Preliminary exploration of the role of fibrinogen-like protein 2 in neuroblastoma

Abstract

Fibrinogen-like protein 2 (FGL2) has been reported to modulate the tumor microenvironment and play critical roles in the initiation and progression of various tumors. However, its role and underlying mechanisms in neuroblastoma remain unclear. This study aimed to investigate the function of FGL2 in regulating the proliferation, migration, and invasion of neuroblastoma cells. FGL2 expression in neuroblastoma specimens was assessed using immunohistochemistry, polymerase chain reaction, and western blotting. The prognostic value of FGL2 in patients with neuroblastoma was evaluated using Kaplan–Meier survival analysis. Cell Counting Kit-8, colony formation, wound healing, and Transwell invasion assays were performed to assess cell proliferation, migration, and invasion following FGL2 knockdown. The potential molecular mechanisms were explored by western blotting to examine changes in signaling molecules after FGL2 silencing. The results showed that both protein and messenger RNA levels of FGL2 were significantly lower in high-risk neuroblastoma samples compared with non-high-risk samples. Low FGL2 expression was significantly associated with advanced International Neuroblastoma Staging System stage, high-risk classification, and poor survival status. Kaplan–Meier analysis further revealed that patients with low FGL2 expression had significantly worse survival outcomes. Functionally, Cell Counting Kit-8 and colony formation assays demonstrated that FGL2 knockdown increased the viability of neuroblastoma cells, whereas wound healing and Transwell assays confirmed that silencing FGL2 significantly enhanced migratory and invasive capacities compared with control cells. Mechanistically, FGL2 suppressed cell proliferation, migration, and invasion by regulating the nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway. These findings suggest that FGL2 may serve as both a prognostic biomarker and a potential therapeutic target in neuroblastoma.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-26272-x.

Introduction

Neuroblastoma (NB) is the most common extracranial solid tumor in children, originating in the adrenal medulla or sympathetic ganglia¹. It accounts for approximately 8–10% of all childhood tumors and 15% of cancer-related deaths². Advances in molecular biology and medical technology in recent years have markedly improved the prognosis of NB³. However, the five-year overall survival rate for high-risk patients remains only 40–50%. This highlights the urgent need to identify novel molecular biomarkers for elucidating regulatory mechanisms, evaluating prognosis, and developing therapeutic targets.

Fibrinogen-like protein 2 (FGL2), located on chromosome 7q11.23 in humans, belongs to the fibrinogen-like protein family⁴. In humans, FGL2 exists in two structural forms: a membrane-associated protein (mFGL2) and a secreted soluble protein (sFGL2)⁵. It has multiple biological functions, including roles in coagulation and immunoregulation. Previous studies have demonstrated that FGL2 plays an important role in various tumors^6–8, influencing patient prognosis by regulating immune surveillance and immunosuppression mechanisms^4,9. Olli-Pekka et al. reported that positive FGL2 expression was associated with favorable survival outcomes in gastrointestinal stromal tumors¹⁰.

Moreover, our previous research demonstrated, through bioinformatics analysis, that FGL2 levels were lower in high-risk NB samples¹¹. However, no experimental studies have yet reported on the prognostic value or molecular mechanisms of FGL2 in NB. Therefore, we aimed to explore the potential roles of FGL2 in this disease.

Materials and methods

Clinical samples

A total of 32 paraffin-embedded tissue samples from patients diagnosed with NB were collected at the Children’s Hospital of Hebei Province between January 1, 2011, and December 31, 2020. Basic patient characteristics, including sex, age, and pathological data, were recorded. The follow-up endpoint was overall survival, defined as the time from initial treatment to death from any cause. Follow-up data were obtained through telephone interviews or outpatient visits. In addition, four pairs of freshly frozen high-risk and non-high-risk NB tissues were collected after surgical resection at the Children’s Hospital of Hebei Province between June 1, 2023, and December 31, 2023. The clinical data for these freshly frozen samples were retrieved between January 1, 2024, and January 31, 2024. This study was approved by the Ethics Committee of the Children’s Hospital of Hebei Province (20,210,794) and conducted in accordance with the ethical standards of the Declaration of Helsinki. Written informed consent for participation was obtained from the parents or legal guardians of the patients.

