Leucine restriction ameliorates Fusobacterium nucleatum-driven malignant progression and radioresistance in nasopharyngeal carcinoma

Songhe Guo; Shan Xing; ZhenYu Wu; Fangfang Chen; Xiaoyun Pan; Qifan Li; Wanli Liu; Ge Zhang

doi:10.1016/j.xcrm.2024.101753

. 2024 Oct 1;5(10):101753. doi: 10.1016/j.xcrm.2024.101753

Leucine restriction ameliorates Fusobacterium nucleatum-driven malignant progression and radioresistance in nasopharyngeal carcinoma

Songhe Guo ^1,³, Shan Xing ^1,³, ZhenYu Wu ², Fangfang Chen ², Xiaoyun Pan ², Qifan Li ², Wanli Liu ^1,^∗, Ge Zhang ^2,^4,^∗∗

PMCID: PMC11513822 PMID: 39357525

Summary

Radiotherapy resistance is the main cause of treatment failure among patients with nasopharyngeal carcinoma (NPC). Recently, increasing evidence has linked the presence of intratumoral Fusobacterium nucleatum (Fn) with the malignant progression and therapeutic resistance of multiple tumor types, but its influence on NPC has remained largely unknown. We found that Fn is prevalent in the tumor tissue of patients with NPC and is associated with radioresistance. Fn invaded and proliferated inside NPC cells and aggravated tumor progression. Mechanistically, Fn slowed mitochondrial dysfunction by promoting mitochondrial fusion and decreasing ROS generation, preventing radiation-induced oxidative damage. Fn inhibited PANoptosis by the SLC7A5/leucine-mTORC1 axis during irradiation stress, thus promoting radioresistance. Treatment with the mitochondria-targeted antibiotics or dietary restriction of leucine reduced intratumoral Fn load, resensitizing tumors to radiotherapy in vivo. These findings demonstrate that Fn has the potential to be a predictive marker for radioresistance and to help guide individualized treatment for patients with NPC.

Keywords: intratumoral microbiota, Fusobacterium nucleatum, radioresistance, nasopharyngeal carcinoma

Graphical abstract

Highlights

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High abundance of F. nucleatum associated with poor radiotherapy efficacy in NPC
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F. nucleatum proliferates in NPC cells and promotes tumor growth and metastasis
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F. nucleatum promotes NPC radioresistance via the SLC7A5/leucine-mTORC1 axis
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Leucine-restricted diet ameliorates radioresistance in NPC

Guo et al. report that F. nucleatum drives radioresistance in nasopharyngeal carcinoma by reducing PANoptosis via the SLC7A5/leucine-mTORC1 axis. These findings reveal a mechanism of host-microbe interactions in nasopharyngeal carcinoma and potential strategies for improving clinical response to radiotherapy.

Introduction

Nasopharyngeal carcinoma (NPC) is a malignant tumor that originates from the epithelial cells of the nasopharynx and displays aggressive metastatic and invasive properties.¹ Currently, radiotherapy serves as the primary treatment modality for NPC.²^,³^,⁴ However, approximately 70% of newly diagnosed patients with NPC present with advanced-stage disease, rendering them insensitive to radiotherapy due to radioresistance, which ultimately contributes to the primary cause of mortality in patients with NPC.⁵^,⁶ Therefore, identifying the molecular mechanism responsible for NPC radioresistance and developing a strategy to improve radiotherapy efficacy are crucial.

Notably, microbial constituents within tumor tissues have been identified in various cancer types.⁶^,⁷^,⁸ The intratumoral microbiota impacts cancer pathology by influencing the processes involved in tumorigenesis, cancer progression, and resistance to therapy.⁹^,¹⁰ Recently, two studies confirmed the presence of an intratumoral microbiome¹¹^,¹² and revealed that a high intratumoral bacterial load is associated with a poor prognosis in patients with NPC. However, the specific functions and mechanisms of intratumoral microbes and key bacteria involved in therapeutic responses in NPC need to be elucidated.

