Lung cancer (LC) is renowned for its high malignancy and mortality rates. Globally, the average annual incidence of LC is approximately 31.5/100,000, with the incidence in China exceeding 40/100,000 (Sung et al., 2021). Non–small-cell lung cancer (NSCLC) constitutes over 85% of lung cancers (Siegel et al., 2023). Despite the development of various medications for NSCLC, few can effectively prevent distant metastasis, particularly brain metastasis (BM). Brain metastasis is well-known for its high invasiveness and poor prognosis (Siegel et al., 2023). The heredity of LC susceptibility poses a significant obstacle, with genetic researchers identifying poor heredity as a key challenge. Although studies have highlighted the importance of inherited familial hazards and somatic mutations (Li et al., 2018; Shen et al., 2019), heritability is only partially explained by genetic effects, and results depend on unique responders.

Conversely, non-genetic factors, including environmental factors, behaviors, and microbiota, are considered essential LC etiological factors. Emerging research suggests that microorganisms may significantly impact LC progression (Jin et al., 2019; Zhuang et al., 2019. Commensal microorganisms, as found by Jin et al. (Jin et al., 2019), stimulate myeloid cells to produce Myd88-dependent IL-1β and IL-23, inducing the activation and proliferation of Vγ6+Vδ1+γδ T cells, ultimately advancing tumor cell proliferation and inflammation. Another study by Lin et al. (Liu et al., 2023) revealed that Aspergillus sydowii residing in lung adenocarcinoma promotes tumor progression by suppressing the activity of cytotoxic T lymphocytes. While many LC microorganism studies focus on lung microbes (Lee et al., 2016; Cameron et al., 2017), recent research has associated oral microorganisms with LC incidence and progression (Christiani, 2021; Hosgood et al., 2021). Hosgood et al. (Hosgood et al., 2021) found that lower alpha diversity of oral microorganisms suggests a higher risk of lung cancer, potentially linked to fundamental processes such as immunoreaction, metabolism, and genotoxicity (Mao et al., 2018).

The most complex microbiome, the intestinal microbiome, significantly affects immunological homeostasis, metabolic balance, and tissue development (Liu et al., 2021a). The intestinal microbiome can influence gastrointestinal tumors (Yang et al., 2022) and has been associated with various extraintestinal tumors (Zhang et al., 2021; Jiang et al., 2022). Recent studies have explored a possible association between the intestinal microbiome and NSCLC. Vernocchi P (Vernocchi et al., 2020) reported that Rikenellaceae, Oscillospira, and Bacteroides plebeians were enriched in NSCLC. Additionally, Zhang (Zhang et al., 2018) found significant differences in eight predominant genera, including Escherichia/Shigella, Dialister, Enterobacter, Kluyvera, Fusobacterium, Veillonella, and Bacteroides, between patients with NSCLC and healthy controls (HCs). Emerging evidence also suggests that specific changes in individual species, such as Prevotella copri and Bifidobacterium longum, and the general diversity of the intestinal microbiome are linked to the response to immunotherapy (Jin et al., 2019). Meanwhile, the intestinal microbiome and its metabolite, particularly short-chain fatty acids (SCFAs), may contribute to NSCLC development. SCFAs have been shown to modulate inflammatory and immunological responses by inhibiting histone deacetylases and activating G-protein-coupled receptors (GPRs). A deficiency in SCFAs may contribute to the progression of various tumors, including distant metastases (Hou et al., 2022). In conclusion, increasing data suggest that the intestinal microbiome and its metabolites significantly affect tumor progression by modulating immunoreaction and inflammation, as well as interacting directly with targeted drugs (Dohlman et al., 2022; Huang et al., 2022).

Despite these substantial achievements, several questions persist in these research endeavors. First, many studies have overlooked the dynamic microbial shifts during the various stages of NSCLC. Second, the variations associated with BM of NSCLC have not yet been adequately described. Thirdly, research on the underlying association between NSCLC and variations in microbiota and its metabolite, particularly SCFAs, is currently lacking. The primary objective of this study was to address these challenges by investigating gut microbiota and SCFAs in patients with NSCLC at various stages (early stage and BM stage). Additionally, the aim was to establish a microbial biomarker panel capable of distinguishing patients with NSCLC, specifically those with BM, from HCs. This approach could enhance our understanding of the progression and metastasis of lung tumors, leading to the identification of novel therapeutic targets and robust microbial biomarkers for clinical management. In the future, fecal microbial transplant and the consumption of SCFAs may emerge as fundamental adjuvant therapeutic techniques for NSCLC and even BM.

