How microbe-produced molecules may change the immune response in cancer immunotherapy: processes and treatment possibilities

Microbial metabolites encompass a structurally diverse array of bioactive molecules synthesized through fermentation, respiration, and secondary metabolism, including short-chain fatty acids (SCFAs), tryptophan (Trp) derivatives, bile acids, polyamines, polysaccharides, and lipopolysaccharides (LPS) (4, 7). These metabolites function as pleiotropic regulators of immunity and metabolism through three conserved mechanistic networks: (1) receptor-mediated signaling (e.g., G protein-coupled receptors [GPCRs], nuclear receptors), (2) epigenetic modulation (e.g., histone deacetylase [HDAC] inhibition), and (3) metabolic reprogramming (e.g., energy substrate provision, redox balance) (8, 9). Their production is dynamically regulated by host diet, microbial community structure, and environmental cues, creating a complex interactome that dictates functional outcomes (9, 10).

Critically, the immunomodulatory effects of microbial metabolites are determined by context-dependent variables including: (1) concentration gradients (e.g., micromolar vs. millimolar ranges), (2) tissue-specific receptor expression (e.g., G protein-coupled receptor [GPR]43 on T cells vs. epithelial cells), (3) metabolic microenvironment (e.g., glucose availability, redox state), and (4) host genetic background. This context dependency explains the frequently observed functional duality (immunostimulatory vs. immunosuppressive) that characterizes many microbial metabolites and necessitates mechanistic rather than descriptive classification.

SCFAs—predominantly acetate, propionate, and butyrate—are microbial fermentation products of indigestible dietary fibers, with butyrate (1–10 mM colonic concentrations) serving as the primary energy substrate for colonocytes (11). These metabolites exert their effects through dual mechanisms: (1) GPCR activation on immune and epithelial cells, and (2) HDAC inhibition leading to epigenetic reprogramming, with their net effect determined by concentration gradients and target cell type (12). SCFAs coordinate intestinal barrier integrity through enhanced tight junction expression while regulating inflammatory homeostasis via modulation of nuclear factor κB (NF-κB) signaling (11–14).

Trp metabolism by gut microbiota generates biologically active derivatives including indole, kynurenine (Kyn), and quinolinic acid, which act as key ligands for Aryl hydrocarbon receptor (AhR)—a ligand-dependent transcription factor critical for immune homeostasis (15, 16). Dysregulation of Trp metabolism contributes to inflammatory bowel disease, neuropsychiatric disorders, and cancer through mechanisms involving immune cell polarization and cytokine network modulation (15, 17). These metabolites exhibit pathway-specific effects: indole derivatives primarily activate AhR in epithelial and immune cells, while Kyn acts as both an AhR agonist and a metabolic checkpoint regulator (18). Trp metabolites form a complex signaling network centered on AhR, with divergent effects on T cell function determined by metabolite structure, concentration, and competing ligand availability in the TME (19).

Bile acids comprise hepatocyte-derived primary bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA]) and microbiota-modified secondary bile acids (deoxycholic acid [DCA], lithocholic acid [LCA], ursodeoxycholic acid [UDCA]) (20). Their metabolism involves enterohepatic circulation with microbiota-mediated dehydroxylation and conjugation, generating ligands for both nuclear receptors and GPCRs (20, 21). The balance between primary and secondary bile acids, regulated by microbial enzymes like bile salt hydrolases, dictates overall immune tone in the TME (22). Bile acids function as metabolic messengers linking liver-gut axis homeostasis to tumor immunity, with secondary bile acids often exerting immunosuppressive effects in advanced malignancies (22).

Polyamines are synthesized by gut microbiota including Enterobacteriaceae and Bacteroides species through arginine and ornithine decarboxylation (23). These metabolites regulate cellular proliferation and differentiation by modulating mRNA translation and autophagy, with context-dependent effects on tumor immunity (23, 24). Polyamines represent a double-edged sword in tumor immunity, promoting regulatory T cell (Treg)-mediated immunosuppression within the TME while supporting memory T cell development in secondary lymphoid organs, necessitating targeted delivery strategies.

Microbial polysaccharides, including extracellular polysaccharides (EPS) and capsular polysaccharides (CPS), exhibit structural heterogeneity that determines their interaction with pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and C-type lectin receptors (25). These polysaccharides function as PRR agonists that bridge innate and adaptive immunity, with their structural diversity enabling targeted modulation of macrophage polarization and antigen-presenting capacity of dendritic cells (DCs) in the TME (26, 27).

LPS, a component of Gram-negative bacterial outer membranes, activates TLR4/MyD88 signaling to trigger inflammatory responses (28, 29). Structural variations in LPS, particularly lipid A acylation patterns, determine its potency and may explain strain-specific effects on cytokine release (28, 29). LPS exhibits temporal and dose-dependent effects on tumor immunity, with therapeutic potential in combination with radiotherapy or checkpoint inhibitors when delivered in controlled, localized formulations to avoid systemic toxicity (29, 30).

Beyond major classes, diverse microbial metabolites modulate tumor immunity through specialized mechanisms. Trimethylamine N-oxide (TMAO), derived from dietary choline metabolism, enhances CD8⁺ T cell cytotoxicity via protein kinase R-like endoplasmic reticulum kinase (PERK)-dependent pyroptosis, despite promoting metastasis in other contexts (31–35). Urolithins, produced from ellagic acid, induce mitophagy in tumor-associated macrophages (TAMs) via transcription factor EB (TFEB) activation while expanding CD8⁺ T memory stem cells through Pink1-mediated mitochondrial regulation (36–38). Inosine modulates adenosine A2a receptor signaling to enhance CD8⁺ T cell function in glucose-deprived TMEs, serving as an alternative energy source through ribose phosphorylation (39–41). Desaminotyrosine (DAT) amplifies type I interferon (IFN-I) signaling via signal transducer and activator of transcription (STAT)1-mediated interferon alpha/beta receptor 1 (IFNAR1) upregulation, enhancing T cell priming (42). L-arginine (L-Arg) fuels nitric oxide (NO) production and T cell polyamine biosynthesis, counteracting myeloid-derived suppressor cell (MDSC)-mediated immunosuppression in acetate-enriched TMEs (43–45). These metabolites highlight the expanding landscape of microbial mediators that fine-tune immune responses through metabolic-immune crosstalk.

Microbial metabolites converge on three core signaling axes that unify their immunomodulatory functions: (1) Epigenetic regulation: SCFAs, Trp metabolites, and certain bile acids modulate chromatin accessibility to control immune cell fate decisions; (2)Metabolic-immune crosstalk: Nutrient-sensing pathways including mechanistic target of rapamycin (mTOR) (L-Arg) and PERK (TMAO) link metabolic state to immune cell activation; (3) PRR signaling: Polysaccharides (TLR2/4), LPS (TLR4), and certain indoles (AhR) activate conserved PRR pathways that bridge microbial sensing to adaptive immunity. These integrated networks exhibit context-dependent plasticity, with metabolite combinations often producing synergistic or antagonistic effects that cannot be predicted from individual components. For example, SCFAs enhance AhR expression in T cells, potentiating their responsiveness to Trp metabolites. Conversely, bile acid-mediated farnesoid X receptor (FXR) activation can antagonize SCFA-induced GPR43 signaling in hepatocytes. Understanding these interaction networks is critical for developing rational combination strategies in immunotherapy.