Immunohistochemical staining

Paraffin-embedded tissue specimens were deparaffinized, dewaxed and rehydrated in a graded ethanol series. Sections were incubated overnight at 4 °C with polyclonal rabbit anti-human FGL2 antibodies (1:50, PA554306, Thermo Fisher, USA). After washing with phosphate-buffered saline, the sections were incubated with a goat anti-rabbit secondary antibody for 60 min at room temperature. The signal was then visualized using a peroxidase substrate 3,3′-diaminobenzidine staining kit (DAB-0031/1031, Miaxim.bio, China). Two independent pathologists, blinded to the clinical outcomes, evaluated the stained sections. The total immunohistochemistry score for FGL2 expression was semi-quantitatively determined based on the proportion of positively stained cells and the staining intensity. The proportion of positive cells was scored as follows: 0 points for 0–5%, 1 point for 5–50%, and 2 points for > 50%. Staining intensity was scored as 0 points for no staining, 1 point for yellow–brown staining, and 2 points for brown staining. The final score was the sum of the proportion and intensity scores, and FGL2 expression was classified as low (0–2) or high (3–4).

Western blot

Tissues and cells were lysed in cell lysis buffer, and total protein was extracted. Protein concentration was determined using a bicinchoninic acid assay (No. 23225, Thermo). Equal amounts of protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% bovine serum albumin for 2 h at room temperature, the membranes were incubated overnight at 4 °C with one of the following primary antibodies: IκBα (1:1000, A19714, ABclonal, China), phospho-IκBα-S32 (1:1000, AP0707, ABclonal, China), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65/RelA (1:1000, A2547, ABclonal, China), phospho-NF-κB p65/RelA-S536 (1:2000, AP1294, ABclonal, China), FGL2 (1:1000, PA554306, Thermo Fisher, USA), IKKβ (1:2000, ab124957, Abcam, USA), phospho-IKKβ (1:1000, ab194519, Abcam, USA), Snail (1:1000, ab2163477, Abcam, USA), E-cadherin (1:1000, ab314063, Abcam, USA), vimentin (1:2000, ab92547, Abcam, USA), and glyceraldehyde-3-phosphate dehydrogenase (1:2000, 10,494–1-AP, Proteintech, USA). Membranes were washed three times with Tris-buffered saline with Tween 20, then incubated for 1.5 h with horseradish peroxidase-conjugated secondary antibodies (sc-18814 / sc-2453,anti-mouse or anti-goat IgG, Santa Cruz). Glyceraldehyde-3-phosphate dehydrogenase served as a loading control. Protein bands were visualized using an enhanced chemiluminescence kit, and band intensities were quantified with ImageJ software.

Quantitative real-time polymerase chain reaction (PCR)

Total RNA was extracted from tissue samples and cell lines using TRIzol (No. 15596026, Invitrogen). For each sample, 1 µg of RNA was reverse-transcribed using the PrimeScript RT reagent kit (RR037A,Takara). Quantitative PCR was then performed with Power SYBR® Green PCR Master Mix (No, 4,367,659, Takara) following the instructions of the manufacturer. The primer sequences for FGL2 were: forward 5’-AAATGTTCAAAGTGTCCCAGCCAAG-3' and reverse 5’-TGCCTATTGCGTAGTAGTCAGAGC-3'. Relative expression levels were normalized, and the 2^-ΔΔCt method was used to calculate the relative messenger RNA (mRNA) expression.

Cell culture and transfection

Human NB cell lines (IMR-32 and SH-SY5Y) were purchased from the American Type Culture Collection and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum in a standard humidified atmosphere containing 5% carbon dioxide at 37 °C. Cells in the exponential growth phase were used for transfection. They were transfected with FGL2-specific small interfering RNA (siRNA) or the corresponding negative control (NC), both purchased from Biowit Technologies (Shenzhen, China), according to the instructions of the manufacturer. The target sequences for FGL2 siRNA were: sense 5’-GCUCUGACUACUACGCAAUTT-3’ and antisense 5’-AUUGCGUAGUAGUCAGAGCTT-3’. The sequences for NC siRNA were: sense 5’-UUC UCC GAA CGU GUC ACG UTT-3’ and antisense 5’-ACG UGACAC GUU CGG AGAATT-3’. After 48 h, western blot analysis was performed to assess the knockdown efficiency.

