Two human NB cell lines [SK-N-AS (AS) and SK-N-BE2 (BE2)] and a mouse fibroblast cell line (NIH3T3) were used in this study. SK-N-BE2 (BE2) represents the MYCN-amplified cell line, whereas SK-N-AS (AS) represents the MYCN-non-amplified cell line. All cell lines were gifts from Dr. Carol J Thiele (Cellular and Molecular Biology Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States). NB cells were cultured in RPMI-1640 medium (Solarbio, China) containing 10% FBS (Gibco, Israel), antibiotics (a mixture of penicillin and streptomycin; working concentration 100 U/mL) (BI, Israel), and 2 mM glutamine (BI, Israel) at 37°C in a 5% CO₂ incubator. NB cells in the logarithmic growth phase were used for the experiments.

Small interfering RNAs (siRNAs) were purchased from Tongyong (Anhui, China). The target sequences of siRNAs are as follows: control siRNA: 5′-UUC​UCC​GAA​CGU​GUC​ACG​UTT-3′; FTO siRNA#1: 5′-CUA​CAA​CGG​ACA​AGA​UGA​ATT-3′; FTO siRNA#2: 5′-CGG​UGG​CAG​UGU​ACA​GUU​ATT-3′; FTO siRNA#3: 5′-GGA​AGA​AGA​UGG​AGG​GUG​UTT-3′. The FTO overexpression plasmids (NM_001080432, pEZ-M39 vector) and empty vector were purchased from GeneCopoeia (United States). AS, BE2, and NIH3T3 cells were seeded into 6-well plates at 1 × 10⁵/well, 5 × 10⁵/well, and 1 × 10⁵/well, respectively, and cell adhesion was maintained overnight. siRNAs or plasmids were transfected into cells using jetPRIME according to the manufacturer’s instructions.

To explore the function of FTO in response to paclitaxel, etoposide, and cisplatin, 16 h after the transfection, cells were seeded into 96-well plates, and then treated with paclitaxel (AS, 3 nM; BE2, 3 nM), etoposide (AS, 1.5–2 μg/mL; BE2, 2 μg/mL), or cisplatin (AS, 0.5 μg/mL; BE2, 2 μg/mL), for 72 h.

The relationship between the expression of FTO and the prognosis of clinical patients with NB was analyzed via the R2 database (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?species=hs) using a dataset of 498 samples from patients with NB across seven countries. The patients’ ages at diagnosis varied from 0 to 295.5 months (median age, 14.6 months). Tumor stages were classified according to the International Neuroblastoma Staging System (INSS). Events were defined according to the revised International Neuroblastoma Response Criteria. The significance of the relationship was determined using Pearson correlation or one-way ANOVA analysis, with p values less than 0.05 considered statistically significant (Zhang et al., 2015).

After 16 h of transfection, AS, BE2, and NIH3T3 cells were seeded into 96-well plates at densities of 0.5 × 10⁴/well, 1.5 × 10⁴/well, and 0.5 × 10⁴/well, respectively. The cell confluence rate in each well was recorded using the IncuCyte ZOOM live-cell dynamic imaging system. After 72 h of drug treatment, cell proliferation and survival were detected using a Cell Counting Kit-8 (CCK-8 assay) (GlpBio, United States). The CCK-8 assay was performed according to the manufacturer’s instructions. Absorbance was measured at 450 nm.

After transfection, AS and BE2 cells were seeded into 6-cm dishes at densities of 3 × 10³/well and 5 × 10³/well, respectively. After 10 days of observation, the cells grew into visible cell clones. The cells were then fixed with 4% paraformaldehyde for 30 min, stained with Giemsa working solution for 30 min, and carefully washed with ultrapure water. After being air dried, the whole 6-cm dish was photographed. Finally, the number of colonies in each group was counted using ImageJ software.

AS and BE2 cells were harvested following transfection with FTO siRNAs (#1, #2, #3) or FTO overexpression plasmid for 48 h. Total protein was extracted using the whole-cell lysis assay kits (Keygen Biotech, China), according to the manufacturer’s protocol. For each sample, 30 µg of protein was loaded onto SDS-PAGE gels, transferred to PVDF membranes, and blocked with 5% non-fat milk to prevent non-specific antibody binding for 2 h at room temperature. The membranes were then incubated overnight at 4°C with specific primary antibodies: FTO antibody (Proteintech, China, diluted at 1:1,000) and β-actin antibody (Proteintech, China, diluted at 1:5,000). Subsequently, the membranes were washed thrice for 5 min each with Tris-buffered saline-Tween 20 (TBST) and incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (Proteintech, China, diluted at 1:5,000) for 2 h at room temperature.

