From chaos to order: optimizing fecal microbiota transplantation for enhanced immune checkpoint inhibitors efficacy

Emerging clinical evidence indicates that FMT may substantially augment the therapeutic efficacy of ICIs in patients with melanoma. In a prospective clinical trial (NCT03341143↗) involving patients with advanced melanoma, investigators administered FMT intervention to 15 subjects concurrently receiving pembrolizumab treatment.²⁸ The results demonstrated that 40% of patients experienced clinical benefit and manifested rapid and sustained alterations in their gut microbiota. The gut microbiota of responders exhibited specific bacterial communities associated with anti-PD-1 response, concomitant with enhanced activation of CD8+ T cells and diminished myeloid cells, indicating that FMT may potentiate ICI efficacy by modifying the immune microenvironment. In a separate phase I clinical trial (NCT03353402↗), investigators administered treatment to 10 patients with anti-PD-1-refractory metastatic melanoma who had previously attained complete response (CR) following nivolumab monotherapy, and assessed the safety and feasibility of nivolumab re-induction.²⁹ Three subjects attained six-month progression-free survival, with two exhibiting partial response (PR) and one achieving CR. Subsequently, the investigators performed comprehensive microbiome analysis and tissue biopsies. Overall, substantial alterations in gut microbiome composition were observed across all FMT recipients; the responder group demonstrated microbial characteristics conducive to immunotherapy, characterized by elevated relative abundances of Enterococcaceae, Enterococcus, and Streptococcus australis, and diminished relative abundance of Veillonella atypica. Advantageous modulations in immune cell infiltration and gene expression profiles were detected in the intestinal lamina propria the TME. A phase I clinical trial (NCT03772899↗) evaluating the combination of FMT from healthy donors (HD) with anti-PD-1 therapy in treatment-naive patients with advanced melanoma demonstrated objective responses in 13 patients (65%), comprising 4 (20%) CR and 9 (45%) PR. After a median follow-up of 20.7 months, the median progression-free survival remained unreached, with 16 patients maintaining survival³⁰ The study also demonstrated durability of treatment responses, with a subset of patients exhibiting sustained clinical responses following the administration of combined FMT and anti-PD-1 therapy.

In a clinical trial (ChiCTR2100046768) focusing on patients with microsatellite stable metastatic colorectal cancer (MSS-mCRC), investigators implemented a triple therapy regimen combining FMT with PD-1 inhibitors and vascular endothelial growth factor receptor (VEGFR) inhibitors.³¹ Preliminary results suggested that this innovative triple therapy regimen effectively mitigated tumor progression, significantly decreased serum tumor marker levels, augmented patient immune function, and facilitated a balanced state of the gut microbiota. Gut microbiome analysis demonstrated that treatment responders harbored higher abundances of Proteobacteria and Lachnospiraceae, while exhibiting relatively lower abundances of Actinobacteria and Bifidobacterium. Although FMT did not significantly modify the overall structure of the peripheral blood T cell receptor (TCR) repertoire, clonally expanded TCR sequences displaying characteristics of antigen-driven immune responses were detected in treatment responders. In the NCT04951583↗ clinical trial, the objective response rate (ORR) post-FMT treatment attained 70%, comprising 2 cases of CR and 12 cases of PR. Further analysis elucidated significant differences in gut microbiome characteristics between responders and non-responders. Presently, multiple clinical trials investigating ICIs in combination with FMT treatment strategies are ongoing, including NCT04038619↗,³²NCT04163289↗,³³NCT04951583↗,³⁴ and NCT05008861↗,³⁵ which will further elucidate the potential of FMT in augmenting ICI efficacy.

A subsequent study focusing on patients with specific solid tumors including metastatic gastric cancer (GC), esophageal squamous cell carcinoma (ESCC), and hepatocellular carcinoma (HCC) (NCT04264975↗) provided additional evidence supporting the positive role of FMT.³⁶ Among 13 patients receiving anti-PD-1 therapy, FMT induced sustained microbiome changes and clinical benefits in 6 patients (46.2%), with one achieving partial response and five maintaining stable disease. The investigators observed significant increases in the levels of cytotoxic T lymphocytes (CTLs) and pro-inflammatory cytokines in both patients’ peripheral blood and the TME. Notably, Prevotella merdae isolated from the fecal samples of FMT responders was shown to enhance T cell activity by promoting CTLs infiltration and to significantly inhibit tumor growth in mouse xenograft models.