As widely acknowledged, the gut microbiota plays a crucial role in maintaining the balance of metabolic function and immunological homeostasis. Microorganism dysbiosis has been implicated in promoting pathological inflammation and potentially contributing to cancer progression (Elkrief et al., 2019). Moreover, the structure and regulation of the intestinal microbiota are emerging as diagnostic and therapeutic tools for patients with cancer. Despite mounting evidence suggesting close associations between the intestinal microbiome and NSCLC, research on the alteration of the intestinal microbiome and its metabolites, particularly SCFAs, in patients with NSCLC at various stages is limited. Additionally, the potential microbial biomarkers of NSCLC malignancy haven’t been adequately explored. Therefore, we conducted a study with 115 subjects, including 40 patients with ELC, 40 patients with BM of NSCLC, and 35 HCs, to examine biomarkers and the role of the gut microbiota in the progression of NSCLC. The findings may provide new insights into the involvement of the intestinal microbiome in the diagnosis and treatment of NSCLC, particularly in cases of BM.

Intestinal microbiome richness and evenness, as measured by three α-diversity indices, were found to be lower in patients with BM compared to ELC and HCs, consistent with prior studies (Liu et al., 2019). The loss of microbial ecology is well-documented in connection with chronic diseases (Budden et al., 2019). Firmicutes and Actinobacteria were less prevalent in the gut microbiota of patients with NSCLC, particularly in the BM stage. Firmicutes have been linked to immunological responses that regulate anti-inflammatory and apoptotic functions (Dalile et al., 2019). Actinobacteria, considered probiotics, has shown a reduction in colorectal cancer (Liu et al., 2017; Liu et al., 2019). Patients with NSCLC exhibited a lower ratio of Firmicutes to Bacteroidetes (F/B). A high F/B ratio typically indicates a healthy condition, and a reduced ratio may generate metabolic imbalance and decrease circulating SCFAs (Liu et al., 2019). This reduction in SCFAs was observed in the NSCLC group, especially in the BM stage, as measured by gas chromatography. SCFAs are known essential modulators of systemic inflammation and immunity, influencing intestinal and blood-brain barrier permeability (Dalile et al., 2019). They have demonstrated protective functions in various cancers by modulating programmed cell death and cell adhesion (Mirzaei et al., 2021).

Fusobacteria and Proteobacteria were more abundant in the gut microbiota of the NSCLC group, particularly in the BM group. Fusobacteria, known for opportunistic infections, may accelerate cancer progression by regulating T regulatory cells and activating autophagy (Liu et al., 2019). Proteobacteria, containing a large number of gram-negative pathogens (Vaz-Moreira et al., 2017), may imply intestinal epithelial barrier damage and susceptibility (Yoo et al., 2020). Variations at the family and genus levels were more complex. All evidence pointed toward a disruption in the balance of the intestinal microbiota in patients with NSCLC, particularly in cases of BM. We hypothesize that NSCLC creates an imbalanced intestinal microenvironment via the “gut–lung axis,” and this disrupted intestinal microbiome could potentially promote the progression of NSCLC. This assumption needs further validation through future animal research.

The potential ELC-associated intestinal microbiome revealed in this study sheds light on the intricate association between gut microbiota and the progression of NSCLC. The presence of the genus Streptococcus, known to promote tumor advancement by degrading tannic acid (Oehmcke-Hecht et al., 2020), suggests a potential contributing factor in early-stage NSCLC. On the contrary, Blautia, enriched in ELC, has been reported to have preventive benefits against carcinogenesis and probiotic qualities by promoting SCFA generation and suppressing inflammatory responses (Liu et al., 2021b). The transient compensating response hypothesis is considered, indicating a complex interplay of microorganisms in response to the early stages of NSCLC.

Four BM-associated microorganisms were identified, including Enterobacteriaceae and Klebsiella, both known for their potential to produce colicins that may damage DNA and contribute to the occurrence and progression of various cancers. Enterobacteriaceae’s association with an imbalanced immunological environment (Yoo et al., 2020) and its potential to alter the gut milieu suggest a compromised immunological and metabolic environment in individuals with BM of NSCLC. These findings highlight the connection between intestinal microbiome profiles and cancer risk. Low-abundance microorganisms in patients with NSCLC, such as Faecalibacterium, Bifidobacterium, and Butyricicoccus, were also revealed. Faecalibacterium and Butyricicoccus, known for their anti-tumor effects through butyric acid (Li et al., 2022) generation, play crucial roles in maintaining gut physiology (Singhal et al., 2021), influencing immunological responses, inhibiting pathogen invasion, and suppressing tumor development (Chen et al., 2020). Additionally, Bifidobacterium, a primary acetate source, effectively resists cancer by maintaining the dynamic equilibrium of metabolism and immunity (Badgeley et al., 2021). The decline of these SCFA-producing microorganisms may serve as a risk factor for the progression of NSCLC. The creation of a diagnostic model comprised of six abundant microorganisms for patients with NSCLC at various stages, with satisfactory predictive performance demonstrated by ROC curves, suggests the potential of the intestinal microbiome as a noninvasive diagnostic tool.