Total RNA was extracted from NB cells, with 1 µg of RNA utilized for cDNA synthesis employing the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). RT-qPCR was performed employing SYBR Premix Ex Taq™ II (Takara, Japan) on a 7500 Fast Real-Time PCR System. Relative quantification of gene expression was performed with the 2 (−ΔΔCt) method. Ct values were standardized to β-actin. The primer sequences are shown below:

Data for this analysis were obtained from 498 NB patient samples from the GSE49710 dataset available in the GEO database. The relationship between the expression of FTO and chemotherapeutic drugs or inhibitor sensitivity was analyzed using R software with the “pRRophetic” package, which was downloaded from Bioconductor. The significance between groups was analyzed using the Wilcoxon rank-sum test, with p values less than 0.05 considered statistically significant.

Data are expressed as mean ± SD. Comparisons between two groups were carried out using the Student's t test. Statistical analyses were performed with GraphPad Prism software. Statistical significance was set as p values less than 0.05.

To explore the correlation between FTO expression and the prognosis and clinical pathologic characteristics of patients with NB, datasets comprising 498 patients with NB were selected from the R2 database (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi). Patients with high FTO expression had better overall survival probability and event-free survival probability than patients with low FTO expression (p < 0.001) (Figure 1A). Patients with a favorable prognosis, characterized by positive histopathology; non-MYCN amplification; INSS stages 1, 2, 3, and 4S; and no high-risk factors, exhibited significantly higher FTO expression (p < 0.001) (Figure 1B). Additionally, patients with no disease progression (p = 0.05), no deaths due to the disease (p = 0.29), or female sex (p = 0.47) demonstrated a trend toward increased FTO expression (Figure 1B). These findings suggest that high FTO expression is associated with better prognosis and favorable clinical characteristics.

To explore the role of FTO in NB cells’ survival and proliferation, we designed an FTO overexpression plasmid (OE-FTO). This plasmid upregulated FTO expression (Figure 2A; Supplementary Datasheet 1). Two NB cell lines (AS and BE2) and NIH3T3 cells were transfected with an empty vector and the OE-FTO plasmid, and then cell confluence, survival, and proliferation were detected. The AS and BE2 cells’ confluence curves of the OE-FTO-transfected cells (red curve) were lower than those of the empty vector-transfected cells (blue curve) (Figure 2B). At 72 h after transfection, the cell confluence of the OE-FTO-transfected cells was 17.5% lower than that of empty vector-transfected cells in AS cells (p < 0.01) (Figure 2B), and 19.1% in BE2 cells (p < 0.05) (Figure 2B). However, in NIH3T3 cells, the confluence curves of OE-FTO and EV overlapped, showing no statistical significance in the 72 h endpoint analysis (Supplementary Datasheet 1). The results of the CCK-8 assay were consistent with the cell confluence data. Compared to the empty vector-transfected cells, the survival of OE-FTO-transfected cells decreased by 11.6% in AS cells (p < 0.05) (Figure 2C) and by 7.4% in BE2 cells (p < 0.01) (Figure 2C), and no statistically significant difference was observed in NIH3T3 cells ((p > 0.05) (Supplementary Datasheet 1). In the colony formation assay, the number of colonies formed by OE-FTO-transfected cells was 36.8% lower than that of empty vector-transfected cells in AS cells (p < 0.05) (Figure 2D), and 24.1% lower in BE2 cells (p < 0.05) (Figure 2D). Representative images of NB cells at 0 h and 72 h after transfection are shown in Figure 2E, indicating fewer cells in the OE-FTO transfection group than in the control group. These data indicate that the upregulation of FTO inhibits NB cells’ survival and proliferation.