Alterations in gut microbial diversity represent one of the key factors underlying variations in ICI treatment efficacy following FMT. Accumulating evidence indicates that the diversity of the gut microbiome is closely associated with the therapeutic response to ICIs. Gut microbial diversity has been demonstrated to potentially enhance the efficacy and prognosis of ICI treatment. Research indicates that high α-diversity of the gut microbiota correlates with favorable responses to ICI treatment, potentially attributed to the ability of diverse microbiota to promote CD8+ T cell infiltration into tumors.^37,38 A longitudinal study in HCC patients demonstrated temporal increases in gut microbiome diversity during immunotherapy treatment,³⁹ though the direct correlation with treatment response requires further investigation. Conversely, patients with low microbial diversity or dysbiosis frequently exhibit poor responses to ICI treatment. However, patients undergoing ICI treatment are susceptible to microbiome dysbiosis, potentially resulting in reduced diversity and a shift toward a Gram-negative enterotype.⁴⁰ FMT has the capacity to reverse microbiome dysbiosis by introducing healthy microbial communities and modifying microbial composition.^41–44 This intervention has the potential to not only augment the effectiveness of immunotherapy but also mitigate adverse reactions to immunotherapy.⁴⁵

Variations in gut microbial composition also significantly influence the efficacy and prognosis of ICI treatment following FMT. Research has demonstrated that FMT can significantly modify the composition of the gut microbiome. Post-FMT, the abundance of dominant intestinal bacteria increases significantly, while enteric pathogens associated with intestinal oxidative stress exhibit a marked decrease.⁴³ More specifically, the enrichment of certain bacterial taxa demonstrates a strong correlation with positive responses to ICI treatment. For instance, increased proportions of Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium correlate with positive responses to immunotherapy.⁴⁶ Similarly, increased abundance of Faecalibacterium, Ruminococcus, and Akkermansia muciniphila is linked to enhanced responses to anti-PD-1 therapy.^39,47–51 These beneficial bacteria may mediate their effects through various mechanisms, including the promotion of T cell activation, enhancement of anti-tumor immune responses, and maintenance of gut health.^52,53 The taxa significantly enriched in ICI responders both pre- and post-FMT predominantly belong to Firmicutes and Actinobacteria, while the depleted bacteria primarily belong to Bacteroidetes .^54–57 These alterations in bacterial strains may contribute to improved ICI efficacy.

Conversely, the presence of certain bacteria may negatively impact the efficacy of ICIs. For instance, the enrichment of Proteobacteria (including Escherichia coli, Shigella, and Klebsiella), Clostridium, and certain members of Actinobacteria has been correlated with adverse reactions to ICI treatment.^58–60 Interestingly, some bacterial species show variable associations with treatment outcomes. For example, Akkermansia muciniphila has been correlated with favorable responses to ICI treatment in some studies,^57,61 while in others it has been associated with resistance.^62,63Bacteroides has also exhibited similar bidirectional effects. This complexity may result in individual variations in ICI treatment efficacy following FMT. It is important to note that different ICI regimens may exert varying effects on the commensal microbiota.^61,64 For instance, responders to nivolumab-ipilimumab demonstrated enrichment of Faecalibacterium, Bacteroides thetaiotaomicron, and Holdemanella filiformis, while patients responding to pembrolizumab showed enrichment of Dorea formicigenerans .⁶¹ This observation may elucidate the differences in FMT outcomes for various ICI treatment regimens.

Gut microbial metabolites are instrumental in modulating the host immune system and the TME, potentially serving as a critical determinant in elucidating the disparities in ICI treatment efficacy following FMT. FMT has the potential to modulate the therapeutic effects of ICIs through alterations in the metabolite profile generated by the gut microbial community. Current evidence indicates that certain microbial metabolites can potentiate the antitumor effects of ICIs, while others may attenuate their action. This dichotomy offers a plausible mechanistic basis for the heterogeneity in patient responses to ICI treatment following FMT.