Fusobacterium nucleatum (F. nucleatum, Fn), a prevalent oral pathogen, is frequently detected in various tumor tissues.¹³ Recently, accumulating evidence has indicated that intratumoral Fn functions as an oncogenic bacterium that exacerbates tumor progression, including in gastrointestinal and nongastrointestinal tumors.¹³^,¹⁴ As a facultative intracellular bacteria,¹⁵ Fn employs multiple virulence factors, such as Fap2, FadA, Fn-Dps, and Fn-MliC, to attach to and invade host cells,¹⁶ facilitating intracellular Fn survival and proliferation and promoting carcinogenesis.¹⁷^,¹⁸ In addition, Fn-infected host cells release exosomes and secrete proinflammatory chemokines to reshape the tumor microenvironment and promote metastasis.¹⁹ Notably, Fn diminishes the presence of CD4⁺ and CD8⁺ T cells within the tumor microenvironment, attenuating immunotherapy efficacy in patients with colorectal cancer (CRC) and esophageal squamous cell carcinoma (ESCC).²⁰^,²¹ Moreover, Fn has been reported to confer chemoresistance by modulating autophagy in CRC and ESCC.²²^,²³ Interestingly, a recent study showed that Fn was significantly enriched in the nasopharynx and reshaped the nasopharyngeal microbiota of patients with NPC.²⁴ However, whether Fn is involved in NPC development, therapeutic responses, and the clinical signature of Fn-positive patients with NPC has been unclear.

In the present study, we evaluated intratumoral Fn and its clinical significance in patients with NPC and described the roles and mechanisms of Fn in influencing NPC progression and radiotherapy efficacy. Moreover, we evaluated the role of dietary restriction combined with antibiotics in sensitizing Fn-infected tumors to radiotherapy in patients with NPC.

Results

F. nucleatum is prevalent in human NPC tissues and is associated with poor radiotherapy efficacy

To determine whether Fn is present in NPC tissues, we stained tissue microarrays containing human NPC tissue samples (cohort 1, n = 81) via fluorescence in situ hybridization (FISH) with an Fn-specific probe. We detected Fn in 80.25% (65/81) of the analyzed NPC tissues and defined the number of fluorescence cells/mm² > 100 as the high abundance of Fn (Figures 1A and 1B). Furthermore, compared with low or undetectable amounts of Fn, a high amount of Fn correlated with remarkably poor prognosis in patients with NPC (p = 0.039) (Figure 1C). Notably, the overabundance of Fn was associated with distant metastasis (p = 0.003) (Figure 1D). Moreover, immunofluorescence (IF) analysis revealed that Fn was more abundant in NPC specimens than in adjacent noncancerous tissues (cohort 2, n = 17; Figure 1E).

Next, we analyzed the differences in tumor tissues between radiosensitive (S) and radioresistant (R) patients with NPC (cohort 3-1, n = 10) using 5R-16S rRNA sequencing (Figure 1F). Principal-component analysis (PCA) and principal coordinates analysis (PCoA) demonstrated the distinctive composition of the bacterial community between S and R patients (Figures 1G and 1H). At the phylum level, the R group had relatively greater abundances of Firmicutes, Bacteroidetes, and Fusobacteria than did the S group (Figure 1I). Analysis of the relative abundances of the genera showed enrichment of Fusobacterium, Capnocytophaga, and Streptococcus (Figure 1J). We used the linear discriminant analysis effect size (LEfSe) algorithm to define the potential differential bacterial patterns between postradiotherapy NPC relapse patients and nonrelapse patients. We found that Fn, Bacteroidetes, Porphyromonas endodontalis, Capnocytophaga sputigena, and Solobacterium were more abundant in the R group than in the S group (Figures 1K and 1L). Importantly, Fn was more enriched in the R group than in the S group according to the qPCR (cohort 3-2, n = 20) and IF (cohort 3-1, n = 10) assay results (Figures 1M and 1N). These results indicated that Fn is prevalent in human NPC tissues and that a high amount of intratumoral Fn may promote NPC radioresistance.

