Chemotherapeutic agents are known to alter the composition of the gut microbiota, and several studies have confirmed significant shifts in specific microbial populations during NSCLC chemotherapy (19–23). Platinum-based drugs exert antitumor effects by inhibiting DNA replication and targeting cellular membranes and mitochondria, forming intra-strand platinum-DNA adducts that lead to double-strand breaks (DSBs) (24). These agents may exert their tumor-suppressive effects through microbiota-dependent pathways, with their efficacy partly relying on the intra-tumoral generation of reactive oxygen species (ROS), which decreases in response to reduced DNA damage. Particularly, an increase in Bifidobacterium species has been observed during platinum-based treatment of NSCLC ( Table 1 , Figure 1 ) (25). Pemetrexed, a multitargeted antifolate, inhibits several folate pathway enzymes—thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase—which are involved in purine and pyrimidine nucleotide synthesis for DNA and RNA production (26–28). In mouse models, pemetrexed treatment altered the gut microbiota, significantly increasing the relative abundance of Enterococcaceae, Lactobacillaceae, and Streptococcaceae (19). Paclitaxel was found to decrease the overall abundance of gut microbiota in lung cancer-bearing mice, with a significant disruption in the Bacteroidetes/Firmicutes ratio (p < 0.01) (29).

For patients with locally advanced NSCLC, concurrent chemoradiotherapy (CCRT) is a standard treatment. Recent studies have shown that alpha diversity of gut microbiota is significantly associated with therapeutic response, indicating that dysbiosis is a key environmental factor affecting prognosis. One study explored dynamic changes in gut microbiota and their predictive value for progression-free survival (PFS) following CCRT in NSCLC patients. It was observed that the abundance of Bacteroidetes and Proteobacteria increased, while Firmicutes decreased after treatment. Patients with longer PFS demonstrated significantly greater diversity in fungi, archaea, and viruses compared to those with shorter PFS. Key metabolic pathways affected included fatty acid metabolism, arginine biosynthesis, lipopolysaccharide biosynthesis, and ascorbate and aldarate metabolism ( Table 1 , Figure 1 ) (30, 31).

Targeted therapy using tyrosine kinase inhibitors (TKIs) has become a cornerstone in NSCLC treatment alongside chemo- and radiotherapy (42). Studies examining the gastrointestinal microbiome in patients with Epidermal Growth Factor Receptor (EGFR)-wild-type versus EGFR-mutant NSCLC revealed a predominance of Proteobacteria, implicating its role in disease mechanisms (43). In EGFR-mutant patients treated with EGFR-TKIs, lower levels of Proteobacteria and higher levels of Bacteroidetes and Firmicutes were observed (44). Despite the efficacy of second-generation TKIs such as afatinib, clinical trials have reported grade ≥3 diarrhea in over 25% of patients, with about 15% discontinuing treatment due to severe diarrhea, compromising therapeutic outcomes (45). Mouse models have shown significant increases in Peptostreptococcus, Staphylococcus, Escherichia-Shigella, and Akkermansia following afatinib treatment ( Table 1 , Figure 1 ) (46). A study investigating the gut microbiota profile in fecal samples from a lung-specific conditional EGFR mutant transgenic mouse model of lung tumorigenesis demonstrated that Lactobacillus, a genus of bacteria known for producing short-chain fatty acids (SCFAs), may serve as a predictive factor for tumor initiation and progression in EGFR mutation-induced lung adenocarcinoma models (47).