SCFAs are pivotal metabolites synthesized by gut microbiota via the fermentation of dietary fiber, predominantly comprising acetate, propionate, and butyrate. In preclinical mouse models, research has demonstrated that SCFA levels are significantly elevated in FMT-treated mice, with notable increases in the concentrations of butyrate, acetate, and caproic acid.^56,65,66 SCFAs augment the therapeutic effects of ICIs via multifaceted mechanisms, encompassing various levels of immune regulation. Empirical evidence suggests that butyrate can substantially potentiate the cytotoxic activity of CD8+ T cells.^65–67 More precisely, in the mouse models, butyrate can augment the antitumor cytotoxicity of CD8+ T cells by suppressing the DNA-binding protein 2 (ID2)-dependent interleukin-12 (IL-12) signaling pathway, consequently enhancing the therapeutic effect of anti-PD-1 therapy.⁶⁸ Additionally, in the mouse models, butyrate has been shown to elevate the levels of histone 3 lysine 27 acetylation (H3K27ac) in the promoter regions of programmed cell death gene 1 (Pdcd1) and CD28 molecules in CD8+ T cells, upregulating the expression of PD-1 and CD28, and modulating TCR signaling pathways. These actions synergistically promote the expression of antitumor cytokines in cytotoxic CD8+ T cells, thus potentiating the effect of anti-PD-1 therapy.^65,67,69 Research has demonstrated that SCFAs can augment the mitochondrial content in antitumor effector T cells and upregulate key metabolic pathways, including glycolysis, oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and fatty acid oxidation (FAO). These metabolic alterations not only ensure adequate energy provision for immune cells but also significantly enhance the therapeutic efficacy of ICIs.^66,70 In the mouse models, SCFAs not only serve as an energy source for immune cells but also facilitate the differentiation of CD8+ T cells into memory T cells and promote their long-term survival.^20,71 This effect is crucial in sustaining a prolonged antitumor immune response.

The impact of FMT on ICI efficacy exhibits considerable heterogeneity, with some patients showing no improvement or potentially diminished responses. This has been demonstrated in the randomized controlled clinical trial NCT03817125↗, where patients receiving SER-401 (a proprietary formulation of bacterial Firmicutes spores isolated and purified from the stool of human donors) showed lower objective response rates (25.0% vs 66.7%) and disease control rates (37.5% vs 83.3%) compared to the placebo group. Furthermore, immunological analysis revealed increased presence of Tregs and effector T cells with high expression of TIGIT and PD-1 in the SER-401 group, suggesting a shift toward an immunosuppressive tumor microenvironment. These patients also exhibited increased activation of signaling pathways associated with immune checkpoint blockade resistance. This heterogeneity in treatment response can be attributed to the following factors. Under specific conditions, SCFAs can elicit immunosuppressive effects, primarily characterized by the following mechanisms: SCFAs, particularly butyrate, have the capacity to suppress immune responses through activating G Protein-Coupled Receptor 43 (GPR43) and promoting the differentiation of Forkhead Box P3-positive (Foxp3+) CD4+ Tregs.^65,72 Excessive proliferation of Tregs can potentially suppress anti-tumor immune responses, thereby diminishing the therapeutic efficacy of ICIs. Elevated concentrations of butyrate and propionate can compromise B lymphocyte DNA recombination capacity via Histone Deacetylase (HDAC) inhibition, resulting in impaired local intestinal and systemic T cell-dependent and T cell-independent antibody responses.⁶⁵ Under specific conditions, SCFAs can potentially facilitate tumor progression through the activation of certain signaling pathways. For instance, in prostate cancer models, SCFAs have been shown to promote tumor growth and metastasis through the activation of the Insulin-like Growth Factor 1 (IGF-1) signaling pathway.⁷³ Following FMT, some patients can develop bile acid metabolism disorders, potentially precipitating a series of adverse effects: Certain secondary bile acids, such as Deoxycholic Acid (DCA), can enhance tumor cell proliferation and survival through the activation of the Wnt/β-catenin signaling pathway.^74,75 Specific bile acids have the potential to suppress the function of anti-tumor immune effector cells, including Natural Killer T cells (NKT cells), thereby compromising the body’s anti-tumor immune surveillance.⁷⁶ Consequently, the variability in ICI treatment efficacy following FMT likely reflects the balance between these beneficial and detrimental factors. For some patients, FMT can result in an increase in beneficial metabolites and a decrease in harmful metabolites, consequently augmenting the efficacy of ICIs. Conversely, for other patients, FMT can induce an increase in harmful metabolites or a decrease in beneficial metabolites, thereby attenuating the efficacy of ICIs.