F. nucleatum invades NPC cells and promotes tumor growth and metastasis in NPC mice

Utilizing Fn and tumor cell coculture system, we further evaluated the ability of Fn to invade NPC cells (Figure 2A). Fn and the CNE2 and 5-8F cells were cocultured at MOI = 10 for 12 h, respectively. Tumor cells were infected with Fn and then incubated with 100 μg/mL gentamycin to eliminate extracellular Fn. Confocal laser scanning microscopy was used to observe the intracellular localization of Fn. Fn was labeled with red fluorescence in the cytoplasm of cancer cells (Figure 2B). Moreover, the amount of intracellular Fn increased steadily for the first 24 to 72 h, as determined by counting the bacterial colonies on brian heart infusion (BHI) (BHI) plates (Figure 2C).

*F. nucleatum* accumulates in mouse NPC tissues and promotes tumor growth, migration, and radioresistance

(A) Schematic diagram: intracellular Fn detection was assessed by gentamycin protection assay.

(B) Immunofluorescence visualization of α-tubulin and Fn in NPC cells. Cells were infected with Fn (MOI = 10:1) for 12 h. Representative images of n = 3 biologically independent replicates showing similar results. Scale bar: 10 μm

(C) Intracellular bacterial proliferation was assessed by gentamycin protection assay. The numbers of viable bacteria per cell were determined by the serial dilution method.

(D) Colony formation assays were performed. Representative images of n = 3 biologically independent replicates showing similar results. Statistical results are presented in the right panels.

(E) Transwell assays were performed. Representative images of n = 3 biologically independent replicates showing similar results. Statistical results are presented in the right panels. Scale bar: 100 μm

(F) Subcutaneous xenograft tumor model was constructed: a flowchart showing the *in vivo* experimental design (n = 5/group).

(G) Representative images showing xenografts in nude mice, 21 days later.

(H) Individual tumor growth curve.

(I) Tumor growth curve.

(J) Tumor weight.

(K) Representative IHC staining of ki67 in mouse tumor tissues. Scale bar: 50 μm

(L) Lung metastasis model was constructed: a flow chart showing the *in vivo* experimental design.

(M) Representative images of the lung metastatic nodule.

(N) Histopathological examination of the lung tissue sections. Scale bar: 100 μm

(O) Statistics analyses of lung metastatic nodule number.

(P) Statistics analyses of lung weight.

(Q) *In vivo* fluorescence images of tumor-bearing mice after intravenous injections of Fn-DiD.

(R) Intracellular Fn assays were performed. Statistical results are presented in the right panels.

(S) A subcutaneous xenograft tumor model was constructed: a flowchart showing the *in vivo* experimental design (n = 5/group).

(T) Representative images showing xenografts in nude mice, 21 days later.

(U) Tumor growth curve.

(V) Individual tumor growth curve.

(W) Tumor weight.

(X) Representative IHC staining of ki67in mouse tumor tissues. Scale bar: 50 μm

(Y) Fn abundance in tumor tissue samples was measured using qPCR. Data are shown as mean ± SD. p values were determined by independent sample t tests (C–E, J, O, P, R, W, and Y), and two-way ANOVA (I and U), ∗p < 0.05, ∗∗p < 0.001, and ∗∗∗p < 0.001.

To evaluate the effect of Fn infection on NPC cells, we performed proliferation and migration assays using the CNE2 and 5-8F cells cocultured with Fn or heat-killed Fn (K-Fn). Compared to no treatment or treatment with K-Fn, Fn significantly promoted the growth of CNE2 and 5-8F cells (p < 0.01 and p < 0.001, respectively) (Figure 2D). Furthermore, Fn treatment markedly enhanced the migration of both CNE2 and 5-8F cells (both p < 0.001) (Figure 2E).