Cell Counting Kit-8 (CCK-8) assay

Transfected NB cells were seeded in 96-well plates at a density of 5 × 10³ cells per well. At 0, 1, 2, and 3 days after seeding, 10 mL of CCK-8 solution (No. C0038,Beyotime, Jiangsu, China) was added to each well, and the cells were incubated at 37 °C for an additional two hours. Optical density at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, USA) to assess cell viability.

Colony formation assay

Cells were seeded in six-well plates at a density of 0.8 × 10³ cells per well and cultured for 14 days. Colonies were fixed with 4% formaldehyde and then stained with 1% crystal violet for 30 s. After washing with running water, the plates were air-dried at room temperature, and the number of colonies was counted using ImageJ software (version 1.52p).

Transwell assay

Transfected IMR-32 cells and SH-SY5Y (~ 2 × 10⁴) were harvested and seeded into the upper chambers of Transwell inserts. For the invasion assay, the upper chamber was pre-coated with 40 mL of Matrigel (354,234, Corning, New York, USA). The lower chamber was filled with culture medium containing 20% fetal bovine serum. After 24 h of incubation, cells remaining on the upper surface of the membrane were removed with a cotton swab. Invaded cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The number of invaded cells was counted in five random fields under a light microscope at × 200 magnification.

Wound healing assay

Cells were seeded into six-well plates at a density of 5 × 10⁵ cells per well and cultured for 48 h until reaching approximately 90% confluence. An artificial wound was then created in the cell monolayer using a pipette tip. Floating cells were removed by washing with phosphate-buffered saline, and the remaining cells were cultured in serum-free RPMI-1640 medium. Wound closure was photographed at 0 and 48 h using an inverted microscope at the same locations for comparison.

Statistical analysis

Statistical analyses were conducted using SPSS software (version 20.0; SPSS Inc., Chicago, IL) and Prism 8 (GraphPad Inc., USA). Continuous variables were presented as mean ± standard deviation, and categorical variables as frequencies or percentages. The chi-square test or Fisher’s exact test was used to assess correlations between FGL2 expression and clinicopathological variables. Survival curves were generated using Kaplan–Meier analysis and compared with the log-rank test. All experiments were performed at least three times, and differences between two groups were analyzed using the Student’s t-test. A P-value < 0.05 was considered statistically significant.

Results

Expression levels of FGL2 protein and mRNA in NB tissues

The expression of FGL2 in NB was evaluated using immunohistochemical staining, western blotting, and PCR (Fig. 1A–C). Immunohistochemical staining showed that FGL2 expression was lower in high-risk NB samples compared with non-high-risk samples. Consistent with these findings, both western blot analysis of protein levels and PCR analysis of mRNA levels demonstrated significantly lower FGL2 expression in high-risk samples than in non-high-risk samples (both P < 0.001).

Fig. 1.

FGL2 expression in neuroblastoma. (A) Immunohistochemistry showed that the expression of FGL2 in high risk NB samples was lower than the expression in non-high risk NB samples (× 200) (B) The protein of FGL2 was signficantly decreased in high risk NB samples compared with non-high risk NB samples by western blot analysis. (C) The mRNA of FGL2 was signficantly decreased in high risk NB samples compared with non-high risk NB samples by RT-PCR analysis. (D) Kaplan–Meier survival analysis was conducted based on FGL2 expression by immunohistochemistry. Low FGL2 expression was associated with the poor overall survival of NB. H: high risk, N: non-high risk, ***P < 0.001.

Association between FGL2 protein levels, clinical characteristics, and survival in patients with NB

To investigate the role of FGL2 in tumor pathogenesis and progression, we examined the association between immunohistochemical FGL2 expression and clinicopathological parameters. As shown in Table 1, FGL2 expression was significantly associated with International Neuroblastoma Staging System stage, risk stratification, and survival status (P < 0.05), but not with age, sex, or Myc-N proto-oncogene (MYCN) amplification. These results suggest that low FGL2 expression is related to malignant invasion and metastasis in NB. To further assess the relationship between FGL2 expression and overall survival in patients with NB, Kaplan–Meier survival analysis was performed. The median patient age was 20 months (range: 1 month–9 years), and the median follow-up time was 28 months (range: 4–91 months). During the follow-up period, seven patients died. The Kaplan–Meier survival curve showed that patients with low FGL2 expression had significantly poorer survival than those with high expression (log-rank test, P < 0.001; Fig. 1D).