To further explore the role of FTO in NB cells’ survival and proliferation, we designed three FTO siRNAs (#1, #2, and #3) that downregulated FTO expression (Figure 3A; Supplementary Datasheet 1). FTO siRNA#2 (FTO siRNA) was selected for subsequent studies because of its significant downregulating effects in both AS and BE2 cell lines. Two NB cell lines (AS and BE2) and NIH3T3 cells were transfected with control siRNA and FTO siRNA, and then cell confluence, survival, and proliferation were evaluated. AS and BE2 cells’ confluence curves of FTO siRNA-transfected cells (red curve) were higher than those of control siRNA-transfected cells (blue curve) (Figure 3B). At 72 h post-transfection, the confluence of the FTO siRNA-transfected cells was significantly increased by 29.0% compared to the control siRNA in AS cells (p < 0.01) (Figure 3B), and by 12.4% in BE2 cells (p < 0.05) (Figure 3B). However, in NIH3T3 cells, the confluence curves of FTO siRNA and control siRNA overlapped, showing no statistical significance at the 72 h endpoint analysis (Supplementary Datasheet 1). The results of the CCK-8 assay were consistent with the cell confluence data; the survival rates of the cells transfected with FTO siRNA showed a significant increase of 20.0% in AS cells (p < 0.01) (Figure 3C), an increase of 11.2% in BE2 cells (p < 0.01) (Figure 3C), and no statistically significant difference in NIH3T3 cells (p > 0.05) (Supplementary Datasheet 1) compared to the control siRNA. In the colony formation assay, the number of colonies formed by FTO siRNA-transfected cells was significantly increased by 66.8% in AS cells (p < 0.01) (Figure 3D) and by 34.7% in BE2 cells (p < 0.05) (Figure 3D), compared to the control siRNA. Representative images of NB cells at 0 h and 72 h after transfection are shown in Figure 3E, illustrating that there were higher number of cells in the FTO siRNA-transfected group than in the control siRNA-transfected group. These results suggest that downregulation of FTO significantly promotes NB cells’ survival and proliferation.

Chemoresistance is the leading cause of treatment failure in patients with NB. To explore the effects of FTO on the sensitivity of NB cells to chemotherapeutic drugs, we selected etoposide, cisplatin, and paclitaxel—three compounds frequently used in the treatment of patients with NB.

To investigate the effect of FTO on the sensitivity of NB cells to etoposide, we transfected FTO siRNA or OE-FTO into AS and BE2 cells. The cells were then treated with etoposide for 72 h, and cell confluence and survival were assessed. After etoposide treatment, the confluence curves of FTO siRNA-transfected cells (purple curve) were lower than those of control siRNA-transfected cells (green curve) (Figure 4A, left). At 72 h post-etoposide treatment, the confluence of FTO siRNA-transfected cells was 16.3% lower than that of control siRNA-transfected cells in AS cells (p < 0.01) (Figure 4A, upper right), and 9.6% lower in BE2 cells (p < 0.01) (Figure 4A, lower right). The confluence of cells transfected with OE-FTO was not significantly different from that of cells transfected with the empty vector after the etoposide treatment (Figure 4B). The results of the CCK-8 assay were consistent with the cell confluence data; under the etoposide treatment, the cell survival of FTO siRNA-transfected cells was 11.9% lower than that of control siRNA-transfected cells in AS cells (p < 0.01), and 7.1% lower in BE2 cells (p < 0.01) (Figure 4C), but OE-FTO had no significant effect on the sensitivity of NB cells to etoposide (Figure 4E). Representative images of NB cells at 0 h and 72 h post-etoposide treatment are shown in Figures 4D, F, which corroborate the cell confluence and the CCK-8 assay. These findings suggest that downregulation of FTO enhances the sensitivity of NB cells to etoposide, whereas overexpression of FTO does not significantly affect cell survival following etoposide treatment.

To explore the effect of FTO on the sensitivity of NB cells to cisplatin, FTO siRNA or OE-FTO were transfected into AS cells and BE2. Subsequently, the cells were treated with cisplatin for 72 h, and cell confluence and survival rates were assessed. After cisplatin treatment, the confluence curves of FTO siRNA-transfected cells (purple curve) were slightly higher than those of control siRNA-transfected cells (green curve) (Figure 5A, left). Meanwhile, after cisplatin treatment, the confluence curves of cells transfected with OE-FTO (purple curve) were slightly lower than those of cells transfected with the empty vector (green curve) (Figure 5B, left). However, there were no significant differences in cell confluence 72 h after cisplatin treatment between cells transfected with FTO siRNA or OE-FTO compared to their respective controls (Figures 5A, B, right). These findings were consistent with the results of the CCK-8 assay, which showed no significant differences in cell survival rates between cells transfected with FTO siRNA or OE-FTO and their controls under cisplatin treatment (Figures 5C, E). Representative images of NB cells at 0 h and 72 h after cisplatin treatment are shown in Figures 5D, F, respectively, and similar trends were observed in the cell confluence and CCK-8 assay results. These findings suggest that FTO expression levels have no impact on the sensitivity of NB cells to cisplatin.