To further confirm the role of Fn in vivo, a xenograft model and an experimental lung metastasis model were established in nude mice (Figure 2F). In accordance with the results of the in vitro experiments, Fn infection accelerated tumor growth and increased tumor weight compared to treatment with PBS (Con) and K-Fn (p < 0.05) (Figures 2G–2J). Moreover, we observed enhanced ki67 staining in Fn-infected xenograft tissues (Figure 2K). In the lung metastasis model, after 14 days, the lungs were surgically removed (Figures 2L and 2M). Hematoxylin and eosin (H&E) staining of lung sections revealed that the Fn-infected groups presented a greater number of nodules than did the control or K-Fn-treated groups (p < 0.05) (Figures 2N and 2O). Moreover, the weight of the lungs in the Fn-infected group was markedly greater than that in the PBS or K-Fn-treated groups (p < 0.05) (Figure 2P). Taken together, our results highlight that intratumoral Fn exerts oncogenic effects on NPC.

F. nucleatum accumulates in mouse NPC tissues and promotes radioresistance

Next, we determined whether Fn in the oral cavity/gut migrates into tumor tissues through systemic blood circulation. We assessed the biodistribution of DiIC₁₈(5) solid (DiD)-labeled Fn after systemic administration via tail vein injection in a nude mouse 5-8F xenograft model. After 3 days, we conducted small animal live optical imaging to determine whether Fn accumulates in tumor tissues (Figure 2Q). To investigate the effect of X-ray irradiation on Fn survival in NPC cells, Fn-infected CNE2 and Fn-infected 5-8F cells were exposed to 2–8 Gy irradiation. Then, we assessed the ability of Fn to multiply inside tumor cells using a gentamycin protection assay. The results showed that the number of intracellular Fn did not obviously differ at 48 h after 2–8 Gy irradiation compared with that after 0 Gy irradiation (Figure 2R).

To determine whether Fn infection increases the radioresistance of NPC tumors in vivo, we subcutaneously inoculated 5-8F cells into nude mice to construct a xenograft model. Mice were preinfected with Fn three times before X-ray treatment; when the tumors reached 200 mm³ after tumor cells seeding, they were locally irradiated (IR) with X-rays at a single dose of 20 Gy, and tumor volume was measured every other day (Figure 2S). Consistent with the in vitro experiment results, Fn infection facilitated tumor growth and reduced the antitumor effect of radiotherapy (Figures 2T, 2U, 2V, and 2W). Moreover, Fn infection significantly increased ki67 expression in tumor tissues (Figure 2X). In addition, there was no significant difference in the Fn DNA level in tumors between the IR and unirradiated mice (Figure 2Y). These results suggest that Fn infection significantly increased the resistance of NPC cells to irradiation.

Intracellular F. nucleatum promotes radioresistance in NPC cells by suppressing host apoptosis and DNA damage

To further explore the function of intracellular Fn in NPC tumor radioresistance, cultured CNE2 and 5-8F cells were exposed to an X-ray radiation source to achieve the desired doses of 2, 4, and 8 Gy. After 24 h, a cell viability assay was conducted to assess the survival rate of the cells. Morphologically, the irradiation-treated CNE2 and 5-8F cells exhibited large bubbles emerging from the plasma membrane and cell swelling. Notably, Fn infection visibly suppressed this phenomenon, whereas E. coli did not (Figure 3A). Moreover, live/dead cell and colony formation assays revealed better survival in CNE2 and 5-8F cells infected with Fn than in control cells after irradiation treatment (Figures 3B and 3C). Flow cytometry analysis revealed that compared with irradiation alone, irradiation accompanied by Fn infection significantly decreased the number of apoptotic cells (Figure 3D). Cell viability was measured by lactate dehydrogenase (LDH) release assays. Similarly, Fn infection significantly attenuated the irradiation-induced increase in LDH release (Figure 3E). Furthermore, a comet assay showed that Fn infection significantly protected NPC cells from irradiation-induced DNA damage (Figure 3F). Consistently, the protein level of the DNA damage marker γH2AX was markedly increased postirradiation in both cell groups. The expression of the γH2AX protein was downregulated in Fn-infected NPC cells (Figure 3G). Taken together, these results indicated that Fn promotes irradiation resistance by suppressing apoptosis and inhibiting the DNA damage response in NPC cells.