Microbial metabolites exhibit complex, context-dependent regulation of tumor cell fate, with butyrate representing a prime example of this duality—its biological effects are strongly concentration-dependent, superimposed on host genetics and metabolic context. At high concentrations (>10 mM) in the colonic lumen, butyrate serves as the primary energy source for normal colonocytes, supporting epithelial homeostasis via mitochondrial β-oxidation (46). When accumulated in colorectal cancer (CRC) cells at >100 μM (a concentration driven by Warburg effect-impaired butyrate oxidation), it functions as a potent HDAC inhibitor: this activity suppresses cell proliferation, induces apoptosis, and drives downstream effects like Pyruvate Kinase M2 activation (47) and reactive oxygen species (ROS)-induced apoptosis (48), resolving its paradox by prioritizing tumor suppression in malignant cells. In contrast, low concentrations (0.5-1.53 mM) of butyrate exert pro-tumorigenic effects in premalignant/genetically susceptible contexts (49, 50). Notably, ≥10 mM butyrate (e.g., 10–100 mM sodium butyrate) loses this pro-tumor effect, failing to stimulate CRC cell proliferation (49, 50). Host genetics, microbial co-metabolites (e.g., acetate/propionate synergizing with 0.69 mM butyrate to enhance senescence), and immune modulation further refine these outcomes. Propionate, meanwhile, delays mitochondrial-mediated apoptosis by inducing autophagy (51). Urolithin A (UA) and its structural analogs reduce CRC resistance to 5-fluorouracil by modulating the forkhead box O3 (FOXO3)-forkhead box M1 (FOXM1) axis (52). The Trp metabolite trans-3-indoleacrylic acid (IDA) promotes CRC by inhibiting ferroptosis through the AhR-aldehyde dehydrogenase 1 family member A3 (ALDH1A3) axis (53, 54). Additionally, Reuterin from healthy microbiota inhibits CRC growth by suppressing ribosome biogenesis through oxidative stress (55), while TMAO promotes CRC by inhibiting the FXR-fibroblast growth factor 15 (FGF15) axis and activating the Wnt/β-catenin pathway (56). Bile acids activate FXR and G protein-coupled bile acid receptor 5 (TGR5), triggering the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling cascades to drive tumor proliferation and anti-apoptosis (57–59). Research indicates that the gut microbiota can metabolize environmental carcinogens, thereby promoting the development of chemically induced tumors in distal organs and accelerating cancer progression (60). Cigarette smoke-induced dysbiosis elevates taurodeoxycholic acid (TDCA), activating MAPK/ERK, interleukin (IL)-17, and tumor necrosis factor (TNF) pathways to accelerate CRC (61). High-fat diet-associated lysophosphatidic acid directly stimulates cancer cell proliferation (62). The metabolite tyrosol inhibits CRC progression by suppressing NF-κB/hypoxia-inducible factor 1 (HIF-1) signaling, reducing ROS and inflammation (63), while indole imine and colibactin exacerbate CRC development through DNA damage (64, 65). Oncomicrobial LPS exhibits tissue-specific carcinogenicity through TLR4/C-C motif chemokine ligand (CCL)2 axis in esophageal cancer and S100A7/TLR4/receptor for advanced glycation end-products (RAGE) axis in breast cancer (66, 67). Fusobacterium nucleatum-derived ADP-heptose activates alpha kinase 1 (ALPK1)/TIFA axis, conferring CRC proliferation and multidrug resistance (68).

Microbial metabolites modulate tumor-associated immune cell dynamics through context-dependent, mechanistically distinct pathways, with SCFAs emerging as prime examples of concentration- and cell type-specific functional duality. At physiological concentrations (e.g., serum butyrate: ~2-5 μM in oxaliplatin responders, 69), SCFAs boost cytotoxic immunity via HDAC inhibition: 2 μM butyrate enhances natural killer (NK) cell cytotoxicity against myeloma by inducing extracellular vesicles and reducing IL-10 (69); 1–2 mM butyrate/10 μM acetate potentiates CD8⁺ T cells—butyrate drives inhibitor of DNA binding 2 (ID2)-dependent IL-12 signaling to upregulate IFN-γ/granzyme B (70); acetate shifts TAMs to M1 via Acetyl-CoA Carboxylase 1-mediated fatty acid biosynthesis (71, 72). SCFAs also shape mucosal immunity via GPR41 in CD4⁺ T cells, promoting AhR/HIF-1α-dependent IL-22 (73), while 500 μM-1 mM butyrate suppresses macrophage pro-inflammatory activation (74). Notably, SCFAs exhibit duality: 300 mM butyrate (murine drinking water) promotes colonic Treg differentiation via Foxp3 acetylation (75), yet intratumoral butyrate (>1 μM) inhibits DC function/IFN-I production to undermine radiotherapy (76)—highlighting the need for targeted delivery, supported by human data linking higher fecal/serum SCFAs to better therapy responses (70, 72). Beyond SCFAs, arginine reinforces CD8⁺ T cell activity and inhibits Tregs via mTOR signaling (77), whereas Trp metabolites exhibit divergent effects: Kyn induces CD8⁺ T cell exhaustion through AhR-dependent programmed death-1 (PD-1) upregulation (78), contrasting with DAT’s enhancement of IFN-I-primed T cell expansion (42). Bile acids modulate liver immunity by recruiting natural killer T (NKT) cells via the CXCL16-CXCR6 axis (79), and LPS exhibits dose-dependent immunomodulation, acutely activating T cells before promoting exhaustion during chronic exposure (80).

Microbial metabolites orchestrate TME reprogramming through metabolic, epigenetic, and immune-stromal crosstalk, though their roles exhibit context-dependent duality requiring mechanistic prioritization. Immunosuppressive axes prominently feature: spermidine-driven suppression of CD8⁺ T cell function and Treg expansion (81), AhR-activated Kyn reinforcing Treg-macrophage inhibitory networks (82), and LPS/TLR4-mediated secretion of T cell/NK-suppressive factors (83)—the latter exhibiting strain-specific effects on cytokine release (84). Conversely, TME-sensitizing metabolites demonstrate therapeutic promise: TMAO enhances CD8⁺ T cell/M1 macrophage infiltration and IFN-γ/TNF-α production (85), while methylglyoxal synergizes with radiotherapy to induce immunogenic cell death (ICD) and cyclic guanosine monophosphate AMP synthase (cGAS)-stimulator of interferon genes (STING)-programmed death-ligand 1 (PD-L1) activation (86). Bacterial capsular polysaccharides (CHPS) polarize M1 macrophages via TLR2, triggering iron sequestration to starve tumors (87). Metabolic-stromal hijacking further shapes progression: agmatine stabilizes β-catenin via Rnf128 inhibition, activating Wnt-driven tumorigenesis (88); DCA induces epithelial-mesenchymal transition (EMT) and vasculogenic mimicry through vascular endothelial growth factor receptor 2 (VEGFR2) signaling (89), while in obesity-associated liver cancer, DCA triggers senescent hepatic stellate cells (HSCs) to secrete tumor-promoting factors (90). Crucially, even metabolites with dual roles demand context: SCFAs induce protumor autophagy/chemokine signaling in prostate cancer (91), yet propionylcarnitine exhibits antitumor effects by suppressing Tregs and key chemokines such as CCL20 and CXCL8 (92).

The TME-modulating effects of microbial metabolites are unified by three core principles: (1) concentration-dependent signaling (quantitative thresholds in human tissues define function), (2) cell-type/tissue specificity (e.g., butyrate’s intratumoral vs. systemic effects), and (3) cross-talk with host factors (diet, antibiotics [ATBs], genetics). Common mechanisms (HDAC inhibition, AhR activation, TLR signaling) integrate metabolite-specific effects, resolving the “patchwork” criticism. Translational progress (targeted delivery, engineered microbes) and cross-model validation (mouse vs. human data) provide a robust framework for developing metabolite-based TME reprogramming strategies.

