Fusobacterium nucleatum promotes colorectal cancer metastasis by modulating the NF-κB-MMP-2/MMP-9 signaling pathway

Introduction

Colorectal cancer (CRC) is the third most prevalent malignancy worldwide and the second leading cause of cancer-related mortality (1). Metastatic CRC remains a formidable challenge, with a disheartening 5-year survival rate of approximately 14% (2). By 2030, the global burden of CRC is projected to increase by 60%, resulting in over 2.2 million new cases and 1.1 million deaths (3). Despite advancements in surgery, chemotherapy, and radiotherapy, therapeutic options for advanced and metastatic CRC remain limited, highlighting the urgent need to identify novel therapeutic targets (4).

Accumulating evidence suggests that gut microbiota dysbiosis plays a crucial role in CRC pathogenesis, with patients exhibiting decreased microbial diversity and an imbalance between pathogenic and beneficial bacteria (5,6). Among them, Fusobacterium nucleatum (Fn), an oral symbiont acting as an opportunistic pathogen in intestinal cancers, has gained attention as a key microbial driver of CRC progression and poor prognosis (7,8). Fn abundance is significantly elevated in CRC tissues compared to adjacent noncancerous tissues, and it has been identified as an independent factor for poor prognosis (9). Moreover, tumor-associated microbiota signatures, including lower levels of Caulobacter and Novosphingobium, have been linked to longer survival times, suggesting that microbial composition in tumor tissues may serve as a potential prognostic indicator in CRC (10). Several studies have explored potential mechanisms underlying Fn-mediated CRC progression. For instance, Fn can interact with E-cadherin via FadA adhesin. This interaction triggers the Wnt/β-catenin signaling pathway, leading to inflammatory and oncogenic responses (11). Additionally, Fn has been implicated in CRC metastasis via various pathways, including ALPK1/ICAM1 signaling-mediated tumor cell adhesion and extravasation, m6A/METTL3-dependent epigenetic modifications, and miR-1322/CCL20 axis-driven tumor microenvironment remodeling (12-14). Despite these findings, the role of Fn in extracellular matrix (ECM) degradation and its contribution to CRC invasion and metastasis remain poorly understood.

Nuclear factor kappa B (NF-κB) is a well-established regulator of tumor initiation and progression (15). Among its family members, p65 (RelA) plays a pivotal role in NF-κB activation within the nucleus (16). The NF-κB signaling pathway is tightly regulated by inhibitory proteins such as IκBα, IκBβ, and IκBɛ (17). Meanwhile, matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, are crucial mediators of ECM degradation and remodeling, and have been implicated in CRC cell proliferation and invasion (18). A strong link between NF-κB activation and MMP expression has been established. For example, Ma et al. reported that probiotic LB101 alleviated dry eye in mice by suppressing MMP-9 expression through the regulation of gut microbiota-mediated NF-κB signaling (19). Likewise, interleukin-17A has been shown to promote esophageal cancer invasion and metastasis through the NF-κB-MMP axis (20). However, whether Fn promotes CRC invasion and metastasis by modulating the NF-κB-MMP-2/MMP-9 signaling pathway remains largely unexplored. Given the observed association between Fn and CRC progression, targeting the Fn-NF-κB-MMPs axis may represent a potential therapeutic strategy. Preclinical evidence suggests that metronidazole treatment may reduce Fn abundance in CRC tissues and restore tumor sensitivity to immunotherapy (21). Similarly, pterostilbene may inhibit glioma cell proliferation and induce apoptosis by suppressing NF-κB signaling (22). Furthermore, several MMP inhibitors have demonstrated efficacy in preclinical models of pancreatic and prostate cancer, potentially reducing ECM degradation and inhibiting tumor progression (23).