Table 1.

Clinicalpathological characteristics between groups.

Clinicopathological characteristics	Number of patients	Low FGL2	High FGL2	χ2	P
Age(months)
< 18	15	4	11	0.744	0.388
≥ 18	17	7	10
Gender
Male	15	6	9	0.396	0.529
Female	17	5	12
INSS stage
1 + 2	22	3	19	13.422	< 0.001
3 + 4	10	8	2
MYCN amplification
Non-Amp	26	7	19	3.413	0.065
Amplified	6	4	2
Risk stratification
Non-high	23	5	18	5.788	0.016
High	9	6	3
Living status
Alive	25	5	20	10.469	0.001
Dead	7	6	1

INSS, Internationa Neuroblastoma Staging System; MYCN, Myc-N proto-oncogene.

FGL2 knockdown promotes proliferation, migration, and invasion of NB cells

To investigate the role of FGL2 in NB, FGL2-silenced NB cell lines were generated. Western blot analysis confirmed effective knockdown, with significantly reduced FGL2 levels in cells transfected with FGL2 siRNA compared with those transfected with NC (P < 0.001; Fig. 2A). CCK-8 assays showed that the FGL2 siRNA group exhibited higher cell viability than the NC group (Fig. 2B). Colony formation assays further demonstrated that FGL2 knockdown significantly enhanced NB cell proliferation (Fig. 2C). Transwell and wound healing assays were performed to assess the effects of FGL2 on cell invasion and migration. FGL2 knockdown markedly increased invasive capacity in the Transwell assay, and the wound closure rate was significantly higher in FGL2 siRNA-transfected cells compared with the NC group (P < 0.001; Fig. 2D, E). In summary, these in vitro findings suggest that FGL2 inhibits the proliferation, migration, and invasion of NB cells.

Fig. 2.

FGL2 inhibits NB cell proliferation migration and the invasion (A) western blot assays demonstrated that FGL2 was effectively knocked down by siRNA in IMR-32.and SH-SY5Y cells. (B) CCK8 assay demonstrated that FGL2 knockdown promoted the cell proliferative ability. (C) Colony formation assay indicated that FGL2 knockdown promoted the proliferation of NB cells. (D) Transwell assays revealed that FGL2 knockdown increased the invasive number of NB cells. (E) The wound-healing assay indicated that FGL2 knockdown significantly increased cell migration rate in NB cells.*P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001.

FGL2 inhibits NF-κB signaling activity in NB cells

To investigate the signaling pathway involving FGL2, NF-κB activation was examined in NB cells by assessing the expression of NF-κB pathway-related proteins using western blot analysis. FGL2 levels did not affect the total expression of p65 or IκBα; however, FGL2 knockdown increased the phosphorylation levels of both proteins. The elevated p-p65/p65, and p-IκBα/IκBα ratios indicated that silencing FGL2 promoted NF-κB translocation into the nucleus, thereby enhancing NF-κB transcriptional activity (Fig. 3A). Moreover, FGL2 downregulation did not significantly alter the total expression of the upstream regulator IKKβ, but it increased the phosphorylation level of IKKβ (p-IKKβ), resulting in a higher p-IKKβ/IKKβ ratio. Consistent with NF-κB pathway activation, the expression of downstream factors Snail and vimentin was upregulated, whereas E-cadherin expression was downregulated (Fig. 3As).

Fig. 3.

FGL2 blocks the activation of NF-Κb signaling pathway. (A) Western blot was performed to show that BAY 11–7082 reversed the expression of NF-κB nuclear translocation -related proteins . (As) The expression levels of the upstream regulator and downstream factors of NF-κB pathway-related proteins (B–E) The effect of siFGL2 on cell proliferation (B) and (C), invasion (D), and migration was (E) reversed by BAY 11–7082. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001.