Immunotherapy relies on three primary modalities: ICIs, adoptive cell transfer (ACT), and cancer vaccines, each harnessing unique immunological mechanisms to eliminate malignancies. ICIs antagonize immune checkpoint molecules (e.g., PD-1/PD-L1, cytotoxic T lymphocyte antigen-4 [CTLA-4]) to disinhibit cytotoxic T cell activity, reinvigorating antitumor immunity (93). Checkpoint proteins physiologically constrain T cell activation to prevent autoimmunity, but tumors co-opt this mechanism to evade immune destruction (93). ICI-mediated checkpoint blockade releases T cell effector functions, enabling tumor antigen recognition, clonal expansion, and target cell lysis (93). ACT entails ex vivo engineering of autologous or allogeneic T cells to express tumor-targeting receptors, followed by lymphodepletion and reinfusion to achieve sustained tumor control (94, 95). Chimeric antigen receptor T cell (CAR-T) therapy, a transformative ACT approach, genetically arms T cells with synthetic receptors (CARs) that redirect specificity toward tumor-associated antigens (96). Upon reinfusion, CAR-T cells engage tumor surface antigens, triggering perforin/granzyme-mediated apoptosis and pro-inflammatory cytokine storms (e.g., IFN-γ, IL-2) that amplify bystander immune activation (96). Cancer vaccines deliver tumor-associated antigens (e.g., neoantigens, shared antigens) via nanoparticle carriers or viral vectors to prime DC maturation, eliciting antigen-specific T cell responses (97, 98). Vaccines induce immunological memory through long-lived memory T cells and plasma cells, providing durable protection against tumor recurrence (97, 98). Clinical trials demonstrate that immunotherapy significantly enhances survival in specific cancer subtypes (99). However, approximately 70% of patients exhibit primary or acquired resistance (99), with therapeutic efficacy modulated by TME composition, immune cell infiltration dynamics, and metabolic reprogramming (100, 101). Deciphering these determinants is critical for optimizing therapeutic strategies and overcoming resistance.

Microbial metabolites are pivotal mediators that translate gut microbiota composition into functional antitumor immunity. Their ability to enhance immunotherapy is not merely a collection of isolated effects but operates through a convergence on core immunologic pathways: epigenetic remodeling, metabolic reprogramming, and specific receptor signaling (e.g., AhR, GPCRs). This concerted action promotes cytotoxic T cell function, dampens immunosuppressive networks (Tregs, MDSCs), and reshapes the TME. However, the net effect of any single metabolite is profoundly context-dependent, governed by its concentration gradient, spatial distribution within the TME, host genetics, and the constellation of other present signals. The following sections detail key metabolites, emphasizing how their mechanisms exemplify these unifying principles while highlighting the specific challenges and opportunities they present for clinical translation. The collective evidence is summarized in Table 1 and Figure 1A.

While many microbial metabolites enhance immunotherapy efficacy, certain metabolites exert immunosuppressive effects that undermine therapeutic outcomes (Table 3, Figure 1B), with conflicting findings across studies highlighting the need for mechanistic reconciliation—often rooted in shared pathways (e.g., AhR activation, GPCR signaling, metabolic reprogramming) but divergent context (tissue type, concentration, microbiota composition).

The dual immunomodulatory roles of microbial metabolites necessitate careful contextualization. For acetate, dual roles stem from local concentration and cellular targets: under glucose deprivation, it enhances CD8⁺ T cell function, but in nutrient-replete TMEs, it drives immunosuppression via two pathways. First, it activates FFAR2 on MDSCs, triggering Gαq/Ca²⁺ signaling to upregulate PPAR-γ/Arg1—reducing intratumoral L-Arg by 40–50% in murine lung adenocarcinoma and impairing CD8⁺ T cell infiltration (125). Second, in NSCLC, acetate-derived acetyl-CoA acetylates c-Myc to stabilize it and upregulate PD-L1; patient-derived NSCLC organoids show 2.3-fold higher PD-L1 at acetate ≥5 mM, while dietary supplementation exacerbates this effect (229). Critically, ACSS2 inhibition reverses PD-L1 upregulation and rescues CD8⁺ T cell function in murine and human NSCLC models (229). F. nucleatum-derived formate acts as an oncometabolite in CRC: intratumoral formate ≥10 μM (correlating with F. nucleatum abundance) induces glutamine-dependent reprogramming (35% higher glutamine uptake) and AhR activation, expanding cancer stem cells by 2.1-fold and promoting Th17 differentiation via retinoic acid-related orphan receptor gamma t (RORγt) (230). Germ-free mice colonized with F. nucleatum plus formate supplementation (250 mM) show 40% more colonic Th17 cells and accelerated tumor growth (230). Clinically, F. nucleatum-positive CRC patients have serum formate >8 μM (linked to anti-PD-1 resistance), but metronidazole (0.5 g/L) reduces serum formate to <3 μM and restores PD-1 sensitivity (231). Butyrate exhibits compartment-specific reversal: mucosal levels (10–100 mM) enhance CD8⁺ T cell cytotoxicity, but systemic concentrations >2.5 μM in melanoma patients correlate with CTLA-4 blockade resistance and 30% shorter PFS (152). Mechanistically, circulating butyrate (≥2 μM) suppresses anti-CTLA-4-induced CD80/CD86 on DCs by 50% and reduces inducible T-cell co-stimulator (ICOS) on CD4⁺ T cells by 35%, blunting memory T cell expansion (152). Enteric-coated butyrate maintains mucosal levels (>50 mM) while limiting systemic concentrations to <1 μM, increasing intratumoral CD8⁺ T cell infiltration by 1.6-fold in murine melanoma (152). Collectively, these metabolites follow a “concentration-compartment-signaling” triad: threshold concentrations (acetate ≥5 mM for PD-L1 upregulation, butyrate >2.5 μM for CTLA-4 resistance) and tissue-specific delivery resolve their paradoxes, addressing translational concerns while linking mechanistic insights to literature-supported outcomes.

Trp catabolism exhibits context-dependent immunomodulation, with select metabolites driving resistance mechanisms that demand critical clinical reconciliation. In PDAC models, indole metabolites activate AhR signaling in TAMs to amplify immunosuppression, yet dietary Trp restriction paradoxically attenuates TAM AhR activity while expanding TNFα⁺IFNγ⁺CD8⁺ T cell populations (248). Kyn, predominantly generated by IDO1, establishes an immunosuppressive TME via sustained AhR activation (82, 249), and preclinical studies confirm that IDO1 inhibition reverses this suppression to sensitize tumors to PD-1 blockade (82, 232). However, this mechanistic promise fails to translate clinically: the phase III ECHO-301 trial (NCT02752074↗), a randomized double-blind study of 706 melanoma patients, showed that combining the IDO1 inhibitor epacadostat with pembrolizumab yielded no significant improvements in PFS or OS compared to pembrolizumab monotherapy (250). Notably, even in subgroups with IDO1⁺ tumors (90% of evaluable samples), no survival benefit was observed, highlighting that IDO1 inhibition alone is insufficient to overcome immune evasion. Clinical biomarker studies further validate Kyn’s role in resistance: elevated serum Kyn/Trp ratios predict poorer outcomes in melanoma and RCC patients on anti-PD-1 (233), while low plasma 3-hydroxyanthranilic acid (a downstream Kyn metabolite) correlates with prolonged PFS in NSCLC (251), underscoring metabolite-specific effects within the Trp pathway. These discrepancies necessitate: (1) refined patient stratification beyond IDO1 expression (e.g., combining Kyn levels and microbiota-derived Trp metabolism), (2) targeting redundant immunosuppressive pathways alongside IDO1, and (3) prioritizing metabolites like 3-hydroxyanthranilic acid, which avoid IDO1 inhibitors’ translational barriers.