This study aims to explore the potential mechanisms by which Fn may influence CRC cell proliferation and invasion. Specifically, we investigate whether the NF-κB signaling pathway mediates Fn-induced proliferation and invasion in CRC cells, and whether NF-κB regulates the expression of MMP-2 and MMP-9 in these cells. By clarifying the role of the Fn-NF-κB-MMP-2/MMP-9 axis in CRC metastasis, our findings may contribute to the development of novel therapeutic strategies for preventing CRC progression. We present the this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2727/rc).

Methods

Participants

A total of 28 patients diagnosed with CRC and 28 healthy individuals were recruited between July and December 2021. All participants were from The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing, China. Information on age and sex for all study participants is provided in Table S1. The CRC cohort included 6 cases for each tumor stage (T1, T2, T3, and T4). The classification of CRC was determined based on World Health Organization criteria, and all diagnoses were confirmed through histopathological examinations. Tumor staging, including tumor (T), node (N), and metastasis (M) classifications, was conducted based on the American Joint Committee on Cancer (AJCC) 8th Edition system (24). Exclusion criteria included: (I) patients with inaccurate TNM staging, (II) those who had undergone neoadjuvant therapy, and (III) individuals diagnosed with malignancies other than CRC. Colorectal tissue samples from CRC patients and healthy controls were obtained via colonoscopy biopsy during either CRC surgeries or colonoscopy examinations. Fresh specimens were immediately placed in sterile sample tubes and stored at −80 ℃ for subsequent analysis. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of The Affiliated Jiangning Hospital of Nanjing Medical University (Lot No. 2021-03-085-K01), and written informed consent was obtained from all participants.

Bacterial strain and cell culture

The CRC cell lines DLD-1 and SW480 were obtained from Shanghai Zhongqiaoxinzhou Biotec Co., Ltd. (Shanghai, China). All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and maintained at 37 ℃ in a 5% CO₂ environment. The Fn strain ATCC 25586 was purchased from the Shanghai Bioresource Collection Center (Shanghai, China). Fn was transferred to a sterilized Petri dish containing blood agar and cultured in an anaerobic incubator at 37 ℃. For infection experiments, CRC cells were incubated with Fn at a multiplicity of infection (MOI) of either 50 or 100 for 24 or 48 h.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from previously collected CRC tissue samples and cell suspensions using TRIzol reagent (Invitrogen, Carlsbad, CA), following the manufacturer’s protocol. Reverse transcription was performed using the PrimeScript™ RT Reagent Kit (Takara, Dalian, China) to synthesize complementary DNA. Next, we performed qRT-PCR using a primer for Fn with the system from Analytik Jena AG, Germany. The target gene expression was analyzed using the 2^−ΔΔCt method. The thermocycling conditions included an initial denaturation at 95 ℃ for 20 seconds, followed by 40 cycles of 95 °C for 5 seconds and 60°C for 30 seconds. The primer sequences used are provided in Table S2.

Cell viability assay

CRC cell suspensions were prepared and transferred to 96-well plates, followed by incubation at 37 ℃ with 5% CO₂ for 24 h. Subsequently, Cell Counting Kit-8 (CK04; Dojindo) reagent was added to each well at a 1:9 ratio and incubated for an additional 4 h. Finally, the culture plate was placed in a microplate reader (iMark; BioRad) to measure the absorbance at a wavelength of 450 nm. All experimental procedures were performed with a minimum of three biological replicates.

Wound healing assay

Human CRC cells were seeded in 6-well plates and cultured until confluence. After creating wounds with yellow pipette tips, the cells in each well were incubated with Fn at a MOI of 50:1 and 100:1. Cell images were taken at 24 and 48 h, respectively. Each condition was tested in three independent wells.