Antibiotics have a significant impact on gut microbiome health, often leading to dysbiosis or the proliferation of harmful flora that can undermine the effectiveness of therapies such as immunotherapy for NSCLC. Antibiotic use has been associated with poor responses to combination therapies, including immunotherapy (48–54). Analysis of bacterial phyla has shown that antibiotic treatment for up to 4 weeks prior to immunotherapy increases the abundance of Bacteroidetes. Systemic use of antibiotics has been linked to an increased Bacteroidetes/Firmicutes ratio, correlating with poorer immunotherapy outcomes (55). Broad-spectrum antibiotic-associated gut microbiome dysbiosis, occurring in patients treated with long-term antibiotics, leads to impaired T-cell immunity ( Table 1 , Figure 1 ) (56). A study sequencing 16S ribosomal DNA (rDNA) from 69 fecal samples of advanced NSCLC patients prior to immune checkpoint blockade (ICB) therapy revealed that antibiotic use was significantly associated with a decrease in gut microbiota diversity (57). An analysis of bacterial diversity and differential abundance of fecal samples from NSCLC patients treated with anti-PD-1/PD-L1 antibodies showed that feces from patients not treated with antibiotics were enriched in Clostridium perfringens, especially within the Rumatococcaceae, UCG13, Clostridium spp., and Agathrobacterium spp. families. In contrast, feces from patients who received antibiotics were enriched in Hungatella (48). Plasma citrulline, a marker of intestinal barrier function, decreases early in NSCLC patients treated with nab-paclitaxel following antibiotic use, affecting citrulline metabolism and, consequently, intestinal microbiome metabolism (58). Vancomycin preferentially targets Gram-positive bacteria, including butyrate-producing species, reducing short-chain fatty acid (SCFA) concentrations in fecal and tissue samples. Vancomycin also eliminates two major families of SCFA-producing Clostridia: Ruminococcaceae and Eubacteriaceae (59). However, further studies are required to determine whether antibiotic use directly alters intestinal flora and metabolism, and to explore the mechanisms by which antibiotic use may reduce the efficacy of NSCLC treatments by modulating intestinal flora and metabolism.

The gut microbiome plays an important role in modulating visceral pain, and recent evidence suggests that it may also be involved in various types of chronic pain, such as inflammatory pain, headaches, neuropathic pain, and opioid tolerance (60). Morphine and other opioids disrupt the gut barrier, alter gut flora and metabolism, and impair function by inhibiting mucus and bicarbonate secretion, disrupting muscle coordination, and increasing the risk of bacterial translocation (61). Long-term morphine use has been shown to significantly alter the gut microbiome, promoting the growth of Gram-positive pathogens while reducing biliary isolates (62). Additionally, activation of the mu-opioid receptor (MOR) by opioid ligands has been found to disrupt tight junction protein (ZO-1) coordination through activation of toll-like receptors (TLR) 2 and 4, thereby affecting intestinal epithelial integrity ( Table 1 , Figure 1 ) (63). Therefore, the rational use of analgesic drugs is also critical in the treatment of NSCLC, and it has been shown that analgesic drugs alter the gut microbiota, which in turn is detrimental to the treatment of NSCLC.

The fundamental theory of Traditional Chinese Medicine (TCM) for treating NSCLC focuses on restoring balance through the principles of ‘strengthening the body’ and ‘eliminating evils’ when the body’s immunity is weak, and the tumor’s growth is overly strong. “Strengthening the body” refers to enhancing the body’s anti-cancer immunity, while “eliminating evil” directly inhibits the growth, proliferation, invasion, and migration of tumor cells. The clinical manifestations of NSCLC are often characterized by lung qi and lung yin deficiency. TCM’s core therapeutic paradigm for lung cancer revolves around the concepts of ‘lung qi deficiency’ and ‘qi-yin deficiency’, with an emphasis on “supporting the positive and curtailing the negative” (4, 64). It has been investigated that combination therapy with monoclonal antibodies remodeled the composition of the gut microbiota and increased the number of SCFAs-producing bacteria Muribaculum to sensitize the antitumor effects of anti-PD-1 therapy and restore the microbial composition of fecal samples from those who did not respond to anti-PD-1 therapy (65). Polysaccharides derived from Spirulina have been shown to increase the abundance of Lactobacillus, Allobaculum, Alloprevotella, and Olsenella, while reducing Bacteroides and Acinetobacter levels. These effects may be linked to the inhibition of lung cancer in mice (66). Fuzi-Li zhong pill (FLP) is a well-validated TCM formula that has long been used in China for gastrointestinal disease and adjunctive therapy for depression (67). Lateralis Radix Praeparata (Fuzi), a traditional Chinese herb, is known for its relatively low toxicity and has been found to improve gut dysbiosis in NSCLC, decreasing the abundance of Proteobacteria while increasing that of Firmicutes (68, 69). Si junzi Decoction (SJZD) is a traditional Chinese medicine formula widely used in the treatment of gastrointestinal disorders. Despite its proven effectiveness, the precise mechanisms by which SJZD operates remain incompletely understood (70). A study evaluating the efficacy of SJZD on quality of life, hematological parameters, and modulation of gut flora in post-surgical NSCLC patients demonstrated that SJZD had a favorable effect on increasing microbial abundance and diversity, promoting probiotic microorganisms, and modulating microbial functions (71).

