The Kyn pathway is the predominant route for Trp metabolism, accounting for approximately 95% of its catabolism (26). This pathway is governed by key rate-limiting enzymes, including IDO1/2, TDO, and kynurenine-3-monooxygenase (KMO) (27). Trp metabolism is initiated by oxidation and cleavage catalyzed by IDO1 or TDO, which gives rise to multiple downstream branches of the Kyn pathway. These reactions generate a range of metabolites, including 3-hydroxykynurenine, 3-hydroxyanthranilic acid, N-formyl Kyn, xanthurenic acid, kynurenic acid, anthranilic acid, and quinolinic acid. Collectively, these intermediates are referred to as Kyn pathway metabolites (28). The core function of this metabolic pathway lies in its potent immunomodulatory effects, which are mediated through two primary mechanisms. First, it rapidly depletes local Trp, leading to T-cell dysfunction and anergy. Second, its key metabolite, Kyn, serves as an endogenous ligand for the AhR. AhR activation drives the differentiation of Tregs and suppresses effector functions, thereby actively fostering an immunosuppressive TME (29–31).

The serotonin pathway is an essential biochemical process involved in regulating serotonin signaling, particularly within the central nervous system (CNS) (32, 33). Although this pathway accounts for only approximately 1-2% of total Trp metabolic flux (34–37), it exerts disproportionate biological effects. This apparent discrepancy reflects the high potency of serotonin as a receptor-mediated signaling molecule, whereby low ligand concentrations are sufficient to activate high-affinity receptors and elicit amplified downstream responses (38), including effects within immune cells and the tumor microenvironment (39).

The pathway begins with dietary Trp absorption into the circulation (40). In the brain, Trp is first hydroxylated by the rate-limiting enzyme Trp hydroxylase (TPH1 or TPH2) to form 5-hydroxytryptophan (5-HTP) (41). 5-HTP is then decarboxylated by aromatic L-amino acid decarboxylase (AADC) to generate serotonin (42, 43). Serotonin synthesis, transport, and turnover are tightly regulated by TPH isoenzymes, serotonin transporters, and multiple receptor subtypes. Newly synthesized serotonin may be released into the synaptic cleft to mediate signaling or stored in vesicles (44), while reuptake is controlled by the serotonin transporter (45). Ultimately, serotonin is oxidatively degraded by monoamine oxidase (MAO) on the outer mitochondrial membrane, producing its primary terminal metabolite, 5-hydroxyindoleacetic acid (5-HIAA) (46), which is subsequently excreted in the urine (47).

The microbial indole pathway comprises multiple enzymatic reactions and represents a major route for Trp metabolism in the gut. In addition to decarboxylation reactions that generate tryptamine, gut microorganisms can convert Trp into a variety of indole derivatives through alternative biochemical processes, including deamination and side-chain cleavage. These pathways give rise to metabolites such as indole-3-propionic acid (IPA), indole-3-acetic acid (IAA), and indole-3-acetaldehyde (IAld) (48). These metabolites exhibit diverse biological activities: indole activates the pregnane X receptor (PXR) and AhR to enhance intestinal epithelial barrier function (49). IAId regulates IL-22 production, aiding mucosal homeostasis (50, 51). And IPA demonstrates potent antioxidant and anti-inflammatory properties (52). As natural AhR ligands, indole and its derivatives activate AhR signaling upon binding. This AhR-ligand complex then translocate to the nucleus and regulates the transcription of downstream genes involved in detoxification, immune regulation, and cellular development, playing a key role in modulating immune cell differentiation and overall immune responses (15).

The microbial indole pathway and the host Kyn pathway do not operate in isolation; rather, they engage in continuous metabolic crosstalk. This creates an interdependent network governed by shared substrates and reciprocal feedback through interactive signaling (53). While intestinal microbiota transforms Trp into indole derivatives, host cells, specifically tumor cells and immune cells expressing IDO1/TDO, predominantly metabolize it through the IDO1/TDO-driven Kyn axis. Kyn pathway metabolites from these two arms bidirectionally regulate each other, demonstrating that systemic Trp metabolism is co-orchestrated by both microbial and host activities (18, 19). For example, bacterial-derived IAA can directly inhibit the enzymatic activity of host IDO1, thereby curtailing the downstream generation of the pro-cancerous metabolite Kyn (54). Conversely, under conditions of tumor or inflammation, host cytokines (e.g., IFN-γ) perform a dual role: they upregulate IDO1 and concurrently remodel the intestinal microbiota. These changes reciprocally influence the composition of microbial indole metabolites, forming a dynamic feedback loop (21).