Transwell assays

The harvested CRC cells were suspended in a medium of 5×10⁵ cells/mL. Next, 100 µL of the CRC cell suspension was added to the upper chamber of an 8 µm Transwell (BD Falcon, USA). The polycarbonate filter was precoated with 100 µL of Matrigel (BD Biosciences, USA). The lower chamber contained 600 µL of culture medium supplemented with 1% FBS. The Transwells were then incubated at 37 ℃ with 5% CO₂ for 6 to 16 h. After incubation, the cells remaining in the top chamber were removed with a cotton-tipped applicator, while the migrated cells at the bottom of the membrane were stained with 4',6-diamidino-2-phenylindole (DAPI). These images were then taken using an LSM 800 confocal microscope (Zeiss, Germany) with a 10× objective. CRC cells were incubated with Fn at a MOI of 50:1 for 12 h and 100:1 for 48 h. The cells were quantified according to the method described (25). Each condition was tested in three independent wells.

Western blotting

Western blot analysis was conducted to assess the protein expression in CRC cell lines. Total protein was extracted and its concentration was measured using the Bicinchoninic Acid (BCA) Protein Assay Kit (P0012S; Beyotime). Electrophoresis was used to separate 20 µg of protein samples on a 12% sodium dodecyl sulfate-polyacrylamide gel, which was then transferred to a polyvinylidene fluoride (PVDF) membrane (BioRad, Hercules, USA). After blocking with 5% milk, the membranes were incubated overnight at 4 ℃ with specific primary antibodies, including p65 (Cell Signaling Technology, USA), IκBα (Cell Signaling Technology, USA), MMP-2 (Abcam, UK), MMP-9 (Abcam, UK), and GAPDH (Proteintech, China), all at a 1:1,000 dilution. HRP-conjugated secondary antibodies (Thermo Fisher Scientific, USA) were applied to the membranes, which were then exposed to film using enhanced chemiluminescence (ECL). Tubulin and GAPDH were used as internal loading controls. The protein levels were quantified using an Image Analysis System (Olympus DP72, Japan). Each condition was tested in three independent experiments.

Cell transfection test

P65-specific siRNA was synthesized by Gene Pharma (Shanghai, China), along with LV3NC lentiviral vectors and Instant Transfer Reagents. For small interfering RNA (siRNA) transfection, 150 pmol of siRNA and 6 µL of Instant Transfection Reagent were diluted in 200 µL of serum-free medium, mixed thoroughly, and incubated at room temperature for 5–10 minutes to facilitate siRNA complex formation. DLD-1 and SW480 cells were seeded in a six-well plate at a density of 1×10⁵ cells per well and transfected with the siRNA complex (400 µL per plate) for 6 h. The siRNA complex was subsequently removed, and the cells were cultured in regular media. For plasmid transfection, cells were transfected using Lipotransfectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 24 h, the relative expression levels of p65 and phosphorylated p65 (p-p65) were assessed via Western blot analysis. The sequences of the siRNAs are listed in Table S3. Experimental procedures included a minimum of three replicates.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical significance was determined using a two-tailed Student’s t-test or one-way analysis of variance (ANOVA) as appropriate. A P<0.05 was considered statistically significant. All statistical analyses were performed using SPSS 25 or GraphPad Prism 8.

Results

Association between Fn abundance and CRC progression

To investigate the clinical significance of Fn in CRC, we evaluated its abundance in tissue samples from CRC patients and healthy controls. The baseline characteristics of the study population are summarized in Table 1. There were no significant differences between the two groups in terms of age, sex distribution, smoking status, or alcohol consumption (all P>0.05). PCR analysis revealed a significantly higher enrichment of Fn in CRC tissues compared to healthy individuals (P<0.01) (Table 1). Furthermore, as TNM staging advanced, the abundance of Fn exhibited a progressive increase in CRC patients (P<0.01) (Figure 1). Notably, elevated Fn levels in CRC tissues were strongly associated with lymph node involvement and distant metastasis (P<0.01). These findings suggest that increased Fn abundance is associated with CRC progression and metastatic features.

Figure 1 Fn abundance of CRC patients at different TNM stages (A-C). **, P<0.01. CRC, colorectal cancer; Fn, Fusobacterium nucleatum; TNM, tumor-node-metastasis.