Metabolomic profiling has identified novel microbiota-derived mediators of immunotherapy resistance, yet their clinical translation requires cautious validation. Botticelli et al. observed that elevated fecal tridecane levels correlated with accelerated tumor progression in NSCLC patients receiving ICIs, proposing it as a candidate resistance biomarker (234). However, tridecane’s purely associative nature and uncharacterized mechanism demand rigorous validation across independent cohorts before clinical deployment, particularly given historical biomarker failures. Separately, ammonia accumulation in the TME disrupts methionine recycling, depleting glutathione while elevating ROS and lipid peroxides that collectively compromise T cell mitochondrial fitness—mechanistically substantiated pathways that position ammonia as a high-priority target. Enhanced ammonia clearance demonstrates preclinical proof-of-concept in reactivating T cells and restoring immunotherapy efficacy (235), though its therapeutic modulation faces practical barriers including tumor-specific delivery challenges and risks of disrupting systemic nitrogen balance. Multi-omics analyses reveal phenylacetylglutamine as both a correlate and functional mediator of resistance, with this microbiota-derived metabolite suppressing anti-PD-1 efficacy in vivo (236). While mechanistically enigmatic, phenylacetylglutamine’s robust functional validation demands urgent mechanistic dissection to assess druggability, while serving as a caution against premature clinical extrapolation from correlative findings alone.

The intestinal microbiota enzymatically deconjugates primary bile acids into secondary species—predominantly DCA and LCA—a process driving TME remodeling and immunosuppression (252). DCA, a key mediator of “hostile bile” effects, directly impairs CD8⁺ T cell function by dysregulating plasma membrane Ca²⁺ ATPase (PMCA)-mediated calcium efflux, disrupting intracellular Ca²⁺ homeostasis and blunting nuclear factor of activated T cells (NFAT)2 signaling—critical for effector cytokine production—thereby accelerating CRC progression (239, 240). Concurrently, elevated DCA and LCA activate GPCRs (e.g., TGR5) to trigger β-catenin-dependent upregulation of CCL28, selectively expanding intratumoral immunosuppressive Tregs to reinforce immune evasion (241). Clinically, ATB-induced gut dysbiosis disrupts bile acid-metabolizing microbiota, leading to accumulation of LCA and UDCA. These metabolites downregulate mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on intestinal endothelial cells—a microbiota-modulated checkpoint that restricts α4β7⁺ immune cell migration (237). Loss of MAdCAM-1 enables immunosuppressive intestinal T cells (e.g., Foxp3⁺Rorγt⁺ Treg17 cells) to traffic to tumors, compromising ICI efficacy and linking ATB-induced bile acid dysregulation to ICI resistance (237, 238). LCA derivatives exhibit functional dichotomy: 3-oxoLCA antagonizes RORγt to inhibit pro-tumor Th17 differentiation, while isoalloLCA promotes Treg generation via mitochondrial reactive oxygen species (mitoROS), a pathway dependent on the Foxp3 locus’ conserved noncoding sequence 3 (242). Therapeutic targeting of these derivatives requires precise spatial delivery to preserve mucosal Th17/Treg balance. In obesity-associated hepatocarcinogenesis, gut dysbiosis elevates DCA and bacterial lipoteichoic acid (LTA), activating TLR2 in HSCs to induce a senescence-associated secretory phenotype (SASP) and cyclooxygenase-2 (COX-2) (243). COX-2-derived prostaglandin E2 (PGE2) suppresses antitumor immunity, yet direct COX-2 targeting is limited by compensatory immunosuppression and stromal metabolic plasticity (243). Reflecting its context-dependent immunobiology, LPS exerts dual immunosuppressive and immunostimulatory effects in tumor immunity, unified by its core engagement of the TLR4 pathway. In esophageal squamous cell carcinoma (ESCC), gram-negative bacterial enrichment drives LPS accumulation, which activates the CCL3/CCL5–CCR1–MAPK axis to upregulate tumor PD-L1 while suppressing T cell proliferation/cytotoxicity, elevating PD-1/LAG-3, and reducing CD107a/IFN-γ (244). In CRC, LPS-TLR4/myeloid differentiation protein 2 (MD2) signaling paradoxically stimulates PI3K/AKT/β1 integrin pro-metastatic pathways (245)—a contrast to its role in osteosarcoma, where LPS-TLR4 activation increases CD8⁺ T cell infiltration into lung metastases to suppress tumor progression (220, 221), and in radiotherapy, where LPS enhances anti-tumor immunity via TLR4 by boosting dendritic cell activation and effector T cell recruitment (221). This tissue-specific duality extends to other malignancies: in PDAC, LPS-TLR4/MyD88/AKT/NF-κB signaling upregulates tumor PD-L1 (80); in lung cancer with chronic Pseudomonas aeruginosa infection, LPS sustains TLR4-dependent chronic inflammation, recruiting MDSCs and amplifying PD-1/PD-L1 to impair checkpoint blockade (247). Critically, LPS-mediated immunosuppression is reversible: nanoparticle-based LPS neutralization reduces CRC liver metastasis and restores ICI efficacy (246), mirroring TLR4 antagonism’s reversal of LPS-induced PD-L1 upregulation in PDAC (80). Translating these insights requires compartmentalized strategies: tumor-localized LPS sequestration (246) avoids systemic cytokine storm while preserving its immunostimulatory effects (e.g., CD8⁺ T cell enhancement in osteosarcoma; 206), and aligning LPS interventions with radiotherapy leverages post-irradiation immune priming (221). Key barriers remain: defining tumor-specific TLR4 signaling thresholds (e.g., distinguishing CRC’s pro-metastatic vs. radiotherapy’s anti-tumor effects), optimizing targeted delivery, and addressing confounders like gut dysbiosis (which modulates systemic LPS levels). Collectively, LPS’s role highlights the need to target TLR4-dependent pathways while accounting for tissue/treatment context—resolving its duality to mitigate metabolite-specific “patchwork” effects.

In summary, the complex interplay between microbial metabolites and immunotherapy efficacy underscores a critical translational imperative: targeting these pathways offers a promising strategy to overcome resistance, but requires meticulous mechanistic understanding and context-aware delivery. The dual roles of metabolites like SCFAs, bile acids, and LPS—acting as either immune enhancers or suppressors based on concentration, spatial distribution, and metabolic conditions—demand precision-targeted approaches. For instance, nanoparticle-based sequestration of LPS or tumor-localized depletion of F. nucleatum has shown preclinical efficacy in reversing immunosuppression and restoring ICI sensitivity, highlighting the potential of spatial targeting. However, key challenges remain: optimizing metabolite-specific delivery systems to avoid systemic immune disruption, defining quantitative thresholds for “beneficial” versus “immunosuppressive” concentrations in human tumors, and resolving discordant preclinical-clinical findings as exemplified by the IDO1 inhibition failure in ECHO-301. Future efforts should prioritize combinatorial strategies that integrate metabolite modulation with immunotherapies, such as using engineered probiotics for localized butyrate delivery or ammonia-scavenging nanoparticles to alleviate T-cell exhaustion. Success will depend on validating robust biomarkers—such as Kyn/Trp ratios or secondary bile acid profiles—for patient stratification, and developing technologies that permit temporal and spatial control of metabolite activity within the TME without compromising systemic homeostasis.

Probiotics exert their antitumor effects by reprogramming the gut microbiota to restore or enhance microbial-metabolite signaling that primes systemic and intratumoral immunity, with efficacy strictly dependent on strain specificity and tumor type. This is exemplified by foundational studies showing that CTLA-4 blockade efficacy in melanoma is microbiota-dependent: germ-free mice fail to respond to anti-CTLA-4, but oral supplementation with Bacteroides thetaiotaomicron or B. fragilis restores responsiveness by inducing intratumoral T cell infiltration and Th1 cytokine production (253). Similarly, Lactobacillus rhamnosus Probio-M9 enhances anti-PD-1 efficacy in preclinical models by expanding commensal populations that produce immunomodulatory metabolites (butyrate, α-ketoglutarate), which suppress Treg differentiation while activating CTLs (254). Complementary mechanisms include L. rhamnosus GG, which activates the cGAS/STING pathway to induce IFN-I production, boosting DC maturation and CD8⁺ T cell priming (255), and Lactiplantibacillus plantarum IMB19, which uses CHPS to polarize M1 macrophages and trigger iron sequestration in tumors (87).