Acting as endogenous ligands, a range of metabolites—including indole, indole-3-acetaldehyde (IAld), and Kyn—can activate the AhR, which functions as a central integrator of downstream signaling. The biological consequences of AhR activation are highly context dependent and are influenced by multiple factors, including ligand identity and affinity, ligand concentration, target cell type, and the surrounding tissue microenvironment (55, 56). Within the tumor microenvironment (TME), AhR signaling is frequently associated with the establishment of immunosuppressive conditions, such as promotion of regulatory T cell (Treg) differentiation and inhibition of effector T cell activity (57, 58). However, under specific circumstances—particularly in the presence of high-affinity ligands—AhR activation has also been reported to exert direct tumor-inhibitory effects.

At the molecular level, AhR signaling follows a defined cascade. Microbiota-derived metabolites, together with certain host-derived ligands (including high-affinity indole derivatives and Kyn), bind to and activate AhR (59, 60). Upon activation, AhR translocates to the nucleus and heterodimerizes with the AhR nuclear translocator (ARNT), enabling the complex to bind xenobiotic response elements (XREs) within target gene promoters. The resulting transcriptional programs regulate genes involved in cell cycle control, apoptosis, metabolic adaptation, maintenance of stemness, and cell adhesion and migration. Through these mechanisms, metabolite-driven AhR signaling can directly influence key malignant phenotypes, including tumor cell proliferation, metabolic reprogramming, and metastatic potential (61, 62).

Thus, the metabolomic network orchestrated by the host and gut microbiota drives breast cancer progression through multifaceted mechanisms. These include remodeling the tumor immune microenvironment, directly interfering with cancer cell behavior, and perturbing systemic inflammation and metabolic homeostasis. To elucidate how gut microbiota exert remote metabolic control, research must shift focus from isolated pathways to the integrated network itself. This systemic understanding is essential for developing targeted therapies, such as precise dietary regimens, probiotic interventions, or small-molecule drugs, aimed at this critical network.

Gut microbiota directly competes with the host for dietary Trp by expressing microbial Trp-degrading enzymes, such as tryptophanase and bacterial-type IDO-like enzymes. For instance, Escherichia coli converts Trp into indole (66), while Clostridium sporogenes can produce beneficial metabolites such as IPA (52, 63, 67, 68). It is important to distinguish host and microbial Trp metabolism. In host cells, Trp is predominantly catabolized through the Kyn pathway mediated by IDO1/IDO2 and TDO2, whereas gut microorganisms utilize distinct and independent catabolic pathways. These host and microbial systems differ fundamentally in their enzymatic origins and metabolic routes. Microbial indole metabolites compete with host cells, particularly tumor cells, for the essential amino acid Trp through both direct and indirect mechanisms. Directly, microorganisms consume and utilize Trp as a metabolic substrate; indirectly, microbial metabolites can modulate the expression or activity of host enzymes such as IDO1, either enhancing or suppressing Trp catabolism. In parallel, microbial indole compounds and their downstream metabolites can enter the systemic circulation, where they influence immune responses and inflammatory states. Through these mechanisms, microbiota-derived indole metabolism can affect physiological and pathological processes in distant organs, including breast tissue (63, 65, 69), hereby forming a metabolic link between the gut microbiota and systemic diseases such as breast cancer.

Indole and its derivatives act as endogenous ligands of the AhR, activating AhR signaling in epithelial cells and promoting the expression of tight junction proteins (e.g., occludin, claudin-1), thereby enhancing gut barrier function (51), inhibiting the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and NOD-like receptor protein 3 (NLRP3) inflammasome activation, and reducing the release of pro-inflammatory cytokines such as TNF-α and IL-6 (70, 71). Limosilactobacillus reuteri alleviated inflammation by producing Indole-3-lactic acid (ILA) and IPA that activate AhR, thereby inhibiting Th2 cytokines (IL-4, IL-5) and thymic stromal lymphopoietin (TSLP) expression (50, 72, 73). These data support the pivotal role of the microbiome-AhR axis as an interface that couples microbial metabolic output to the regulation of epithelial integrity and immune responses.