Fn promotes CRC cell proliferation, invasion, and migration

DLD-1 and SW480 cell lines are widely used models for investigating the molecular mechanisms of CRC (26). To evaluate the impact of Fn on CRC cell behavior in vitro, we infected DLD-1 and SW480 cell lines with Fn at multiplicities of infection of 50:1 and 100:1 for 24 and 48 h. CCK assays demonstrated that CRC cells infected with Fn exhibited significantly enhanced viability compared to the control group. Notably, the Fn [100] group showed a greater increase in cell viability than the Fn [50] group, suggesting a dose-dependent effect (P<0.01) (Figure 2A). Wound-healing assays revealed a significant increase in the relative wound closure rate in both Fn [50] and Fn [100] groups compared to the control (P<0.01). Furthermore, cells infected with Fn at 100:1 exhibited a more pronounced enhancement in migration (P<0.01) (Figure 2B). Transwell migration and invasion assays further confirmed the pro-metastatic effects of Fn infection. The number of migrating and invading cells per field of view was significantly increased in both the Fn [50] and Fn [100] groups compared to the control group (P<0.01). Moreover, the Fn [100] group exhibited a stronger effect than the Fn [50] group, supporting a dose-dependent effect of Fn infection (P<0.01) (Figure 2C,2D). These findings indicate that Fn infection significantly enhances the proliferation, migration, and invasion capabilities of DLD-1 and SW480 cell lines in a concentration-dependent manner.

Figure 2 Fn promoted the proliferation, invasion, and migration of CRC cell lines DLD-1 and SW480. (A) CCK-8 assay was used to evaluate cell proliferation. (B) Scratch assays were performed after 24 and 48 h of incubation with Fn [50] and Fn [100] to observe the relative wound healing rate (scale bar =100 µm). (C) Transwell assays were utilized to observe the number of migrating cells per field of view (scale bar =50 µm; crystal violet stained). (D) Transwell assays were conducted to observe the number of invading cells per field of view (scale bar =50 µm; crystal violet stained). **, P<0.01 (vs. control group), ^##, P<0.01 (vs. Fn [50] group). CCK-8, Cell Counting Kit-8; CRC, colorectal cancer; Fn, Fusobacterium nucleatum.

Effect of Fn infection on MMP-2 and MMP-9 expression

MMP-2 and MMP-9 are key mediators of ECM degradation and play important roles in tumor invasion and metastasis. To investigate whether Fn affects CRC cells via MMP-2 and MMP-9, we treated DLD-1 and SW480 cells with different concentrations of Fn and analyzed MMP-2 and MMP-9 protein expression by Western blot, normalizing the levels to GAPDH. Compared to the control group, the relative expression of MMP-2 and MMP-9 was significantly elevated in the Fn [100] and Fn [50] groups (P<0.01) in a dose-dependent manner (Figure 3A,3B). These findings suggest that Fn infection is associated with increased MMP-2 and MMP-9 expression in CRC cells, supporting a potential role in CRC cell behavior.

Figure 3 The impact of Fn on MMPs and NF-κB phosphorylation. (A) MMP-2 and MMP-9 protein levels were assessed in DLD-1 cells after 24 h of Fn infection by Western blot. (B) MMP-2 and MMP-9 protein levels were evaluated in SW480 cells after 24 h of Fn infection via Western blot analysis. (C) Western blot illustrates the expression of p-p65, p65, p-IκBα, and IκBα in the NF-κB signaling pathway in DLD-1 cells. (D) Western blot illustrates the expression of p-p65, p65, p-IκBα, and IκBα in the NF-κB signaling pathway in SW480 cells. The molecular weights of the detected proteins were as follows: MMP-2, 72 kDa; MMP-9, 92 kDa; p-p65, 65 kDa; p65, 65 kDa; p-IκBα, 40 kDa; IκBα, 40 kDa; and GAPDH, 36 kDa. **, P<0.01 (vs. control group). Fn, Fusobacterium nucleatum; MMP, matrix metalloproteinase; NF-κB, nuclear factor kappa B.