Intracellular F. nucleatum promotes host mitochondrial fusion and inhibits ROS generation during irradiation stress

Since irradiation-induced DNA damage is largely mediated by the generation of reactive oxygen species (ROS), we investigated the effect of Fn on the irradiation-induced accumulation of ROS in NPC cells. Notably, Fn infection attenuated irradiation-induced ROS accumulation (Figure 4A). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is crucial for ROS generation, and gp91phox and gp47phox are the predominant subunits of NADPH oxidase.²⁵ Therefore, we examined whether Fn regulates the expression of gp91phox and gp47phox. Fn infection decreased the irradiation-induced increase in cytosolic gp91phox and gp47phox protein expression (Figure 4B). The IF results confirmed the western blot data (Figure 4C).

Intracellular *F. nucleatum* promotes NPC cells mitochondrial fusion and inhibits ROS generation during irradiation stress

(A) Flow cytometric analysis of ROS levels in NPC cells.

(B–F) Fn treatments on mitochondrial phenotypes in NPC cells with 4 Gy irradiation. (B) Western blot analysis of gp91phox and p47phox cytosolic protein expression in NPC cells. The grayscale values of the protein bands were analyzed using ImageJ (right). Data are mean values of three biology repeats. (C) Representative immunofluorescence analysis of gp91phox and gp47phox (red) and DAPI nuclei staining (blue fluorescence). Scale bar: 5 μm. (D) Representative confocal images showing mitochondrial phenotypes. Scale bar: 5 μm. (E) Representative electron microscopy (EM) images showing mitochondrial phenotypes. Scale bar: 1 μm. (F) Representative images and statistical analyses showing the effect of intracellular Fn on mitochondrial fusion in NPC cells with 4 Gy. Data are mean values of three biology repeats. Scale bar: 5 μm. Data are shown as mean ± SD. p values were determined by independent sample t tests (B and F), ∗p < 0.05, ∗∗p < 0.001, and ∗∗∗p < 0.001.

Mitochondrial dynamics are known to affect oxidative phosphorylation (OXPHOS).²⁶ To investigate whether intracellular Fn regulates OXPHOS by affecting mitochondrial dynamics, we examined mitochondrial morphology in 5-8F and CNE2 cells. Fn infection dramatically reduced the irradiation-induced fragmentation of mitochondria, as observed through confocal and electron microscopy (Figures 4D and 4E). These results suggest that intracellular Fn may promote mitochondrial fusion. To test this hypothesis, we conducted a mitochondrial fusion assay as described previously.²⁷ As shown in Figure 4F, Fn infection significantly attenuated the irradiation-induced impairment of fusion activity in 5-8F and CNE2 cells. These results indicated that Fn infection might maintain the balance between mitochondrial fusion and fission to protect mitochondria in NPC cells from over-fragmentation during irradiation stress.

Intracellular F. nucleatum maintains the host’s OXPHOS and reduces PANoptotic cells during irradiation stress