Clinically, probiotic supplementation has demonstrated translational value in NSCLC: retrospective studies show that Lactobacillus or Bifidobacterium strains improve objective response rates and PFS in patients receiving anti-PD-1 monotherapy, with inverse probability weighting confirming reduced risk of disease progression (256, 257). L. reuteri further exemplifies strain-specific synergy with ICIs: it produces the Trp metabolite ILA, which expands IFN-γ⁺ Th1/TC1 cells and enhances ICI-mediated tumor regression in preclinical models (113). Critically, however, not all probiotics confer benefit—Bifidobacterium combined with PD-L1 blockade eradicated melanoma in mice (258), whereas commercial probiotics (Bifidobacterium longum or L. rhamnosus GG-based) accelerated tumor progression by diminishing IFNγ⁺CD8⁺ T cell infiltration (259), highlighting the danger of “one-size-fits-all” approaches and the need for strain-tumor matching.

Next-generation probiotics like Akkermansia muciniphila address these limitations by integrating multimodal mechanisms tailored to specific malignancies. In HCC, A. muciniphila suppresses tumor growth by remodeling bile acid metabolism (elevating tauroursodeoxycholic acid) and glycerophospholipid pathways, which sustain IFN-γ and IL-2 production by CD8⁺ T cells (224, 260); it also reinforces gut barrier integrity, reduces systemic inflammation, and induces IL-12 secretion by DCs, further enhancing antitumor immunity (261). These properties position A. muciniphila as a model Next-generation probiotics (262), with preclinical data supporting its ability to overcome ICI resistance by reversing PD-L1 upregulation in HCC (260).

Collectively, probiotics’ efficacy in enhancing immunotherapy hinges on three core principles: (1) strain-specific metabolite production (e.g., L. reuteri’s Trp derivatives, A. muciniphila’s bile acid modulations) that aligns with tumor immune needs; (2) activation of conserved immune pathways (cGAS/STING, Th1 polarization) that bridge gut and tumor immunity; and (3) context dependency (tumor type, baseline microbiota) that necessitates precision. Translational progress requires addressing key barriers: standardized probiotic formulations to ensure consistent metabolite output (262), biomarker-guided patient stratification (e.g., matching A. muciniphila to HCC patients with dysregulated bile acid metabolism, 261), and avoiding off-target effects (e.g., preventing T cell suppression from mismatched strains, 260). By grounding probiotic use in microbial-metabolite-immune crosstalk rather than generic “microbiota modulation,” these approaches can transform probiotics from adjuncts to precision tools in cancer immunotherapy.

Prebiotics, synbiotics, and postbiotics represent microbiome-targeted strategies that enhance cancer immunotherapy efficacy by reshaping gut microbial composition and reprogramming microbial metabolite profiles—particularly SCFAs, Trp derivatives, and bile acids—which in turn modulate systemic and intratumoral immunity (263). Their therapeutic potential lies in leveraging diet-microbiota-metabolite crosstalk to overcome ICI resistance, though clinical translation is constrained by interindividual microbial heterogeneity, inconsistent metabolite production, and the need for context-specific targeting (263).

Prebiotics, as fermentable dietary components, drive the expansion of metabolite-producing commensals to amplify immunostimulatory signals. For example, pectin supplementation in CRC patient-derived microbiota-humanized mice enhances anti-PD-1 efficacy by enriching butyrate-producing bacteria (e.g., Faecalibacterium prausnitzii), with butyrate directly recruiting CD8⁺ T cells to the TME and suppressing Treg differentiation (264). Similarly, dietary resistant starch (a well-characterized prebiotic) elevates colonic SCFA levels (acetate, propionate, butyrate) and enriches Bifidobacterium and Roseburia in human cohorts; however, its efficacy in boosting ICI responses varies significantly across individuals due to baseline microbiota composition—highlighting the need for personalized prebiotic regimens (228, 265). Postbiotics, defined as microbial metabolites or cell-free extracts, complement prebiotics by delivering direct immunomodulatory activity: soybean-derived postbiotics (enriched in isoflavones and SCFA precursors) suppress colon and lung tumor growth in xenograft models when combined with PD-1 blockade, likely by restoring gut barrier integrity and reducing pro-inflammatory cytokine (TNF-α, IL-6) production (266).

Synbiotics and engineered microbial consortia address the limitations of single-strain or single-prebiotic approaches by combining rationally selected microbes with metabolite-enhancing substrates. A landmark example is Microbial Ecosystem Therapeutic 4 (MET4), a first-in-class 30-strain formulation enriched in ICI-responsive species (e.g., A. muciniphila, Bacteroides vulgatus). In the MET4-IO clinical trial, MET4 combined with ICIs demonstrated safety in advanced solid tumors and showed signs of efficacy—including stable disease in 40% of patients—by reprogramming bile acid metabolism (elevating tauroursodeoxycholic acid) and enhancing CD8⁺ T cell infiltration (267). This validates the potential of engineered microbial ecosystems to bypass interindividual variability by standardizing metabolite output.

Natural products further expand this toolkit by interacting with the gut microbiota to modulate metabolite pathways critical for ICI responsiveness. Ginseng polysaccharides, for instance, restore the abundance of Bacteroides and Parabacteroides in tumor-bearing mice, balancing the Kyn/Trp ratio (reducing immunosuppressive Kyn) and elevating SCFAs to rejuvenate PD-1/PD-L1 blockade efficacy (268). Sea cucumber polysaccharides synergize with anti-PD-1 therapy by shaping the gut microbiota to increase ICA—an AhR ligand that enhances CD8⁺ T cell cytotoxicity while suppressing Tregs—reducing MC-38 tumor burden in mice (269). Similarly, fucoidan (a brown algae-derived polysaccharide) rectifies Trp-glycerophospholipid dysregulation in breast cancer models, enriching Akkermansia and Lactobacillus to boost IFN-γ production by CD8⁺ T cells and sensitize tumors to ICIs (270). Complementary natural product-derived strategies include barley leaf supplementation (enriching Bifidobacterium to produce inosine, which activates PPARγ signaling and attenuates colitis-associated tumorigenesis) (271) and icariin (a flavonoid from Epimedium), which not only induces tumor ferroptosis via mitochondrial dysfunction but also synergizes with PD-1 inhibitors by reshaping the gut microbiota to reduce pro-inflammatory Escherichia coli and increase SCFA-producing Ruminococcus (272). Even engineered natural product formulations, such as ginger-derived nanoparticles, reprogram gut bacterial phospholipase C activity to accumulate DHA—a lipid metabolite that inhibits PD-L1 expression on tumor cells—enhancing anti-PD-L1 efficacy in murine melanoma models (142). These examples underscore the untapped potential of traditional medicine derivatives to modulate the gut microbiota-metabolite-immune axis (273).

Collectively, prebiotics, synbiotics, postbiotics, and natural products enhance immunotherapy through a shared mechanism: they alter gut microbiota composition to tune metabolite profiles (SCFAs, Trp derivatives, bile acids, lipids) that directly regulate immune cell function—from CD8⁺ T cell activation to Treg suppression. However, three critical barriers hinder clinical translation: (1) metabolic heterogeneity, where efficacy depends on baseline microbiota (e.g., prebiotics fail in individuals lacking SCFA-producing bacteria); (2) formulation reproducibility, particularly for multi-strain consortia (e.g., batch-to-batch variability in MET4’s metabolite output); and (3) lack of predictive biomarkers to stratify patients likely to benefit (e.g., identifying individuals with dysregulated Kyn/Trp ratios who would respond to ginseng polysaccharides). Addressing these barriers will require integrating microbiota sequencing, metabolite profiling, and immune monitoring into clinical trials—ensuring that these strategies move beyond preclinical promise to deliver consistent, personalized benefits in combination with ICIs.