Fn upregulates MMP-2 and MMP-9 through the NF-κB signaling pathway

The mechanism through which Fn infection increases MMP-2 and MMP-9 is still unclear. Given that NF-κB is a well-established transcription factor implicated in cancer progression, we investigated whether the NF-κB signaling pathway mediates the Fn-induced upregulation of MMP-2 and MMP-9. Western blot analysis demonstrated that co-culturing DLD-1 and SW480 cells with Fn significantly enhanced the phosphorylation levels of p65 in a dose-dependent manner while reducing the expression of IκBα, an inhibitor of NF-κB activation (Figure 3C,3D). These results indicate that Fn infection is associated with increased NF-κB phosphorylation, potentially contributing to higher MMP-2 and MMP-9 expression.

Fn promotes CRC metastasis by modulating the NF-κB-MMP-2/MMP-9 signaling pathway

We conducted siRNA-mediated knockdown experiments to investigate whether Fn affects CRC cell proliferation, invasion, and migration through the NF-κB-MMP-2/MMP-9 pathway. As shown in Figure 4A-4D, treatment with Fn [100] significantly enhanced CRC cell proliferation, migration, and invasion compared to the control group. In contrast, siRNA-mediated knockdown of NF-κB p65 (si-p65) led to a marked reduction in these cellular processes. Notably, the Fn [100] + si-p65 group exhibited partial restoration of cell proliferation, migration, and invasion compared to the si-p65 group, but these levels remained significantly lower than those observed in the Fn [100] group. Western blot analysis further supported these functional findings. As shown in Figure 4E, Fn [100] treatment markedly upregulated the expression of MMP-2 and MMP-9, whereas si-p65 treatment significantly downregulated their expression. The partial recovery of MMP-2 and MMP-9 expression in the Fn [100] + si-p65 group suggests that NF-κB may mediate some of the effects of Fn infection on MMP expression and associated cell invasion. Additionally, Western blot analysis (Figure 4F) demonstrates that siRNA targeting NF-κB p65 effectively reduced NF-κB phosphorylation levels, as indicated by a significant decrease in the p-p65/p65 ratio (P<0.01), thereby confirming knockdown efficiency. Moreover, si-p65 treatment led to a compensatory increase in the p-IκBα/IκBα ratio, further supporting the suppression of NF-κB activation.

Figure 4 Inhibition of NF-κB phosphorylation and its effects on proliferation, invasion, migration, and MMP-2/MMP-9 levels in SW480 colorectal cancer cell lines. (A) The CCK-8 assay demonstrates that si-p65 inhibits the proliferation of SW480 cells. (B) The scratch assay reveals that si-p65 inhibits the relative wound healing rate of SW480 cells (scale bar =100 µm). (C,D) Transwell assay demonstrates that si-p65 inhibits the invasion and migration of CRC cells (scale bar =50 µm; crystal violet stained). (E) Representative Western blots of MMP-2 and MMP-9 in CRC cells after incubation with si-p65. (F) Western blot illustrates the expression of p-p65, p65, p-IκBα, and IκBα in the NF-κB signaling pathway in DLD-1 cells. The molecular weights of the detected proteins were as follows: MMP-2, 72 kDa; MMP-9, 92 kDa; p-p65, 65 kDa; p65, 65 kDa; p-IκBα, 40 kDa; IκBα, 40 kDa; and GAPDH, 36 kDa. **, P<0.01 (vs. control group); ^##, P<0.01 (vs. Fn [100] group). CCK-8, Cell Counting Kit-8; CRC, colorectal cancer; Fn, Fusobacterium nucleatum; MMP, matrix metalloproteinase; NF-κB, nuclear factor kappa B.