FMT has the potential to augment the antitumor effects of ICIs through multiple pathways associated with the TME. First, FMT can potentially reconfigure the composition of immune cells in the TME. Accumulating evidence has conclusively demonstrated that following FMT, the TME consistently exhibits significantly enhanced infiltration of CD8+ T cells, Th1 cells, and antigen-presenting cells in responding patients. Concomitantly, a significant decrease is observed in the infiltration of myeloid-derived suppressor cells (MDSCs), RORγ+Th17 cells, and CD11b+CD11c+ suppressive myeloid cells within the TME following FMT intervention.^7,77–80 Studies have indicated that following FMT, patients exhibit elevated levels of CD4+ and CD8+ T cells, while Tregs levels diminish.⁸¹ The ratio of intratumoral CD8+ T cell to Treg infiltration is elevated in patients following FMT.⁸² This modification in immune cell composition promotes the formation of an anti-tumor immune microenvironment. Specifically, CD8+ T cells function as the primary effector cells in anti-tumor immune responses, and their increased abundance can directly augment the ability to eliminate tumor cells.⁸³ Th1 cells, conversely, indirectly potentiate anti-tumor immunity by releasing cytokines such as IFN-γ to stimulate macrophages and NK cells. The elevated number of antigen-presenting cells can facilitate the presentation of tumor antigens, thereby inducing more T cell activation. The diminished presence of MDSCs attenuates the suppression of effector T cells. The reduction in Tregs mitigates the suppression of effector T cells. These alterations following FMT collectively establish an immune microenvironment more favorable to the efficacy of ICIs.^78,79,84

Second, FMT has the potential to augment the function of immune cells. Research has demonstrated that post-FMT, patients display enhanced activation states of mucosa-associated invariant T (MAIT) cells, characterized by elevated CD69 expression and reduced PD-1 expression.⁸⁵ This alteration in activation state potentiates the immune surveillance function of MAIT cells. Furthermore, following FMT, the maturation and antigen presentation capacity of dendritic cells (DCs) are augmented.^15,86,87 Mature DCs exhibit enhanced efficacy in presenting tumor antigens, thereby inducing more robust T cell activation.⁷⁹ NK cell activity is potentiated following FMT.⁸⁸ NK cells function as crucial anti-tumor effector cells in the innate immune system, and their augmented activity can directly intensify the cytotoxic effect on tumor cells.⁴³ Additionally, FMT can facilitate the polarization of M1-type macrophages,^66,89 which exhibit pro-inflammatory and anti-tumor properties.⁹⁰

Furthermore, FMT has been demonstrated to regulate the expression of cytokines and chemokines. Research has demonstrated that serum levels of IFN-γ and IL-2 are elevated following FMT,⁸⁰ and these cytokines exhibit significant antitumor effects. IFN-γ enhances the activity of CD8+ T cells, NK cells, and macrophages, while concurrently inhibiting tumor cell growth directly. Conversely, IL-2 stimulates the proliferation and activation of T cells and NK cells. Moreover, FMT has been shown to upregulate the expression of CXCL9 and CXCL10,^91,92 chemokines that facilitate T cell recruitment into the TME.