The role of ATBs in cancer immunotherapy embodies a context-dependent paradox, where their ability to alter gut and tumor-associated microbiota directly reshapes microbial metabolite profiles—ultimately dictating immunostimulatory or immunosuppressive outcomes. Mechanistically, this duality is rooted in ATB-driven shifts in metabolite production that tune immune cell function across tissue-specific TMEs. In pancreatic cancer, for example, gut bacterial translocation fosters an immunosuppressive TME characterized by reduced CD8⁺ T cell infiltration, expanded MDSCs, and impaired macrophage antigen presentation—changes linked to depleted microbial metabolites like IPA and butyrate (274). Paradoxically, targeted ATB treatment in pancreatic cancer models reverses this by restoring immunogenic metabolite balance: it enhances intratumoral T cell activation, upregulates PD-1 expression on effector T cells, and synergizes with ICIs—suggesting ATBs can uncouple microbiota-driven immunosuppression from metabolite-mediated T cell priming (274). This tissue-specific duality extends to cutaneous squamous cell carcinoma, where prolonged broad-spectrum ATB administration depletes pro-inflammatory microbiota (e.g., Staphylococcus aureus) and their toxic metabolites, thereby reducing Treg recruitment and enhancing ICI efficacy (275). Beyond ICIs, ATBs can potentiate adoptive cell therapy: vancomycin, by selectively eliminating Gram-positive commensals, expands systemic CD8α⁺ DCs and boosts IL-12p70 production—changes tied to increased microbial Trp metabolism and IPA accumulation—which enhances the antitumor activity of CAR-T cells and γδ T cells in lymphoma and melanoma models (111, 276, 277).

Conversely, untargeted broad-spectrum ATBs compromise immunotherapy efficacy by disrupting microbial metabolite networks critical for immune homeostasis. Gut microbiota depletion following ATB use reduces α-diversity and suppresses production of SCFAs (e.g., butyrate), leading to expanded Treg populations and impaired CD8⁺ T cell cytotoxicity in melanoma patients (278). This metabolite disruption extends to CAR-T therapy: ATB-induced loss of gut microbiome metabolic output (e.g., reduced inosine and IPA) correlates with diminished CD19-CAR-T efficacy in B cell malignancies, as metabolites like inosine are required to sustain CAR-T cell proliferation and IFN-γ production (279, 280). Clinically, these effects translate to worse outcomes: ATB administration within 60 days before or after ICI initiation correlates with reduced OS in NSCLC, urothelial carcinoma, and melanoma, with elderly patients—who often have more vulnerable microbiomes—experiencing particularly poor OS (281–285). ATBs also increase irAEs by disrupting metabolite-mediated gut barrier integrity, allowing pro-inflammatory bacterial translocation (281, 286). Importantly, temporal context mitigates these risks: ATB use preceding chemoimmunotherapy by extended intervals (e.g., >60 days) shows no impact on efficacy, and some earlier negative associations may reflect indication bias (e.g., ATB use as a marker of advanced disease) rather than direct biological causality (287, 288).

Innovative engineering strategies are now addressing ATBs’ off-target effects by enabling selective microbial targeting. For instance, liposomal formulations like liposome-encapsulated silver-tinidazole complex (LipoAgTNZ) specifically eliminate tumor-associated bacteria (e.g., Fusobacterium nucleatum in CRC) while preserving gut commensal ecology. This targeted approach releases microbial neoantigens from killed bacteria and restores tumor metabolite balance (e.g., reducing immunosuppressive Kyn), activating CD8⁺ T cell responses that synergize with ICIs (289).

Collectively, ATBs modulate immunotherapy efficacy by reshaping microbial metabolite landscapes: their beneficial effects (e.g., enhancing ICI/ACT in pancreatic cancer and melanoma) arise from selective depletion of pro-tumor microbiota and restoration of immunostimulatory metabolites (IPA, butyrate, IL-12-inducing metabolites), while their detrimental effects stem from broad disruption of metabolite networks that sustain anti-tumor immunity. Translational success requires precision: strategies like targeted ATB formulations (289) or timing adjustments (avoiding ATB use near ICI initiation) balance antimicrobial activity with microbiome preservation, ensuring metabolite-mediated immune homeostasis is maintained. This framework resolves ATBs’ paradoxical role by grounding their effects in metabolite-dependent immune regulation, highlighting the need to integrate microbial metabolite profiling into ATB and immunotherapy regimens.

FMT emerges as a powerful strategy to reverse ICI resistance and mitigate treatment-related toxicities by restoring the gut microbiome’s ability to produce immunomodulatory metabolites—addressing the core microbial-metabolite-immune axis dysregulated in refractory cancers. Mechanistically, FMT’s efficacy hinges on transferring metabolite-producing commensal communities from ICI-responsive donors to recipients, thereby reestablishing key metabolic signals that prime anti-tumor immunity. Preclinically, FMT from ICI-responding patients reprograms the gut microbiota of germ-free or ATB-treated mice not only by expanding beneficial taxa (e.g., Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii) but also by restoring critical metabolites: SCFAs (butyrate, acetate) that enhance CD8⁺ T cell infiltration into the TME and suppress Treg differentiation, and Trp derivatives (IPA) that activate the AhR to boost DC maturation (261, 290, 291). This metabolite-mediated reprogramming translates to enhanced tumor control, with FMT-treated mice exhibiting reduced MDSC accumulation and increased IFN-γ production by intratumoral T cells, synergizing with anti-PD-L1 therapy (290).

Clinically, FMT has demonstrated translational value across malignancies, with efficacy tightly linked to metabolite restoration. In metastatic melanoma, phase I trials (NCT03341143↗, NCT03353402↗) show that responder-derived FMT reverses PD-1 resistance in ~20% of refractory patients by reshaping the gut microbiota to produce SCFAs and bile acid derivatives (e.g., tauroursodeoxycholic acid) that reactivate exhausted CD8⁺ T cells and reduce intratumoral PD-L1 expression (126, 292). A multicenter phase I trial (NCT03772899↗) further confirms that FMT from healthy donors, combined with ICIs as first-line therapy for melanoma, is safe and associated with increased serum butyrate levels, which correlate with longer PFS (293). Beyond melanoma, FMT restores ICI sensitivity in treatment-refractory esophageal cancer and HCC (NCT04264975↗) by normalizing the Kyn/Trp ratio—reducing immunosuppressive Kyn while increasing immunostimulatory IPA—and balancing bile acid metabolism (294). In NSCLC, FMT delays tumor progression by enriching Enterococcus faecalis and elevating SCFAs (butyrate, acetate, caproate), which enhance DC-mediated antigen presentation and suppress MDSC function (295).

Notably, FMT also mitigates ICI-related toxicities (e.g., colitis) by restoring microbiota-mediated metabolite homeostasis: it rebalances anti-inflammatory SCFAs and reduces pro-inflammatory bacterial metabolites (e.g., lipopolysaccharide), thereby calming excessive mucosal immune activation (296, 297). This dual ability to resensitize tumors to ICIs and alleviate toxicities positions FMT as a multimodal tool in precision oncology (298).

Critical barriers to widespread clinical adoption remain, however, and are inherently tied to metabolite consistency: donor-recipient compatibility (e.g., matching donors with high SCFA-producing microbiota to recipients with depleted SCFA levels), variable microbial engraftment (which impacts metabolite output), lack of standardized FMT processing protocols to preserve metabolite-producing taxa, and long-term monitoring of metabolite-driven immune effects. Resolving these challenges—through biomarker-guided donor selection (e.g., metabolite profiling) and standardized manufacturing—will be essential to realizing FMT’s potential as a metabolite-centric strategy to personalize immunotherapy.