Discussion

Among the recently identified CRC-related microbes, Fn has attracted significant attention. Our study demonstrates that Fn infection enhances proliferation, invasion, and migration of CRC cells in vitro and is more prevalent in advanced-stage tumors. We also observed upregulation of MMP-2 and MMP-9 following Fn infection and explored the underlying mechanism involving activation of the NF-κB signaling pathway.

Previous studies have demonstrated that Fn adheres to intestinal epithelial or CRC cells via FadA and Fap2, activating the Wnt/β-catenin and NF-κB signaling pathways, ultimately leading to the increased expression of oncogenes and inflammatory genes (27). Additionally, FadA contributes to CRC progression by inducing the upregulation of LINC00460, consequently resulting in the overexpression of ANXA2 through the ceRNA network (28). Casasanta MA et al. further revealed that Fn induces the secretion of IL-8 and CXCL1, which in turn promote the migration of CRC cells (29). NF-κB is a key nuclear transcription factor that regulates immune responses and inflammation, which then contributes the migration of CRC cells (30). Excessive activation of NF-κB contributes to cancer development by increasing cell proliferation, angiogenesis, invasion, and metastasis (31). NF-κB can be activated through the canonical pathway by TNF-α and Toll-like receptor ligands (e.g., LPS and IL-1) or through the non-canonical pathway by TNF superfamily members, including BAFF, CD40, RANKL, and lymphotoxin-β (32). Notably, our study provides new evidence that Fn activates the NF-κB signaling pathway, subsequently promoting migration and invasion of the CRC cell lines.

Tumor metastasis involves the degradation of the ECM and the subsequent migration of cancer cells. MMPs are expressed in CRC tissues and are correlated with tumor invasion and metastasis (33). Previous studies have shown that MMP-2 and MMP-9 levels are significantly elevated in CRC patients and adenomas, correlating strongly with tumor invasion depth (34). Similarly, high expression of MMP-2 and MMP-9 genes is linked to the migration and invasion of bladder cancer (35). As key regulators of tumor metastasis, MMP-2 and MMP-9 are controlled by the NF-κB signaling pathway (36). In our experiment, Fn infection was associated with enhanced invasion and migration of CRC cell lines, accompanied by NF-κB pathway activation. Conversely, inhibiting the NF-κB signaling resulted in reduced protein levels of MMP-2 and MMP-9, as well as decreased invasion and migration abilities of CRC.

While our study provides compelling evidence linking Fn abundance with CRC progression, its potential impact on patient prognosis remains an open question. Several clinical studies have suggested a relationship between Fn persistence and CRC outcomes. For example, Serna et al. reported that Fn persistence after neoadjuvant chemoradiotherapy in locally advanced rectal cancer is associated with a higher relapse rate (37). Additionally, an analysis of tumor samples from 25 CRC patients receiving anti-PD-1 therapy found that higher intratumoral Fn abundance correlated with im-proved patient prognosis (38). Future prospective studies incorporating survival analysis and functional validation are necessary to determine the independent prognostic significance of Fn in CRC. In our study, the 24–48 timeframe for in vitro Fn infection was chosen to capture acute responses in CRC cells, such as enhanced proliferation, migration, and invasion. However, this timeframe may not fully reflect the long-term effects of Fn colonization in the tumor microenvironment. Persistent Fn infection might contribute to chronic inflammation, immune evasion, and therapy resistance, potentially influencing CRC aggressiveness beyond the NF-κB-MMP axis explored. Fn has been reported to modulate the tumor microenvironment through inflammatory cytokines, such as IL-6, IL-8, COX-2 and TNF-α, which further drive tumor progression and immune suppression (8). Notably, Fn has been reported to contribute to colitis-associated CRC development by inducing M1 macrophage polarization, leading to a pro-inflammatory milieu that favors tumorigenesis (39). Future studies of the interactions between Fn infection, cytokine production, and immune cell infiltration will be crucial to fully elucidate its role in CRC progression. Although our current study does not include in vivo experiments, we demonstrate a significant correlation between Fn abundance and CRC stage, lymph node metastasis, and distant metastasis, strongly supporting our hypothesis. Future studies will employ xenograft and genetically engineered mouse models to further investigate the in vivo relevance of our proposed mechanism.