Fourth, FMT has been shown to augment the efficacy of ICIs through the modulation of specific gut microbiota. Specifically, an increase in Bifidobacterium species has been demonstrated to enhance the efficacy of anti-PD-L1 therapy by promoting DCs functionality and facilitating the infiltration and accumulation of CD8+ T cells within the TME.^15,86 Elevated levels of oral Akkermansia muciniphila have been shown to restore the anticancer effects of PD-1 blockade by facilitating the recruitment of CCR9+CXCR3+CD4+ T cells to the tumor site.^89,93,94 Elevated abundance of Faecalibacterium exhibits a positive correlation with CD8+ T cell infiltration in tumors and a negative correlation with suppressive myeloid cells (CD11b+CD11c+). Furthermore, it may potentiate antitumor immune responses by augmenting antigen presentation and enhancing effector T cell function.^7,87Bacteroides fragilis facilitates the maturation of DCs and potentiates IL-12-dependent Th1 antitumor immune responses. Additionally, Bacteroides fragilis induces macrophage phenotype polarization toward M1 and upregulates the expression of CD80 and CD86 on these cells, thereby enhancing innate immunity.⁶⁶Lactobacillus plantarum significantly upregulates the expression of natural cytotoxicity receptor (NCR) proteins and enhances NK cell activation, consequently stimulating innate immunity.⁶⁶ Studies in mouse models have demonstrated that Lactobacillus rhamnosus enhances immune responses by activating macrophages and NK cells, thereby initiating innate immunity.⁸⁸

While FMT can enhance the efficacy of ICIs in numerous instances, it may not universally improve ICI efficacy across all clinical scenarios. Firstly, FMT may induce an expansion of specific immunosuppressive cell populations. Recent studies have demonstrated that following FMT, some patients exhibit increased proportions of Tregs and MDSCs within the TME.^20,55,95 Tregs are known to suppress effector T cell function, whereas MDSCs exhibit broad immunosuppressive capabilities, inhibiting various immune cells, including T cells, NK cells, and DCs. The expansion of these immunosuppressive cell populations may potentially attenuate the antitumor efficacy of ICIs. Secondly, an expansion of specific gut microbial populations may suppress antitumor immune responses. For instance, research has demonstrated that an expansion of Bacteroides species is associated with increased Treg and MDSC populations, potentially resulting in diminished antigen presentation capacity and reduced lymphocyte infiltration within tumors.^20,87 These alterations in the microbial community composition may compromise the therapeutic efficacy of ICIs. Thirdly, FMT may modulate the migratory patterns of specific immune cell populations. Research has revealed that post-FMT, some patients exhibit upregulation of the CCL2/CCR2 axis, resulting in enhanced myeloid cell migration to the liver.⁹⁶ This alteration in cellular trafficking patterns may influence the therapeutic efficacy of ICIs in specific organ-localized tumors.⁹⁷

In conclusion, the impact of FMT on ICI efficacy is multifaceted, encompassing both beneficial and potentially detrimental effects. The beneficial effects primarily manifest as enhanced effector T cell function, improved antigen presentation, modulation of favorable cytokine and chemokine expression profiles, and enrichment of beneficial microbial communities. The potentially detrimental effects are predominantly characterized by the expansion of immunosuppressive cell populations and other immunomodulatory alterations.

Accumulating evidence indicates that the variations in ICI efficacy and prognosis following FMT are likely attributed to the modulation of various signaling pathways by the post-FMT gut microbiome. The beneficial effects of FMT on ICI efficacy are predominantly mediated through the modulation of TLR signaling pathways, resulting in amelioration of inflammatory conditions. A preclinical study has demonstrated that FMT can safely mitigate inflammation, with its mechanism of action primarily involving the TLR-MyD88-NF-κB signaling pathway.⁹⁸ Among the various inflammatory and immune pathways modulated post-FMT, the restoration of TLR signaling pathway homeostasis (balance between pro-inflammatory pathways such as TLR4 signaling and anti-inflammatory pathways associated with TLR2 activation) represents a key advantage of FMT application in cancer.⁹⁹ This homeostasis potentially facilitates the creation of a more favorable immune microenvironment, enhancing the efficacy of ICIs.