To study the mechanism of Fn-mediated radiation resistance in NPC cells, we performed RNA sequencing (RNA-seq). A volcano plot of the overall gene expression data showed that 5,607 genes were differentially expressed, including 2,448 upregulated and 2,159 downregulated DEGs in Fn-infected 5-8F cells at 24 h after irradiation with 4 Gy (Figure 5A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene set enrichment analysis (GSEA) of the RNA-seq results suggested that the OXPHOS pathway was activated by Fn infection during irradiation stress (Figures 5B and 5C). The top 20 enriched KEGG terms in this study included “cellular senescence,” “apoptosis,” and “necroptosis” signaling pathways (Figure 5D). Fn may mediate radiation resistance through the aforementioned pathways. The top 22 upregulated genes (log2-fold change > 2; fragments per kilobase million (FPKM) > 50; p value < 0.0001) in Fn-treated 5-8F cells at 24 h after irradiation with 4 Gy are presented in Figure 5E. Interestingly, changes in the mitochondrial electron transport chain proteins were rather distinct (Figure 5E). Western blot analysis indicated that MT-CO1, MT-ND1, and MT-ATP6 expression significantly increased after Fn treatment in 5-8F and CNE2 cells at 24 h after irradiation with 4 Gy (Figure 5F). The main function of mitochondria is to produce ATP through OXPHOS. To determine the functional impact of Fn infection on cellular metabolism during irradiation stress, we examined the two main bioenergetic pathways, OXPHOS and glycolysis, which were measured using the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. Our results showed that only Fn infection promoted glucose metabolism in NPC cells (Figure 5G). Under irradiation stress, OXPHOS and glycolysis decreased in NPC cells (Figure S1) but increased in Fn-infected NPC cells (Figure 5G). Moreover, ATP production and glucose uptake increased in Fn-infected NPC cells following 4 Gy irradiation (Figures 5H and 5I). Hence, these results suggested that infection with Fn maintained NPC cell’s survival by altering energy metabolism under irradiation stress (Figure 5J).

Intracellular *F. nucleatum* maintains the host’s oxidative phosphorylation and reduces PANoptotic cells during irradiation stress

(A) Volcano plot of whole-transcriptome RNA sequencing of Fn-infected 5-8F cells compared to 5-8F cells 24 h after 4 Gy irradiation, n = 2 biological replicates. Red, increased expression; green, decreased expression; and gray, no difference. p < 0.05 is considered significant.

(B) Bubble graph of KEGG enrichment analysis.

(C) GSEA plots for the indicated upregulated OXPHOS-related pathways in Fn-infected 5-8F cells with 4 Gy irradiation.

(D) Bar chart of KEGG enrichment analysis.

(E) The heatmap is based on the expression of mRNAs for the set of the top 22 significant genes.

(F–K) Fn-infected and uninfected NPC cells were exposed to 4 Gy irradiation. (F) The protein expression of MT-CO1, MT-ND1, and MT-ATP6 was determined by western blot in NPC cells. Statistical results are presented in the below panels. Data are mean values of three biology repeats. (G) Representative traces showing the change in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) during mitochondrial and glycolysis stress tests. OCR levels were measured following sequential treatments with oligomycin (O), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP)-activated (F), and rotenone/antimycin (R + A). ECAR levels were measured following sequential treatments with glucose (Glc), oligomycin (O), and 2-deoxy-glucose (2-DG). Right panels: statistical analyses of OCR or ECAR were assessed. Data are mean values of three biology repeats. (H) ATP content was detected by the ATP Assess Kit. (I) Glucose uptake ability was determined in NPC cells using a glucose uptake assay. (J) A schematic representation of the energy metabolism reprogramming process. (K) The protein expression of BCL2, BAX, cleaved BAX, caspase-3, cleaved caspase-3, PARP, cleaved PARP, GSDME, GSDME-N, GSDMD, GSDMD-N, p-MLKL, MLKL, RIPK1, and cleaved RIPK1 was determined by western blot in NPC cells. Statistical results are presented in the right panels. Data are mean values of three biology repeats. Data are shown as mean ± SD. p values were determined by independent sample t tests (F–I and K), ∗p < 0.05, ∗∗p < 0.001 and ∗∗∗p < 0.001.

PANoptosis is a newly defined form of programmed cell death (PCD) triggered by a series of stimuli, and it involves three well-known PCD pathways (pyroptosis, apoptosis, and necroptosis) simultaneously. We found that irradiation induces PANoptosis in NPC cells (Figure S2). However, B-cell lymphoma-2 (BCL-2) increased and BCL-2-associated X (BAX) and cleaved caspase-3 decreased after Fn treatment in NPC cells exposed to 4 Gy irradiation (Figure 5K). Moreover, the necroptosis-associated proteins phosphorylates mixed lineage kinase domain-like(p-MLKL) and cleaved receptor-interacting protein kinase 1 (Cle-RIPK1) decreased after 4 Gy irradiation in Fn-treated NPC cells (Figure 5K). Next, we analyzed the expression of pyroptosis effector proteins and found that cleaved gasdermin-D (GSDMD) expression decreased after 4 Gy irradiation in Fn-treated NPC cells (Figure 5K). Taken together, these results showed that Fn infection attenuated radiation-induced PANoptosis in NPC cells.