Probiotics have previously been shown to alter gut microbiota composition, thereby influencing cancer treatment outcomes. A study on the anticancer potential of probiotics suggests that gut probiotics exert tumor-suppressive effects through the gut-lung axis microecological regulation (72). Probiotics have been positively associated with overall survival (OS) and progression-free survival (PFS) in NSCLC patients treated with ICIs (73). Further research has shown that probiotics did not affect PFS but identified two dynamic types of gut flora during immunotherapy: one type exhibited the lowest relative abundance at the response time point, while the other showed the highest abundance at the response time point (74). Specific changes in intestinal flora, such as those induced by Clostridium butyricum, have been found to influence clinical outcomes in non-squamous NSCLC (NS-NSCLC) patients receiving bevacizumab combined with platinum chemotherapy, significantly reducing adverse events in patients (21). Clostridium butyricum MIYAIRI 588 (CBM588) has been shown to potentiate the efficacy of PD-1 blockade in NSCLC by modulating gut microbiota diversity and immune responses. CBM588 supplementation enhanced IL-10 secretion by lamina propria monocytes, improved intestinal homeostasis, and facilitated CD8⁺ T-cell activation, thereby helping to overcome resistance to immunotherapy ( Table 2 , Figure 2 ) (75, 76).

Live biotherapeutic products (LBPs), as defined by the U.S. Food and Drug Administration (FDA), are biological agents intended for disease prevention, treatment, or cure (77). Recently, LBPs designed to modulate the gut microbiota, such as Bifidobacterium lactis and Lactobacillus acidophilus, have shown potential therapeutic benefits, including in colorectal cancer [NCT03072641]. Of particular relevance to NSCLC, CJRB-101—an LBP containing Catenibacterium mitsuokai—has shown antitumor activity in humanized NSCLC mouse models when combined with pembrolizumab. Mechanistically, CJRB-101 reprograms M2 macrophages into M1 macrophages co-expressing CXCL9 and CXCL10, thereby enhancing CD8⁺ T-cell activation and augmenting antitumor immune responses (78).

Characterizing the microbiota and metabolomic profiles of patients provides opportunities to target microbiota-derived metabolites that modulate the tumor microenvironment (TME) (79). These metabolites can influence NSCLC progression and shape the antitumor activity of host immune cells. For example, the gut microbiota generates short-chain fatty acids (SCFAs), which not only exert direct antitumor effects but also enhance immune system function (80).

To date, microbiome-metabolite (ME) research in oncology has primarily focused on the impact of gut microbiota composition on the efficacy of ICIs. Variations in the relative abundance of individual microbial strains and overall microbial diversity in patients undergoing ICI therapy appear to correlate with treatment outcomes (97). FMT has potential therapeutic benefits, particularly in converting non-responders to NSCLC treatment into responders. Fecal transplantation from anti-PD-1-responsive patients to non-responsive NSCLC patients increased treatment response rates without increasing toxicity (43, 57, 98). Akkermansia muciniphila (Akk) has been associated with the clinical benefits of ICIs in NSCLC patients, and the relative abundance of Akk may serve as a reliable biomarker for predicting good or poor prognosis in patients receiving PD-1 blockade immunotherapy, refining patient stratification in future studies (99). Akk enrichment modulation enhances immune responses through fecal microbiota transplantation in patients benefiting from immune checkpoint blockade (100). In animal experiments, the relative abundance of Akk predicted clinical response to PD-1 blockade in NSCLC patients. Mice receiving FMT, negative for Fusobacterium tachyzoites, exhibited tumor resistance to PD-1 blockade (15). RNA later preserved stool samples were collected from 65 pre-treatment (baseline) and post-treatment stage III/IV NSCLC patients treated with ICI and classified as responders or non-responders according to RECIST criteria. Mixed and individual responder and non-responder microbiota were transplanted into a gnotobiotic mouse model of lung cancer and treated with ICI, while patient fecal samples were subjected to 16S rDNA and RNA sequencing, which demonstrated that responding patients had a different microbial community structure (P=0.004) and a different bacterial transcriptome (PC2=0.03) at baseline. Taxa significantly enriched in responders included amplicon sequence variants (ASVs) belonging to the genera Rumococcus, Akkermansia muciniphila, and E. faecalis. Transplantation of mixed and individual responding microbiota into gnotobiotic mice reduced tumor growth compared to non-responding colonized mice after ICI use (P=0.023, P=0.019, P=0.008, respectively), showing an increased tumor CD8 ⁺ IFN γ ⁺ T-cells and CD4⁺ CXCR3⁺T-cells phenotype after ICI treatment. Responding mice were enriched with ASV belonging to the genera Mycobacterium, Blautia, Akkermannia and E. faecalis (101). However, many FMT studies have reported only limited methodological descriptions, details of mouse cohorts, and statistical methods. One study performed human-to-germ-free mouse FMT using fecal samples from NSCLC patients with pathological response or no response after neoadjuvant ICI treatment, which produced greater anti-tumor responses in R-FMT mice in combination with anti-PD-L1 therapy compared to NR-FMT, detailed study of the mouse microbiota after FMT using 16S rRNA amplicon sequencing, and use of models for the biological variables were classified and corrected, revealing that the most abundant taxa were shared between human inoculum and mice, although low abundance human taxa were more variable in post-FMT colonized mice. Multiple Clostridium spp. were also associated with tumor outcome in individual anti-PD-L1-treated R-FMT mice (102).