Inhibition of NF-κB with BAY 11–7082 reverses the effects of FGL2 silencing on NB cell progression

To determine whether NF-κB is involved in FGL2-mediated NB progression, cells were treated with the NF-κB inhibitor BAY 11–7082. This inhibitor suppressed the phosphorylation of IKKβ, resulting in reduced p-IKKβ levels. Consequently, IκBα was not phosphorylated, and the expression levels of p-p65 and p-IκBα decreased (Fig. 3A). This was accompanied by downregulation of the downstream factors Snail and vimentin and upregulation of E-cadherin expression (Fig. 3As), indicating that BAY 11–7082 reversed FGL2 knockdown-induced NF-κB pathway activation in SH-SY5Y cells. Functional assays, including CCK-8, colony formation, Transwell, and wound healing assays, further demonstrated that BAY 11–7082 counteracted the FGL2 knockdown-induced enhancement of cell proliferation, migration, and invasion (Fig. 3B–E). In summary, these results indicate that NF-κB signaling is involved in FGL2-mediated suppression of NB progression.

Discussion

As the most common pediatric extracranial solid tumor, NB exhibits is characterized by marked biological heterogeneity and variable clinical severity³. Approximately 50% of children present with metastases at initial diagnosis. Although non-high-risk patients have an overall survival rate exceeding 90%, the five-year overall survival rare for high-risk patients remains only 40–50%. Despite significant advances in the treatment of high-risk NB over recent decades, many patients in this group continue to experience poor outcomes. In recent years, increasing evidence has shown that the tumor microenvironment, along with immune components and immune responses, can influence tumor initiation, progression, and metastasis. Riyue Bao et al. reported that interactions between tumor cells and components of the microenvironment can shape the phenotype of NB, and that high-risk cases with a strong T cell-inflamed signature have a more favorable prognosis¹². Similarly, Yong-Liang Sha et al. found that β-1,3-galactosyltransferase-4 can modulate the tumor microenvironment of NB, and that lipid raft inhibitors can enhance the efficacy of anti-GD2 immunotherapy¹³. However, more than 40% of patients with NB exhibit resistance to immunotherapy and continue to have poor prognoses. Therefore, this study was conducted to investigate the molecular mechanisms underlying immunoregulation in NB.

FGL2 is a member of the fibrinogen superfamily of proteins and exists in two structurally distinct isoforms: mFGL2 and sFGL2^14,15. mFGL2 is a type II transmembrane protein containing both an N-terminal and a C-terminal domain and is expressed on the surface of epithelial cells, macrophages, neutrophils, and other cell types. Numerous studies have shown that mFGL2 acts as an immune coagulator by cleaving prothrombin in the context of innate immunity. It has been implicated in inflammatory bowel disease, sepsis, and severe acute pancreatitis^16–18. In contrast, sFGL2 contains only the C-terminal fibrinogen-related domain and is secreted into the plasma by endothelial cells, macrophages, and regulatory T cells. sFGL2 has been demonstrated to function as a pivotal mediator of inflammation, playing a crucial role in T cell proliferation and in the immunosuppressive activity of regulatory T cells. The structural differences between mFGL2 and sFGL2 result in distinct biological functions, and they may participate in different physiological and pathological processes. An important but unresolved aspect of our work is the potential differential role of these two isoforms in NB. Although we have demonstrated that FGL2 suppresses NB progression by inhibiting NF-κB signaling and epithelial–mesenchymal transition (EMT), we did not distinguish between the contributions of mFGL2 and sFGL2. Previous studies suggest that mFGL2 exerts procoagulant and pro-inflammatory effects, whereas sFGL2 is involved in immune modulation, particularly in suppressing T cell responses within the tumor microenvironment. These isoforms may therefore plausibly exert distinct, or even opposing, effects on NB cell behavior and the surrounding immune landscape. Future studies using isoform-specific antibodies or genetic constructs will be necessary to delineate their individual roles, which could provide new mechanistic insights and help refine therapeutic strategies targeting FGL2. Given its importance in immune regulation, FGL2 has attracted increasing attention in cancer research in recent years⁴. It has been shown to modulate the immune microenvironment and influence prognosis. Several studies have reported decreased FGL2 expression in various tumors, where it appears to play a tumor-suppressive role—findings consistent with our results. For example, Olli-Pekka et al. found that positive FGL2 expression was associated with favorable survival outcomes in gastrointestinal stromal tumors in a multicenter study with follow-up¹⁰, and Feng et al. reported that FGL2 expression was significantly reduced in breast cancer and positively correlated with infiltration of anti-tumor immune cells¹⁹. In our study, both protein and mRNA levels of FGL2 were lower in high-risk NB samples compared with non-high-risk samples. Moreover, low FGL2 expression was significantly associated with advanced International Neuroblastoma Staging System stage, high-risk classification, and poor survival status. Kaplan–Meier survival analysis confirmed that patients with low FGL2 expression had significantly worse survival. Taken together, our experimental findings and statistical analyses suggest that FGL2 may serve as a novel prognostic marker for NB and support its potential role as a tumor suppressor. However, findings on the role of FGL2 in cancer have not been consistent across tumor types. For instance, Khatri Latha et al. reported that FGL2 promotes glioma progression, with patients with low-grade gliomas exhibiting shorter survival when FGL2 expression was high²⁰. Two possible explanations may account for these discrepancies. First, as noted above, mFGL2 and sFGL2 differ structurally and may regulate distinct pathophysiological processes and biological functions, leading to variable effects in different cancers. Second, cancer is inherently heterogeneous and tissue-specific, affecting diverse functions and signaling pathways; therefore, FGL2 may act through different mechanisms depending on the tumor type. Further studies are needed to clarify the precise role of FGL2, particularly the individual contributions of mFGL2 and sFGL2, across various cancers.