Intracellular F. nucleatum promotes radioresistance via the SLC7A5/leucine-mTOR axis in NPC cells

Transcriptome sequencing revealed that SLC7A5 expression was significantly upregulated in Fn-infected 5-8F cells after irradiation with 4 Gy (Figure 5E). Western blot analysis indicated that SLC7A5 expression was significantly increased in 5-8F and CNE2 cells 24 h post-Fn infection after irradiation with 4 Gy (Figure 6A). Analysis of the gene expression profiling interactive analysis (GEPIA) database revealed that SLC7A5 was highly expressed in a variety of tumors (Figure S3A). SLC7A5 expression in head and neck squamous cell carcinoma (HNSC) tissues was greater than that in adjacent tissues (Figure S3B). Kaplan-Meier survival analysis revealed that HNSC patients with higher SLC7A5 expression exhibited shorter overall survival (p = 0.015; Figure S3C). Consistent with our findings, SLC7A5 expression was also found to be significantly upregulated in a Gene Expression Omnibus (GEO) RNA-seq dataset of 18 paired NPC samples (Figure S3D). To further study the function of the SLC7A5 gene, we established stable SLC7A5-overexpressing cells using a retroviral construct. SLC7A5 was knocked down with small interfering RNA (siRNA). Western blotting was used to verify SLC7A5 knockdown or overexpression (Figures S3E and S3F). The overexpression of SLC7A5 promoted tumor growth in vivo (Figures S3G–S3J). The results of a colony formation assay suggested that SLC7A5 overexpression significantly promoted the survival of NPC cells after irradiation treatment (Figure 6B). Consistent with the results of the in vitro experiments, SLC7A5 overexpression reduced the antitumor effect of radiotherapy (Figures S3K, S3L, S3M, and S3N). Furthermore, we found that the expression of SLC7A5 was increased in NPC tissues from patients who experienced a radioresistance response (Figure S3O). Fn infection did not affect the expression of SLC7A5 and p-S6K1 protein in NPC cells under non-IR conditions (Figure S4).

Intracellular *F. nucleatum* promotes radioresistance via the SLC7A5/leucine-mTORC1 axis in NPC cells

(A–J) Fn-infected and uninfected NPC cells were exposed to 4 Gy irradiation. (A) The protein expression of SLC7A5 was determined by western blot in NPC cells. Statistical results are presented in the below panels. Data are mean values of three biology repeats. (B) Clone formation assays of cells with or without overexpression of SLC7A5. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (C) Leucine transport was examined in the presence of cells. (D) Clone formation assays of cells were transfected with or without an SLC7A5-siRNA. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (E) Clone formation assays of cells treated with normal medium or low-leucine (Leu) medium. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (F) The expression of SLC7A5 showed a positive relationship to that of mTOR in the public database (GEPIA). (G) The protein expression of SLC7A5, S6K1, and p-S6K1 was determined by western blot after 4 Gy irradiation in NPC cells. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (H) Representative photographs of colony formation assays. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (I) The apoptosis rates were determined by flow cytometry. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (J) OCR levels were measured following sequential treatments with oligomycin (O), FCCP-activated (F), and rotenone/antimycin (R + A). Right panels: statistical analyses of OCR were assessed. Data are mean values of three biology repeats.

(K) ATP content was detected by the ATP Assess Kit.

(L) Representative images and statistical analyses of mitochondrial fusion in NPC cells. Data are mean values of three biology repeats. Scale bar: 5 μm.

(M) Flow cytometric analysis of ROS levels in NPC cells. Data are shown as mean ± SD. p values were determined by independent sample t tests (A–E and G–L), Pearson correlation analysis (F), ∗p < 0.05, ∗∗p < 0.001, and ∗∗∗p < 0.001.