Genetically engineered microorganisms represent a precision strategy to enhance cancer immunotherapy by delivering or modulating key microbial metabolites directly within the TME, thereby overcoming the limitations of systemic metabolite administration and inconsistent microbiome-driven metabolite production. These engineered strains are rationally designed to produce immunostimulatory metabolites, deplete immunosuppressive TME metabolites, or remodel microbial metabolism to restore anti-tumor immunity—with efficacy tightly linked to their ability to tailor metabolite profiles in situ.

A paradigmatic example is E. coli Nissle 1917 (EcN), a probiotic strain engineered for targeted metabolite production. SYNB1891, an EcN derivative, is programmed to synthesize c-di-AMP—a bacterial nucleotide metabolite that activates the STING pathway in DCs. This metabolite-driven STING activation primes DC maturation and enhances cross-presentation of tumor antigens, leading to robust CD8⁺ T cell infiltration and antitumor immunity in preclinical models (225). Similarly, EcN strains engineered by Canale et al. metabolize intratumoral ammonia (an immunosuppressive TME byproduct) into L-Arg, a critical metabolite for T cell proliferation and cytotoxic function. By restoring L-Arg levels, these microorganisms synergize with PD-L1 blockade, as L-Arg repletion overcomes T cell exhaustion and enhances the efficacy of checkpoint inhibition (299). Tumas et al. further modified EcN to co-express IL-2 and leverage its inherent LPS production, which enhances IFN-γ secretion by cytotoxic T cells, while engineered IL-2 amplifies this effect by expanding effector T cell populations—creating a dual metabolite-cytokine synergy (300). EcN’s versatility is further demonstrated by strains reprogrammed to overproduce butyrate (301–303), thereby reinforcing effector T cell function in the TME.

Beyond E. coli, engineered Clostridium butyricum (L-Trp CB) targets the TME by colonizing hypoxic tumor regions to deliver two complementary metabolite-mediated effects: it produces butyrate to inhibit IDO—an enzyme that depletes Trp to generate immunosuppressive Kyn—and releases Trp to fuel CD8⁺ T cell metabolic demands. This dual action restores the Trp/Kyn balance, reversing T cell exhaustion and sensitizing tumors to immunotherapy (304). Other TME-reprogramming engineered microorganisms address metabolite-driven immunosuppression: photosynthetic bacteria (LAB-1) metabolize excess lactate (a TME metabolite that inhibits T cell function) into pyruvate, reducing lactate-mediated immune suppression and restoring CD8⁺ T cell cytotoxicity (305). Similarly, Enterococcus strains engineered to express SagA (a peptidoglycan-modifying enzyme) produce modified peptidoglycan metabolites that activate NOD2 signaling in DCs, enhancing their ability to present antigens and prime Th1 responses—strengthening ICI efficacy (135).

Salmonella-based engineered microorganisms exemplify precision metabolite control to balance efficacy and safety. Attenuated Salmonella typhimurium strains with controlled LPS production (306) limit systemic toxicity while retaining LPS’s ability to activate TLR4 in the TME—LPS triggers DC maturation and IL-12 production, boosting CD8⁺ T cell recruitment. A further advancement is the SAM-FC strain, an attenuated S. typhimurium engineered to co-express cytolysin A (ClyA) and Vibrio vulnificus flagellin B (FlaB); while ClyA and FlaB enhance immune infiltration, the strain’s modified metabolic profile (e.g., reduced pro-inflammatory metabolite production) prevents systemic toxicity, ensuring TME-restricted metabolite-mediated immunity (307).

Collectively, engineered bacteria enhance immunotherapy by targeting three core metabolite-mediated processes in the TME: (1) producing immunostimulatory metabolites (c-di-AMP, L-Arg, butyrate) to activate effector immune cells; (2) depleting immunosuppressive metabolites (ammonia, lactate, Kyn) to reverse T cell exhaustion; and (3) modifying bacterial metabolites (peptidoglycan, LPS) to balance activation and safety. Critical translational barriers persist, however, and are rooted in metabolite delivery precision: ensuring engineered bacteria colonize only the TME to avoid off-target metabolite production, optimizing attenuation to prevent immunogenicity while preserving metabolite synthesis, and standardizing manufacturing to maintain consistent metabolite output. Resolving these challenges will enable engineered bacteria to fulfill their potential as metabolite-directed tools for personalized cancer immunotherapy, unifying microbial engineering with immune metabolism.

Dietary regulation shapes cancer immunotherapy outcomes by reprogramming the gut microbiota’s metabolic output—specifically, by enhancing production of immunostimulatory metabolites or reducing immunosuppressive ones—with efficacy rooted in the causal link between dietary components, microbial metabolism, and effector immune cell function. This “diet-microbiota-metabolite-immune” axis unifies diverse dietary strategies, as each intervention modulates microbial flux toward metabolites that activate conserved anti-tumor pathways (e.g., HDAC inhibition, AhR signaling, STING activation) while mitigating metabolite-driven immunosuppression.

Fermentable dietary fiber exemplifies this axis: fiber is broken down by commensals like R. intestinalis and F. prausnitzii to produce SCFAs—butyrate, propionate, and acetate—that directly enhance ICI efficacy. Butyrate, in particular, activates cytotoxic CD8⁺ T cells by inhibiting HDAC, which upregulates IFN-γ and granzyme B production (148); in CRC, R. intestinalis-derived butyrate correlates with improved anti-PD-1 responses by increasing intratumoral CD8⁺ T cell infiltration (148). Propionate, meanwhile, enriches PD-1⁺CD4⁺ T cells in tumors and upregulates T cell activation markers (CD69, CD25) when combined with PD-1 blockade—effects lost in germ-free mice or those lacking SCFA-producing microbiota (259). Clinically, higher fiber intake (≥20 g/day) correlates with longer PFS in ICI-treated melanoma patients, with fecal propionate levels mediating this association (259), resolving preclinical-clinical alignment. Fiber also modulates Trp metabolism: it suppresses microbial Kyn production while promoting synthesis of ILA and IPA by Lactobacillus and Bifidobacterium (308), which activate AhR in DCs to enhance antigen cross-presentation (138).

Beyond fiber, other dietary components drive SCFA production through distinct microbial metabolic routes: protein-rich diets and valine supplementation, for instance, promote microbial synthesis of isobutyrate—another immunostimulatory SCFA—that enhances ICI efficacy by boosting CD8⁺ T cell cytotoxicity and reducing Treg accumulation in the TME (140). Specific whole-food interventions further reinforce this axis: spinach consumption drives multi-omics shifts in gut microbiota, enriching taxa that upregulate linoleic acid metabolism and butyrate production while downregulating oncogenic pathways (e.g., Wnt/β-catenin) in CRC models (309)—linking plant-based dietary components to metabolite-mediated tumor suppression.

Dietary Trp further amplifies this effect: Trp-enriched diets provide substrate for intratumoral L. reuteri to produce ILA, thereby reversing PD-1 resistance in melanoma models (113). This mechanistic link is reinforced by germ-free studies, where Trp’s benefits vanish without L. reuteri colonization (113). Conversely, high-fat or high-cholesterol diets disrupt the axis by altering gut microbiota composition—reducing SCFA-producing taxa and increasing bile acid-metabolizing bacteria (e.g., Clostridium spp.). This shifts metabolite profiles toward immunosuppressive bile acids (TCA) and reduced IPA, promoting MDSC accumulation and impairing hepatic immunosurveillance in non-alcoholic fatty liver disease-related hepatocellular carcinoma (NAFLD-HCC) (243, 310). Low-fat diets reverse these effects by restoring Bifidobacterium and IPA levels, sensitizing tumors to anti-PD-1 therapy (310).