Conversely, the potential adverse effects of FMT on ICI efficacy warrant careful consideration. Initially, fecal samples from colorectal cancer patients utilized for FMT can potentially counteract the therapeutic effects of ICIs through the activation of signaling pathways that promote tumor growth or metastasis. In the mouse model, they demonstrated that the FMT with feces of colorectal cancer patients can induce the activation of the Wnt signaling pathway, which is instrumental in various stages of tumor development.¹⁰⁰ Furthermore, investigations have revealed a mechanistic link between the post-FMT gut microbiome and cancer metastasis mediated by the IL-11/circRNA/miRNA axis.¹⁰¹ Additionally, certain FMTs can potentially compromise ICI efficacy through the activation of pro-inflammatory signaling pathways. Research has demonstrated that FMT from liver metastasis (LM) patients significantly enhances liver metastasis compared to FMT from HD.⁹⁶ Following FMT with LM feces, plasma lipopolysaccharide (LPS) concentrations are elevated, resulting in the activation of the LPS/TLR4 signaling pathway, which plays a pivotal role in gut microbiome-mediated liver metastasis.⁹⁶ This pro-inflammatory state can potentially disrupt the normal function of ICIs, consequently diminishing their therapeutic efficacy.

In conclusion, the impact of FMT on ICI efficacy is multifaceted and involves complex modulation of various signaling pathways. This intricate interplay potentially elucidates the heterogeneous responses to FMT in ICI therapy, ranging from enhanced efficacy to negligible changes or even adverse reactions. Future investigations should elucidate the regulatory mechanisms of various signaling pathways modulated by the post-FMT gut microbiome, potentially facilitating the development of more precise combination treatment strategies to optimize the synergistic anti-tumor effects of FMT and ICIs. Moreover, considering the complexity of the gut microbiome and inter-individual variability, the development of more sophisticated gut microbiome analysis and prediction models is imperative for improved prognostication and optimization of FMT’s impact on ICI efficacy in clinical settings.

FMT has demonstrated significant promise in enhancing ICI therapy; however, the processes for donor screening and optimization continue to pose substantial challenges. Donor screening represents a crucial step in ensuring the safety and efficacy of FMT. Recent cases of pathogen transmission resulting from inadequate screening have underscored the critical importance of rigorous donor selection.^107,108 A comprehensive array of factors must be meticulously evaluated when establishing screening criteria. Currently, individuals exhibiting high-risk behaviors, those with severe obesity, autoimmune diseases, malignancies, and neurological disorders are typically excluded from donor consideration.^109–111 However, considerable debate persists within the academic community regarding the inclusion of factors such as age restrictions, BMI thresholds, family history of relevant diseases, or recreational drug use in the donor exclusion criteria. Furthermore, disparities in regional regulations and persistent uncertainties regarding the long-term safety of FMT exacerbate the challenges in establishing uniform standards.

Optimal FMT screening protocols for patients receiving ICI therapy encompass multiple key components that surpass standard FMT protocols. The screening process adheres to a standardized three-tier approach: comprehensive medical history assessment, physical examination, and thorough laboratory testing. Laboratory screening encompasses both standard and advanced diagnostic panels. Standard screening includes analysis for common pathogens (Clostridioides difficile, norovirus, rotavirus), blood-borne viruses (HIV, hepatitis B and C), and antimicrobial-resistant organisms.

It is noteworthy that screening criteria for specific microorganisms remain a subject of ongoing debate in the scientific community. For example, while Giardia is generally recognized as potentially pathogenic, recent studies have demonstrated that FMT containing Giardia did not elicit significant adverse reactions in patients with rCDI.¹¹² The viability of Helicobacter pylori in fecal samples remains a point of contention, resulting in diverse screening strategies adopted by various institutions.¹¹³ Moreover, the screening protocols for emerging pathogens necessitate continuous adaptation to address evolving threats. For instance, despite the absence of conclusive evidence supporting SARS-CoV-2 transmission via FMT, regulatory agencies have mandated its inclusion in screening protocols.¹⁰⁸ The recent emergence of the monkeypox virus further underscores the necessity to enhance screening protocols for sexually transmitted infections.¹⁰⁸