Gene silencing is a critical experimental approach in cancer research. In our study, functional assays were performed to investigate the mechanisms by which FGL2 influences NB progression. CCK-8 and colony formation assays showed that silencing FGL2 increased the viability and proliferative capacity of NB cells. Wound healing and Transwell invasion assays further demonstrated that FGL2 knockdown significantly enhanced cell migration and invasion compared with control groups. This is the first study to reveal that FGL2 suppresses aggressive behaviors of NB cells in vitro, findings that are consistent with our earlier observations in clinical samples. To further elucidate the underlying mechanisms, relevant signaling pathways potentially involved in FGL2-mediated regulation were examined. The role of FGL2 in modulating the NF‐κB signaling pathway in NB has not been reported previously. Our findings identify a novel regulatory mechanism in which FGL2 inhibits the proliferation, invasion, and metastasis of NB cells through suppression of NF-κB signaling and consequent inhibition of EMT. We analyzed upstream regulators of NF-κB, such as IKKβ and p-IKKβ, and downstream EMT-related effectors, including Snail, E-cadherin, and vimentin. FGL2 knockdown increases IKKβ phosphorylation, which in turn enhanced NF-κB activation by promoting phosphorylation of p65 and IκBα, facilitating nuclear translocation of NF-κB transcription factors. Conversely, FGL2 expression limited these phosphorylation events, thereby restraining NF-κB activation. This suppression of NF-κB signaling by FGL2 downregulated the EMT-associated transcription factor Snail, upregulated E-cadherin, and reduced vimentin expression. Because EMT enhances tumor cell plasticity, migration, and metastatic potential, its inhibition by FGL2 ultimately attenuates NB cell aggressiveness. These findings align with the established role of NF-κB in promoting EMT and tumor progression across multiple cancer types^21–23, as well as its contribution to creating an immunosuppressive microenvironment that favors metastasis²². Our data extend these insights to NB, a pediatric solid tumor in which EMT-associated mechanisms remain incompletely understood. Importantly, pharmacologic inhibition of NF-κB reversed the EMT phenotype induced by FGL2 knockdown, confirming that FGL2 modulates NB progression through the NF-κB–EMT axis. Functional assays further supported this link, demonstrating that loss of FGL2 significantly enhanced NB cell proliferation, colony formation, migration, and invasion. Taken together, these results indicate that FGL2 functions as a tumor suppressor in NB by inhibiting NF-κB-driven EMT and suggest that targeting this pathway may offer therapeutic potential for aggressive forms of the disease.