Surprisingly, the observed alterations in the gut microbiota of patients with ELC and BM suggest close associations between these stages. The deficiency of butyrate-producing bacteria, such as Butyricicoccus in Firmicutes, could promote the colonization of pathogenic Enterobacteriaceae (Proteobacteria) by upregulating intestinal pH (Yoo et al., 2020). The reciprocal association, where increasing Enterobacteriaceae inhibits SCFA-producing bacteria (Yoo et al., 2020), creates a potential vicious circle. Furthermore, the expansion of gram-negative bacteria like Enterobacteriaceae and Klebsiella may promote facultative anaerobic pathogens by creating lipopolysaccharide (Yoo et al., 2020), contributing to the drastic reduction of strict obligatory anaerobes of Firmicutes and Bifidobacteriaceae (Actinobacteria) due to the disruption of the intestinal oxygen environment. This holistic dysbiosis of the microbiome, rather than specific pathogens, may be tightly associated with the development of NSCLC. These findings highlight the intricate interactions within the gut microbiota and their potential impact on the progression of NSCLC.

The KEGG prediction pathways uncovered in this study provide insights into the complex interactions between gut microbiota and the host in patients with NSCLC. The disruption of various vital physiological processes, including lipid metabolism and genetic information processing, indicates a multifaceted impact on cancer progression. Despite the decrease in SCFAs, an increase in fatty acids, especially in BM patients, suggests a potential role of long fatty acids produced by Enterobacteriaceae (Mitchell and Ellermann, 2022). This could be significant, as fatty acid biosynthesis derived from acetyl-coenzyme A is critical for cancer cell signaling molecules and membrane biosynthesis. Furthermore, the disturbed pathways related to genetic information processing, such as DNA replication and mismatch repair, point to aberrant epigenetic processes in NSCLC.

The complexity of interactions between the intestinal microbiome and the host is highlighted, suggesting that NSCLC is not only a genetic but also a metabolic illness with concurrent anomalies in the intestinal microbiome. The microbiome may play a ruling role in the NSCLC system, impacting and being impacted by alterations in metabolism and genetic information. The study suggests the initiation of the process by carcinogenic metabolites or toxins that modulate metabolism and genetic information.

The potential influence of microbial community regulation by drugs on NSCLC development (Huang et al., 2022). and the enhancement of anti-cancer therapy efficacy through microbial community variation opens avenues for novel therapeutic approaches (Badgeley et al., 2021). Considering the potential superior clinical efficacy of targeted therapies, the suggestion to use probiotics or SCFAs to combat functional dysbiosis and even BM in chemoradiation-treated patients is noteworthy. This study, being the first to investigate the association between intestinal microbiota and its metabolites in ELC and patients with BM, provides fresh insights into the underlying roles of the microbiome in diagnosing and treating NSCLC.

However, the study acknowledges significant drawbacks, including a relatively small sample size, a broad grouping scope, and the absence of specific subtyping for ELC. The limitation of amplification bias in the 16S rRNA gene sequencing method is recognized. While the data suggest an association between dysbiosis of the intestinal microbiome and disruptions in metabolism and immunology in patients with NSCLC, the study emphasizes the need for more extensive and rigorous experiments to address these questions.

In the context of NSCLC, this study revealed a notable lack of microbial diversity and SCFA-producing bacteria, particularly evident in patients with BM. Furthermore, the identification of a microbial biomarker panel consisting of Faecalibacterium, Bifidobacterium, Butyricicoccus, Klebsiella, Streptococcus, and Blautia presents a promising avenue for noninvasive diagnostic tools in patients with ELC and BM. The findings underscore the potential of gut microbiota-based biomarkers in the diagnostic landscape. The KEGG pathways analysis highlighted aberrations in multiple lipid metabolism-related and genetic information processing pathways in patients with NSCLC, especially those with BM. The substantial correlation observed between fecal microbiota and SCFAs with the malignant degree of NSCLC suggests a potential association between the reduction of SCFA-producing bacteria and the development and progression of NSCLC, including the process of BM. However, the study acknowledges the possibility that these microbial alterations may be a consequence of tumor progression. As a final note, the study emphasizes the need for more extensive and rigorous research in the future to delve deeper into the intricate association between gut microbiota, SCFAs, and the progression of NSCLC.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, PRJNA957735.

The studies involving humans were approved by The Affiliated Hospital of Yangzhou University’s Ethics Committee. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

DC, AP, and YL conceived and designed this project. YW, XZ, and WZ collected the samples and performed the data analysis. HJ, WZ, and XZ conducted the experiments and wrote the manuscript. All authors read and approved this manuscript.