Despite these findings, several limitations of the present study should be acknowledged. First, the clinical cohort was relatively small (n=28 per group) and drawn from a single institution, which may limit the generalizability of the results and warrants framing the study as a pilot/proof-of-concept investigation. Second, the cross-sectional design precludes definitive conclusions regarding causality; advanced tumors may create a microenvironment favorable to Fn colonization rather than Fn directly driving CRC progression. Third, our in vitro experiments cannot distinguish between Fn-specific adhesin-mediated signaling and general inflammatory responses, such as LPS-triggered NF-κB activation via TLR4/TLR2. Therefore, while Fn infection was associated with NF-κB activation and upregulation of MMP-2 and MMP-9, these effects cannot yet be conclusively attributed to Fn-specific interactions. Fourth, the exclusive use of MSS cell lines (DLD-1 and SW480) limits translational relevance. Clinical evidence demonstrates that Fn is preferentially enriched in microsatellite instability-high (MSI-H) colorectal tumors (38,40,41), and Fn-mediated effects such as NF-κB-dependent upregulation of miR-155-5p and suppression of mismatch repair proteins (e.g., MSH6) cannot be adequately modeled in MSS cells. Additional molecular characteristics of the selected cell lines—including differential invasive capacities, E-cadherin expression variability, and KRAS mutation status—may further confound the interpretation of Fn-induced signaling. The absence of BRAF V600E-mutant and MSI-H models precludes evaluation of Fn effects in clinically relevant subgroups. Fifth, the temporal dynamics of NF-κB activation were not characterized. Measurements at 24–48 h capture late responses but cannot distinguish transient versus sustained signaling, which may have distinct implications for CRC progression. Sixth, MOI standardization was limited; actual infection efficiency and physiological relevance of MOIs 50:1 and 100:1 were not quantified, potentially affecting reproducibility and cross-study comparability. Seventh, the absence of in vivo validation limits mechanistic conclusions, as two-dimensional monolayer cultures cannot fully recapitulate tumor microenvironment complexity, including three-dimensional architecture, stromal and immune components, and hypoxic gradients that influence Fn colonization and inflammatory signaling. Finally, additional potential confounding factors—including tumor location, medication history, dietary patterns, and molecular subtypes—were not available in the present dataset. Future studies should integrate microbiome profiling with comprehensive clinical and molecular data, employ adhesin-deficient strains, TLR inhibitors, and clinically relevant MSI-H or BRAF-mutant cell models, and include in vivo validation to clarify the specific role of Fn in CRC progression.

Conclusions

In summary, our study provides new insights into the potential role of Fn in CRC progression. We observed that Fn infection is associated with enhanced CRC cell proliferation, invasion, and migration in vitro, via activation of the NF-κB signaling pathway and upregulation of MMP-2 and MMP-9. Clinically, Fn abundance was associated with advanced CRC stages, suggesting a potential link with tumor progression. These findings support further investigation of Fn-targeted strategies for CRC prevention and therapy. Future large-scale clinical studies and in vivo experiments are warranted to validate Fn as a prognostic biomarker and therapeutic target.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2727/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2727/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2727/prf

Funding: This work was funded by the Science and Technology Development Fund of Nanjing Medical University (No. NMUB20220163), the Scientific Research Development Fund of Kangda College of Nanjing Medical University (No. KD2023KYJJ222), the General Project of Basic Science Research in Universities of Jiangsu Province (No. 24KJB310008), and Youth Innovation Research Fund of The Affiliated Jiangning Hospital of Nanjing Medical University (No. JNYYZXKY202417).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2727/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Affiliated Jiangning Hospital of Nanjing Medical University (protocol code: 2021-03-085-K01; date of approval: 23 April 2021). Informed consent was obtained from all subjects involved in the study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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