Trp metabolism acts as a critical link between gut microbiota and host health. Its dysregulation is implicated in various pathologies, including cancer, neurodegenerative diseases, and autoimmune disorders (48). As the primary site of Trp metabolism, the liver is significantly influenced by gut-derived microbial metabolites (74). Under homeostatic conditions, beneficial gut bacteria convert Trp into indole derivatives, such as IPA. These metabolites reach the liver via the portal vein and function as ligands for the AhR. AhR activation exerts immunostimulatory effects, including the promotion of Th17 cell differentiation, which supports intestinal barrier integrity and host defense (7, 75).

During dysbiosis—for example, during Enterococcus overgrowth—the Trp metabolic pathway alters. Production of beneficial metabolites like IPA declines, while harmful products such as indoxyl sulfate accumulate. This shift promotes hepatic inflammation and fibrosis (69, 76–79). Furthermore, broad-spectrum antibiotic treatment can remodel the gut microbiota, increasing the diversion of Trp into the Kyn pathway (80–82).

Notably, the immunomodulatory role of AhR is highly context-dependent and exhibit marked cell-type specificity. The outcomes of AhR signaling depend not only on the ligand type, local microenvironment, and host physiological status (56), but are also strongly influenced by cell type (e.g., tumor cells, T cells, myeloid cells), the nature and affinity of ligands, the local cytokine milieu, and the stage of disease (55, 59, 83–85). In chronic inflammatory settings (e.g., autoimmune diseases), the Trp metabolite Kyn activates AhR to drive Tregs differentiation and induce a tolerogenic phenotype in dendritic cells (DCs), thereby promoting immunosuppression and anti-inflammatory responses (56, 75, 86, 87). Similarly, within the tumor microenvironment, AhR signaling may enhance immunosuppression under certain conditions, such as Treg polarization or the induction of tolerogenic DC phenotypes (85, 88). Conversely, in chronically stimulated CD8⁺ T cells, the same signaling axis can lead to effector dysfunction or exhaustion, with specific outcomes varying depending on the experimental model and tissue site. These effects are not mutually exclusive and can coexist within the same TME, collectively shaping the overall immune landscape (89). Under homeostasis, microbiota-derived indole compounds activate AhR to enhance Th17 responses and support immune surveillance (88).

This bidirectional regulation indicates that microbial metabolites not only directly affect liver inflammation and fibrosis through pathways involving AhR, but also modulate AhR activity in hepatocytes and the expression of cytochrome P450 enzymes. Consequently, they influence drug metabolism and toxicity (90–92). Thus, the gut microbiota exerts systemic effects beyond the intestine, shaping metabolic and immune responses across multiple organs.

Within the TME, microbial and host-derived metabolites activate the AhR signaling pathway to regulate immune cell differentiation and function. AhR activation drives CD8+ T cell exhaustion and suppresses antitumor immunity, thereby facilitating immune escape and tumor progression. Specifically, tumor-initiating cells can exploit the Kyn-AhR axis to upregulate PD-1 expression on CD8+ T cells, thereby amplifying immunosuppression and protumor effects (30). Thus, by modulating the production and signaling of Trp metabolites, the gut microbiota directly influences systemic immune balance and shapes antitumor immune responses.

In summary, a deeper understanding of these pathways (especially their tissue-specific effects) is crucial for advancing microbiome-based therapeutic strategies targeting the “ microbiota-metabolism-immune” axis. Figure 2 summarizes the overview of how the gut microbiota regulates the host’s availability of Trp, signaling, and immune response through the aforementioned coordinated mechanisms.

Trp metabolism is a key interface in shaping the immunosuppressive TME. In the TME of breast cancer, the uptake of Trp mainly relies on L-type amino acid transporter 1 (LAT1), which is upregulated in both primary and metastatic breast cancer lesions and is associated with poor prognosis (93–95). After entering the cells, Trp is mainly oxidized and degraded through the Kyn pathway. The rate-limiting enzyme of this process, IDO, is highly expressed in various tumors, including breast cancer, and its level is closely related to tumor growth, metastasis, and poor prognosis (13, 14, 96). This metabolic reprogramming directly leads to Trp depletion and accumulation of Kyn in the TME, which in turn suppresses antitumor immunity through multiple mechanisms: Trp depletion activates the General Control Nonderepressible 2 (GCN2) kinase pathway in CD8⁺ T cells, causing cell cycle arrest and functional exhaustion (97); while accumulated Kyn can induce antigen-presenting cells such as DCs to express immune checkpoint molecules like PD-L1 and promote the differentiation of Tregs, thereby comprehensively suppressing the immune response (30, 98). The activation of AhR in T cells and dendritic cells not only promotes the differentiation of Tregs, but also enhances their inhibitory ability and phenotypic stability after maturation, thereby more effectively suppressing the activity of effector T cells and natural killer (NK) cells (98). IPA has been demonstrated to enhance the antitumor function of CD8+ T cells by promoting histone acetylation and increasing the chromatin accessibility at effector gene loci, thereby facilitating epigenetic reprogramming (99). This complex interaction reveals a crucial “gut-tumor” metabolic axis that affects immune evasion (99, 100).

