P. vulgatus relative abundance and plasma IAA and ILA concentrations in patients (n = 48). (G) PFS (left) and OS (right) of patients with high (n = 24) or low (n = 24) concentrations of IAA in plasma. Data are shown as mean ± SEM. Statistical significance was determined by t test (A–C and E), Spearman correlation (F), and log rank test (G). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05; ns, not significant.

Figure 6

Mechanistic dissection of IAA depletion by P. vulgatus and its functional consequences (A) Schematic of the tryptophan (Trp) metabolic pathway and key enzymes for IAA and ILA biosynthesis. P. vulgatus lacks indolepyruvate decarboxylase (IPDC) and instead encodes indolepyruvate oxidoreductase (IOR), redirecting IPyA toward indole-3-acetyl-CoA. (B) Relative abundance of Trp-metabolizing genes (iorA, iorB, and ipdC) in fecal metagenomes of responders (n = 50) vs. non-responders (n = 15). (C) Concentrations of IAA in P. vulgatus culture medium compared with BHI medium (n = 3). (D–G) Tryptophan-deprived tumor model (n = 6 mice/group). (D) Experimental design: mice received Trp-deprived or control diets, followed by Pv/PBS gavage and anti-PD-1/isotype treatment. (E) Tumor growth curves. (F) Representative tumor images. (G) Tumor weights. (H and I) IAA rescue model. (H) Experimental design. (I) Tumor growth curves showing that IAA supplementation restored anti-PD-1 efficacy in Pv-treated mice (n = 6 mice/group). Data are presented as mean ± SEM. Statistical tests: t test (B and C) and ANOVA (E, G, and I). ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05; ns, not significant. Pv, P. vulgatus; IAA, indoleacetic acid; IPyA, indolepyruvic acid; IPDC, indolepyruvate decarboxylase; IOR, indolepyruvate oxidoreductase.

Figure 7

Indoleacetic acid enhances CD8⁺ T cell effector function and potentiates anti-PD-1 therapy (A) Experimental design for IAA intervention model. (B) Tumor growth curves in the IAA intervention model (n = 6 mice/group). (C) Frequencies of IFN-γ⁺ (left) and GzmB⁺ (right) CD8⁺ T cells in tumors from mice treated with PBS_isotype, PBS-PD1, IAA_isotype, or IAA-PD1. (D–G) CD8⁺ T cell depletion model (n = 6 mice/group). (D) Experimental design: CD8⁺ T cells were depleted using anti-CD8 antibody to assess dependency of IAA effect. (E) Tumor growth curves. (F) Final tumor weights. (G) Representative tumor images. (H and I) Frequencies of mouse (H) and human (I) CD25⁺, CD44⁺, CD69^+, GzmB⁺, and IFN-γ⁺ CD8⁺ T cells cultured with increasing concentrations of IAA (n = 3). (J) Cytotoxicity of human CD8⁺ T cells against liver cancer cells upon IAA treatment (0, 10, 100, and 1,000 μM) (n = 3). Data are shown as mean ± SEM. Statistical significance was determined by ANOVA. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05; ns, not significant. IAA, indoleacetic acid.