Research on Fecal Microbiota Transplantation (FMT) would benefit from well-designed large-scale studies that incorporate extensive metadata and standardized bio-sample collection. Such efforts are crucial to minimize noise in downstream multi-omics analyses and to ensure robust long-term follow-up to address potential safety concerns. The results of these studies could guide targeted experimental designs to explore the underlying mechanisms of FMT clinical outcomes and may ultimately lead to the personalized matching of donor and recipient characteristics to achieve optimal therapeutic success (103). Although FMT may be a promising therapeutic option, the risk of bacterial translocation (including antibiotic-resistant bacteria) and sepsis in patients remains a significant safety concern, and studies have been conducted that discuss sepsis due to FMT (104–106). The most feasible translational approach from whole-stool FMT toward safer and standardized microbiome-based therapies involves progressive refinement from complex donor-derived consortia to defined microbial communities and, ultimately, purified microbial metabolites with validated bioactivity (107, 108). Microbial ecosystem therapy (MET), comprising selected, well-characterized commensal strains, represents a key intermediate strategy that maintains ecological functionality while improving safety and reproducibility (109, 110). Preserving therapeutic efficacy along this continuum requires retention of crucial host–microbe interactions that regulate immune homeostasis (111). Therefore, future microbiota-targeted interventions should emphasize context-dependent functionality to achieve both safety and sustained immunotherapeutic benefit in NSCLC treatment.

Castalagin is a polyphenol that enhances resistance to PD-1. In their study, Messaoudene et al. reported that oral supplementation with polyphenol-rich berry camu-camu (CC; Myrciaria dubia) altered the gut microbial composition, leading to antitumor activity and a stronger anti-PD-1 response. Castalagin improved the CD8+/FOXP3+CD4+ ratio in the tumor microenvironment. Moreover, castalagin induced metabolic changes, resulting in an increase level of taurine-conjugated bile acids. Ruminococcus-rich NSCLC responders were found to be able to metabolize castalagin (112, 113). Patients should be advised to minimize animal meat intake and increase plant intake where possible, aiming for 30 plants per week. High fiber intake (>30 g/day) is thought to increase the chances of response to immunotherapy in NSCLC (114). Methionine regulates tumor immunity by modulating the activity of cyclic GMP-AMP synthase (cGAS), so the tumor immune response can be improved by controlling dietary methionine intake (115). Ginseng polysaccharides (GPs) are one of the most abundant constituents of ginseng, and GPs increase the antitumor response to αPD-1 mAb by inhibiting the metabolite kynurenine/tryptophan ratio, which contributes to the suppression of regulatory T-cells and the induction of Teff cells following combination therapy, enhancing the antitumor effects of immunotherapy (116). Specific diets directly or indirectly alter the intestinal flora and metabolism of NSCLC patients and increase the clinical efficacy of NSCLC.