The impact of Trp metabolites on breast cancer extends beyond shaping the immunosuppressive microenvironment. As the common receptor for various Trp metabolites such as Kyn, the AhR signaling pathway plays a direct and multifaceted driving role in breast cancer cells (101, 102). Whether it is indole derivatives from microorganisms or Kyn from the host, they can activate AhR, thereby promoting the proliferation, EMT (103), and maintenance of cancer stem cell characteristics of breast cancer cells (50, 104). Mechanistically, after AhR activation, it translocates to the nucleus and forms a heterodimer with ARNT, regulating the expression of downstream genes such as CYP1A1 and CYP1B1 (105, 106). This activation has subtype-specific effects. For example, in the triple-negative breast cancer (TNBC) model, the AhR signal enhances the invasiveness of tumor cells through the TWIST1/SNAIL1 axis. In estrogen receptor-positive (ER+) breast cancer, the crosstalk between AhR and ER affects the sensitivity to endocrine therapy (such as tamoxifen) (107).

It is worth noting that the role of the AhR signal in breast cancer is highly dependent on the environment, exhibiting both tumor-promoting and potential tumor-suppressing properties (108, 109). This effect depends on the type of ligand, activation level, duration, and cellular background. Although the studies and most evidence from TMEs support its oncogenic role, certain specific ligands (such as some dietary ligands) or in the context of detoxification of exogenous toxins, the activation of AhR may also trigger cell cycle arrest or apoptosis programs, demonstrating tumor-suppressive potential (110). In the pathological context of breast cancer, especially in the microenvironment dominated by the continuous activation of the IDO/Kyn axis, the AhR signal typically tends to promote tumor progression and immune escape (111). The comprehensive understanding of this complexity is crucial for the design of therapeutic strategies targeting AhR.

The influence of the Trp metabolic network is also amplified by the simultaneous activation of different receptor signaling pathways. Besides AhR, metabolites of the Kyn pathway can also transmit signals through G protein-coupled receptor 35 (GPR35) and other receptors, further regulating cellular functions. This multi-receptor synergy is particularly significant in ER+ breast cancer, forming a close interaction between the microbiota-regulated Trp metabolism and estrogen signaling networks. On one hand, the intestinal microbiota expressing β-glucuronidase can hydrolyze estrogen-glucuronide conjugates, promoting the intestinal-portal circulation reabsorption of free estrogen, thereby increasing estrogen exposure in breast tissues (112, 113). On the other hand, Trp metabolism and estrogen signaling can directly interfere with each other through multiple mechanisms: AhR can form a protein complex with ERα, mutually regulating each other’s transcriptional activity (114); Trp metabolic enzyme CYP1B1 participates in the hydroxylation of estrogen, generating 4-hydroxy estrogen metabolites with genotoxicity (115); Kyn pathway metabolites can also regulate the phosphorylation state of ER through activating the GPR35 receptor. Preclinical studies have shown that dietary supplementation of Trp can weaken the estrogen sensitivity of breast tumors by altering the microbiota composition and reducing the activity of β-glucuronidase, providing a new idea for dietary intervention in ER+ breast cancer (116).

The above mechanisms collectively form a complex network of the microbiota-Trp axis influencing the pathogenesis of breast cancer. It is notable that different breast cancer subtypes may exhibit distinct metabolic dependencies: for example, the activation of the IDO1/Kyn/AhR axis is usually more significant in TNBC (28), while the interaction between Trp metabolism and estrogen signaling is more prominent in ER+ breast cancer (52). Identifying these subtype-specific metabolic features is crucial for designing precise treatment strategies, such as combining IDO inhibitors with immune checkpoint blockers for TNBC (116, 117), or using probiotics/dietary intervention to regulate the activity of microbial β-glucuronidase as an adjunctive treatment for ER+ breast cancer endocrine therapy (118). Future research should focus on elucidating the heterogeneity of the Trp metabolic network in different breast cancer subtypes and individual patients, and on evaluating the clinical translational potential of microbiota-targeted intervention measures based on this.