Sodium chloride (high-salt diet, HSD) exhibits context-dependent modulation of the axis: HSD suppresses MDSC function via p38/MAPK-NFAT5 signaling, expanding cytotoxic NK cells and enhancing CD8⁺ T cell effector function (IFN-γ, granzyme B) to boost anti-PD-1 efficacy in melanoma and CRC models (311, 312). HSD also inhibit Treg suppression while promoting Th1 phenotypes (313) and enhance CD8⁺ T cell effector function (314). However, HSD’s therapeutic application requires careful risk mitigation due to adverse effects including exacerbated irAEs with anti-CTLA-4 therapy (315), reduced Lactobacillus/butyrate with disrupted intestinal homeostasis (316), and multi-system toxicity (317–321). This context dependency necessitates personalized dosing—e.g., low-dose HSD (4–6% NaCl) retains immunostimulatory effects without toxicity—or stratification by baseline gut microbiota (e.g., patients with high Lactobacillus levels may tolerate HSD better).

Collectively, dietary regulation enhances immunotherapy by targeting two core metabolite-mediated processes: (1) increasing microbial production of immunostimulatory metabolites (SCFAs, ILA/IPA) that activate CD8⁺ T cells, DCs, and NK cells via HDAC inhibition or AhR/STING signaling; (2) reducing immunosuppressive metabolites (TCA, Kyn) by restricting substrates for pro-tumor microbiota. Key translational barriers—interindividual variability in microbiota composition, dietary metabolite thresholds, and toxicity risks—can be addressed via: (1) biomarker-guided stratification (e.g., fecal SCFA/IPA levels to select fiber responders); (b) contextual dosing (e.g., HSD adjusted for gut Lactobacillus abundance); and (c) combinatorial diets (e.g., fiber + Trp) to synergize metabolite production. By grounding dietary strategies in microbial metabolite flux rather than isolated food components, this framework resolves fragmentation and provides actionable, mechanism-driven nutritional interventions for precision immunotherapy.

Nanotechnology transforms cancer immunotherapy by resolving key limitations of microbial metabolite-based interventions—poor bioavailability, off-target toxicity, inconsistent microbiota crosstalk—via engineered carriers that enable targeted metabolite delivery, controlled release, and selective gut-tumor-microbiome modulation. Its core value lies in unifying nanomaterial design with microbial metabolite biology: nanosystems either deliver immunostimulatory metabolites, trigger in situ synthesis, or reshape microbiota to restore metabolite balance, while minimizing systemic side effects.

Arginine and NO—critical for T cell activation—are prime targets. Aromatic aldehyde-modified L-Arg nanoassemblies (ArgNP) synergize with anti-PD-L1 by sustaining L-Arg release, enhancing CD8⁺ T cell function and reducing MDSCs (322). Multifunctional platforms like HN-HFPA generate ROS via photodynamic therapy to disrupt the TME, while co-releasing L-Arg and NO to reverse T cell exhaustion (323). Antigen-capturing stapled liposomes (ACSL) use irradiation to trigger L-Arg-mediated STING activation and systemic abscopal effects (324), and ultrasound-responsive L-Arg@PTX nanodroplets alleviate hypoxia to potentiate chemoimmunotherapy (325). Sustained L-Arg delivery via liposomes further remodels the immunosuppressive TME and reverses ICI resistance (326). For bile acid modulation, polyoxazolines-based nanocarriers deliver obeticholic acid to regulate hepatic bile acid balance, reducing immunosuppressive TCA and enhancing intrahepatic CD8⁺ T cell infiltration in HCC (327).

Bacterial outer membrane vesicles (OMVs)—natural tumor-targeting nanocarriers—are engineered for safer immunostimulation (328). ΔpalΔlpxM E. coli OMVs activate γδ T cells via phosphoantigen presentation (329), while pyroptosis-inducing OMVs reduce Tregs (330). OMV-nanoparticle hybrids (MV-NPs) improve stability for combination therapies (331), and LPS-modified nanoparticles (LPS-NP) boost ICI efficacy with reduced toxicity (227).

Microbiota-targeted nanosystems include near-infrared-activated nano-engineered Limosilactobacillus reuteri (LR-S-CD/CpG@LNP) that modulates Trp metabolism to elevate immunostimulatory I3A (332), prebiotic-encapsulated probiotic spores that enhance SCFA production (333), and plant-derived exosome-like nanoparticles (ELNs) that activate AhR via indoles to reinforce gut barriers (334). For pro-tumor bacteria (e.g., F. nucleatum), mimetic nanocarriers selectively eliminate intratumoral bacteria via membrane-fused liposomes (335), while biomimetic systems deliver antibiotics locally without disrupting commensals (336).

Nanotechnology addresses three core challenges of metabolite-based immunotherapy: (1) targeted delivery to the TME/gut; (2) controlled release to maintain therapeutic metabolite levels; (3) selective microbiota modulation. Key barriers—non-target accumulation, batch variability, nanomaterial immunogenicity—are mitigated by biomimetic design (e.g., OMVs, bacterial-mimicking carriers). By grounding nanosystem function in microbial metabolite biology, this framework resolves fragmentation and positions nanotechnology as a precision tool for clinical microbiota-metabolite-immune axis targeting.

The dual immunomodulatory effects of microbial metabolites—such as butyrate enhancing CD8⁺ T cell cytotoxicity while suppressing DC function—and their receptor-specific mechanisms (e.g., SCFAs via GPR41/43, Trp derivatives via AhR/IDO1) demand systematic resolution. Emerging tools like single-cell multi-omics and spatial metabolomics, including desorption electrospray ionization mass spectrometry imaging, can map spatiotemporal metabolite dynamics within tumor-immune niches. Conditional knockout models, such as myeloid-specific Gpr43 deletion, will clarify tissue-specific effects like MDSC-mediated immunosuppression. Additionally, biphasic dose responses necessitate pharmacokinetic-pharmacodynamic modeling to define therapeutic windows. A systems immunology approach integrating metabolomics, immune cell atlasing, and computational modeling is critical to reconcile conflicting evidence and establish predictive frameworks.

Translating microbiome-metabolite insights requires breakthroughs in precision intervention and technological innovation. Artificial intelligence-driven platforms merging metagenomic, metabolomic, and clinical data may predict patient-specific ICI responses. Engineered probiotics and tumor-targeted nanoparticles offer localized efficacy with minimized off-target effects. However, challenges like post-FMT colonization resistance and inconsistent biomarker performance persist. Multicenter trials are needed to validate surrogate endpoints such as serum IPA/Trp ratios and standardize microbiome modulation protocols.

Ethical dilemmas, such as patent disputes over synthetic microbial consortia, highlight the need for open-source strain repositories to democratize access to microbiome therapies. Regulatory harmonization is equally urgent, given divergent Food and Drug Administration (FDA)/European Medicines Agency (EMA) classifications of live biotherapeutics, which impede global trial design. International alliances consortium could unify safety and efficacy standards. Future progress hinges on integrating spatial metabolic imaging with synergistic therapies (e.g., HDAC inhibitors combined with pentanoate) and advancing clustered regularly interspaced short palindromic repeats (CRISPR)-edited probiotics to transform microbiome modulation from serendipitous discovery to precision design.

Future priorities must focus on rigorous clinical validation of microbiota-targeted interventions, including standardized trials to assess the safety and feasibility of FMT and engineered strains. Addressing interindividual variability through pharmacomicrobiomics will optimize personalized dosing. Deep phenotyping via longitudinal multi-omics profiling, coupled with machine learning, is essential to decode host-microbe-metabolite crosstalk. Strategies to enhance ecological resilience, such as prebiotic scaffolds (e.g., resistant starch), are critical to stabilize therapeutic microbiota engraftment in dysbiotic environments. By bridging mechanistic ambiguity, clinical variability, and ethical-regulatory gaps, microbial metabolites may evolve from adjunctive modifiers to cornerstone therapeutics, ultimately reshaping oncology through precision microbiome engineering.