Furthermore, we investigated whether FTO affected the sensitivity of NB cells to paclitaxel. FTO siRNA or OE-FTO was transfected into AS and BE2 cells. Subsequently, the cells were treated with paclitaxel treatment for 72 h, after which the cell confluence and survival rates were detected. After paclitaxel treatment, the confluence curves of the cells transfected with FTO siRNA (purple curve) were significantly higher than those of cells transfected with control siRNA (green curve) (Figure 6A, left). Conversely, the confluence curves of the cells transfected with OE-FTO (purple curve) were significantly lower than those of cells transfected with the empty vector (green curve) after paclitaxel treatment (Figure 6B, left). At 72 h post-paclitaxel treatment, the confluence of the FTO siRNA-transfected cells was 16.1% higher than that of control siRNA-transfected cells in AS cells (p < 0.01) (Figure 6A, upper right), and 8.3% in BE2 cells (p < 0.05) (Figure 6A, lower right). Conversely, after paclitaxel treatment, OE-FTO-transfected cells exhibited significantly lower confluence than empty vector-transfected cells (for paclitaxel treatment, in AS cells, OE-FTO vs. empty vector: 66.3% and 85.1%, p < 0.01; in BE2 cells, OE-FTO vs. empty vector: 66.8% vs 84.3%, p < 0.01) (Figure 6B). The results of the CCK-8 assay were consistent with the cell confluence data; after paclitaxel treatment, the cell survival of the FTO siRNA-transfected cells was 7.9% higher than that of the control siRNA-transfected cells in AS cells (p < 0.05) (Figure 6C, left), and 7.1% in BE2 cells (p < 0.01) (Figure 6C, right). The survival rate of OE-FTO-transfected cells was significantly lower than that of empty vector-transfected cells (after paclitaxel treatment, in AS cells, OE-FTO vs. empty vector: 55.6% vs. 70.6%, p < 0.01; in BE2 cells, OE-FTO vs empty vector: 66.3% vs. 74.5%, p < 0.01) (Figure 6E). Representative images of NB cells at 0 h and 72 h after paclitaxel treatment are shown in Figures 6D, F, respectively, which corroborate the trends observed in cell confluence and CCK-8 assay results. These data collectively indicate that downregulation of FTO expression significantly reduces the sensitivity of NB cells to paclitaxel, whereas upregulation of FTO enhances the sensitivity of NB cells to paclitaxel.

As FTO diversely influences the sensitivity of NB cells to etoposide, cisplatin, and paclitaxel, we explored the correlation between FTO expression and the sensitivity of patients with NB to different chemotherapeutic drugs and small-molecule inhibitors. We first downloaded a dataset from the GDSC that can be used for drug sensitivity analysis. We then imported this dataset into the “pRRophetic” package to train a predictive model for drug sensitivity. Subsequently, we applied this predictive model to the data of 498 patients with NB. The data were downloaded from the GEO database to evaluate the relationship between FTO gene expression levels and drug sensitivity. The results revealed distinct sensitivities based on FTO expression levels across different treatments (Figures 7A–C). Consistent with our results, low FTO expression was more sensitive to etoposide (Figure 7A). We did not obtain information about sensitivity to cisplatin and paclitaxel via the GDSC database, but low expression of FTO was less sensitive to 5-fluorouracil (an antimetabolite that inhibits thymidylate synthase) (Figure 7A), another commonly used chemotherapeutic drug, and sorafenib (Figure 7A), a multi-kinase inhibitor that is widely used in many types of cancer treatment. Anaplastic lymphoma kinase (ALK) and Aurora are two commonly mutated genes in NB, and low expression of FTO was more sensitive to TAE684 (ALK inhibitor), VX-680 (pan-Aurora inhibitor), and BMS-754807 (IGF-1R/IR inhibitor, which also inhibits Aurora) (Figure 7B). Furthermore, low expression of FTO exhibited different sensitivities to PI3K inhibitor, AKT inhibitor, MAPK inhibitor, PKCβ inhibitor, JNK inhibitor, Ras inhibitor, NF-κB inhibitor, and P21 inhibitor (Figure 7C).