EMT is increasingly recognized not only as a driver of tumor cell invasion and metastasis, but also as a key modulator of the tumor microenvironment²⁴. Numerous studies have shown that EMT promotes immune evasion by reprogramming the local immune milieu to favor tumor progression. FGL2 has also been reported to regulate the biological functions of colorectal carcinoma cells through the EMT pathway. EMT-inducing transcription factors such as Snail, ZEB1, and Twist1 can upregulate PD-L1 expression on tumor cells, thereby suppressing cytotoxic T cell activity and facilitating immune escape²⁵. ZEB1, for example, directly binds to the PD-L1 promoter and enhances its transcription in lung and breast cancer models²⁶. Similarly, Snail can reduce natural killer cell-mediated lysis of tumor cells by inhibiting the expression of NK cell receptor ligands, such as ULBP1 and MICA²⁷. EMT can also stimulate the secretion of immunosuppressive cytokines, including TGF-β, IL-10, and CCL2, which recruit tumor-associated macrophages, regulatory T cells, and myeloid-derived suppressor cells to the tumor site. These immune cells further suppress anti-tumor immunity, creating a feedback loop that sustains the mesenchymal state and promotes tumor progression²⁸. In our study, FGL2 knockdown led to NF-κB activation and subsequent EMT induction in NB cells, as evidenced by increased Snail and vimentin expression and decreased E-cadherin levels. Based on the mechanisms described above, these EMT-related changes may contribute not only to enhanced cell motility and invasiveness, but also to the development of a more immunosuppressive tumor microenvironment. This study represents a preliminary exploration focused on survival outcomes and molecular regulatory mechanisms related to FGL2 expression. Although we did not directly assess immune cell infiltration, the molecular alterations we observed suggest a potential link between FGL2/NF-κB-driven EMT and immune evasion. Future studies examining immune cell composition and cytokine profiles in FGL2-deficient tumors will be valuable in clarifying this relationship. Overall, our findings support the concept that EMT serves as a bridge between tumor cell plasticity and immune modulation, and they identify FGL2 as a potential upstream regulator of this axis in NB.

Nevertheless, we acknowledge several limitations in our study. First, because the incidence of pediatric tumors is lower than that of adult tumors, our sample size was relatively small. However, in our previous work, we performed prognostic survival analysis of FGL2-related risk scores using external datasets, and those results support our current findings. Large-scale, multicenter, prospective studies will be necessary in the future to increase the robustness of statistical analyses. Second, this study was preliminary, and we assessed the expression levels of only a limited number of NF-κB-related proteins. The specific regulatory mechanisms and the expression of additional NF-κB-related proteins will require more in-depth investigation in future work. Third, our current research primarily focused on survival outcomes and molecular regulatory mechanisms related to FGL2 expression. Future studies will investigate immune cell infiltration within the tumor microenvironment to explore the immunological aspects of FGL2 function. Fourth, rescue experiments were not performed following FGL2 knockdown. Nonetheless, we minimized potential off-target effects by using multiple siRNA sequences and confirming FGL2 knockdown efficiency at the protein level. We plan to include rescue assays in follow-up studies to strengthen causal inference. Fifth, we selected a rapid detection method (CCK-8 assay) to measure tumor cell proliferation. In future studies, we plan to incorporate more direct assays, such as BrdU or EdU incorporation, to validate these findings. Sixth, we used two NB cell lines—IMR-32 (MYCN-amplified) and SH-SY5Y (MYCN–non-amplified)—to represent biologically relevant subtypes. We intend to expand the panel of cell lines in subsequent studies to further verify the robustness of our conclusions. Seventh, our study lacked in vivo experiments in mouse models. To address this, we included additional clinical correlation analyses to support the biological relevance of our findings. Nevertheless, animal studies are essential to confirm these results in a complex tumor microenvironment. In future work, we plan to use orthotopic NB mouse models to investigate the role of FGL2 in tumor progression and immune regulation in vivo.

Conclusions

In summary, our study demonstrates that FGL2 inhibits NB cell proliferation, migration, and invasion by regulating the NF-κB signaling pathway. These findings provide new insights into the potential molecular mechanisms of FGL2 in NB and suggest that FGL2 may serve as a promising therapeutic target for this disease.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

GCD and LW proposed the idea of the article and supervised its completion. CQ and GCD drafted and reviewed the article. CQ, XH,HZ and YYM completed the experiments related to the article. HZZ, ZYW and YZ helped to interpret the data and prepare the figures. All authors read and approved the final manuscript.

Funding

This work was supported by the Clinical Medicine Talent Training Project funded by Hebei Province (ZF2024183) and the Introduced Foreign Intelligence Projects of Hebei Province in 2023.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no potential conflicts of interest with respect to the research, authorship, or publication of this article. This study was based on open-source data and involves no ethical issues or other conflicts of interest. All methods were conducted in accordance with relevant guidelines and regulations.

Ethical approval

This study was approved by the Ethics Committee of the Children’s Hospital of Hebei Province (20210794) and conducted in accordance with the ethical standards of the Declaration of Helsinki. Written informed consent for participation was obtained from the parents or legal guardians of all participants.