In recent years, relevant clinical trials have been conducted around simulated fasting diets, high-fiber diets, nutritional supplements and other related trials, which have either maximized the therapeutic efficacy of NSCLC treatments or maximized the benefits for patients (117).

As previously discussed, immunotherapy plays a crucial role in the treatment of NSCLC. Numerous studies have collected plasma and stool samples from patient cohorts prior to initiating immunotherapy, performing metabolomic and microbiome analyses (118, 119). The results obtained after enrichment are referred to as baseline microbiome characteristics. From a metagenomic perspective, enrichment of Akkermansia may be indicative of favorable prognosis in patients undergoing PD-1 blockade immunotherapy, offering potential for improved patient stratification in future studies (99). Akkermansia muciniphila, a mucin-degrading commensal bacterium, exemplifies the context-dependent functionality of microbiota-based interventions. Under conditions of adequate mucin renewal and minimal antibiotic disturbance, it reinforces gut barrier integrity and immune regulation (120, 121). However, in states of mucin depletion, chronic inflammation, or dysbiosis, its activity may shift from protective to deleterious, aggravating intestinal damage and immune dysregulation (122). These findings underscore the necessity of designing microbial consortia and derived metabolites that maintain host–microbe symbiosis and ensure both safety and therapeutic efficacy in diseases such as NSCLC (120, 122).

Additionally, metabolomic analyses suggest that patients enriched with baseline short-chain fatty acids (SCFAs) may derive long-term benefits from immunotherapy (90). The close link between microbiota and metabolites at baseline not only provides novel insights for clinical stratification but also opens new avenues for clinical translation (86). For instance, some studies have demonstrated SCFA enrichment via metabolomic profiling and verified the presence of microbiota producing SCFAs as a baseline marker for therapeutic efficacy. In parallel, other studies have enriched baseline microbiota and subsequently identified corresponding metabolites, confirming the therapeutic benefits of these metabolites at baseline enrichment (92).

In summary, baseline microbiome and metabolite characteristics have been explored in existing research, and many clinical trials have since emerged, contributing to this growing field.

Numerous clinical trials have focused on the role of gut microbiota and metabolites in various treatment modalities for NSCLC, examining their impact on treatment efficacy and prognosis. These studies underscore the importance of analyzing dynamic changes in the gut microbiota throughout the course of treatment, with specific microbial and metabolic alterations observed after a certain duration of therapy. By exploring the modulation of specific microbiota, these studies suggest that manipulating the microbiome could enhance NSCLC treatment outcomes. Such findings are poised to drive major breakthroughs in future NSCLC therapies.

[NCT03068663] A study grouped 40 patients with NSCLC, 20 patients were treated with surgery only, while the other half also received chemotherapy. The main aim was to explore changes in the lung, upper respiratory tract and intestinal microbiota and potentially find an association between the flora and the prognosis of patients treated. [NCT06221800] Another study was conducted to collect data on the dynamics of the gut microbiome of 82 subjects with advanced/metastatic NSCLC (NSCLC) during treatment with NSCLC. The subjects were classified into 3 groups: anti-PD-1 monotherapy, anti-PD-1 combination chemotherapy and TKIs. therapy, anti-PD-1 combination chemotherapy therapy and TKIs therapy and analyzed the diversity and composition of the gut microbiome of the subjects during the treatment of NSCLC to provide reference for the efficacy of NSCLC treatment [NCT05669846]. Suitable patients will be identified at progression on PD-1 monotherapy or PD-1-containing regimens and patients will undergo a 35-day screening assessment. Following enrolment, patients will be serum matched to suitable donors and patients will receive R-FMT (induction) via colonoscopy on C1D1 and C3D1. R-FMT (maintenance) by sigmoidoscopy on C4D1 will be repeated every 9 weeks. All patients will receive an additional 200mg of pembrolizumab every 3 weeks and patients will be treated until disease progression or intolerance of toxicity or completion of 2 years of treatment ( Table 3 , Figure 3 ).

These clinical trials involve large cohorts and feature robust experimental design, with open results providing valuable insights into the relationship between gut microbiota, treatment efficacy, and patient outcomes. For further details and updates, clinicaltrials.gov is a useful resource.