SLC7A5 is a major transporter for leucine. An increase in leucine uptake was observed following SLC7A5 overexpression (oe-SLC7A5) in NPC cells (Figure 6C). We then investigated whether SLC7A5-mediated leucine influx is involved in the regulation of Fn-mediated radioresistance. As expected, Fn-mediated radioresistance was diminished when SLC7A5 was knocked down in Fn-infected NPC cells (Figure 6D). Moreover, leucine limitation (Leu-L) significantly impaired the ability of Fn to promote the survival of NPC cells after irradiation treatment (Figure 6E). These data demonstrated that Fn-mediated radioresistance might be dependent on SLC7A5-mediated leucine influx. Moreover, SLC7A5 expression was positively correlated with mammalian target of rapamycin (mTOR) expression (p = 0.0003; r = 0.16) (Figure 6F). SLC7A5 has been reported to regulate mTOR levels; thus, we investigated the correlation between SLC7A5 and mTOR in NPC cells. Western blot results indicated that p-S6K1 levels were increased in oe-SLC7A5 cells (Figure 6G). Next, treatment with an mTORC1 inhibitor (rapamycin) significantly decreased the Fn-mediated increase in NPC cells survival after 4 Gy irradiation (Figure 6H). Cell apoptosis assay results supported the aforementioned findings (Figure 6I). Moreover, rapamycin treatment significantly decreased the Fn-induced increase in OXPHOS, ATP production, and mitochondrial fusion (Figures 6J–6L) and reversed the decreased cells ROS induced by Fn in NPC cells after 4 Gy irradiation (Figure 6M). Taken together, these results suggest that Fn promotes NPC cells survival by upregulating SLC7A5-mediated leucine influx, which activates the mTORC1 pathway during irradiation stress.

Intratumoral F. nucleatum clearance and isoleucine restriction improve NPC tumor radiotherapy sensitivity in vivo

To validate the involvement of leucine in radioresistance, we created a leucine-rich diet (1.5% leucine in water) to feed NPC model mice (Figure 7A). These results reveal that the high-leucine diet reduced the antitumor effect of radiotherapy (Figures 7B–7E). Moreover, IHC assay results confirmed that ki67 and p-S6K1 staining was stronger in xenograft tumors from mice fed a leucine-rich diet than in xenograft tumors from mice fed a standard chow diet (Figures 7F and 7G).

Isoleucine restriction improves NPC tumor radiotherapy sensitivity *in vivo*

(A) A Schematic diagram of xenograft and subcutaneous tumorigenicity in mice (n = 5/group).

(B) Representative images showing xenografts in nude mice, 21 days later.

(C) Individual tumor growth curve.

(D) Tumor growth curve.

(E) Tumor weight.

(F and G) Representative IHC staining of ki67 (F) and p-S6K1 (G) in mouse tumor tissues. Scale bar: 50 μm

(H) A Schematic diagram of Fn-positive xenograft and subcutaneous tumorigenicity in mice (n = 5/group).

(I) Representative images showing xenografts in nude mice, 21 days later.

(J) Individual tumor growth curve.

(K) Tumor growth curve.

(L) Tumor weight.

(M and N) Representative IHC staining of ki67 (M), SLC7A5, and p-S6K1 (N) in mouse tumor tissues. Scale bar: 50 μm

Data are shown as mean ± SD. p values were determined by two-way ANOVA (D and K), and independent sample t tests (E and L), ∗p < 0.05, ∗∗p < 0.001, and ∗∗∗p < 0.001.

Additionally, the absence of leucine in the diet of animals adversely affects their health, leading to increased mortality. However, it was found that the intake of leucine by the animals could be reduced without causing toxicity by implementing a rhythmic diet that included leucine-poor intervals, i.e., the “4 + 3” dieting mode (leucine-deficient diet [LDD]-4/3), which involved four days of a standard diet followed by three days of an LDD. This dietary approach did not have a significant impact on weight or mortality. Interestingly, we observed that the LDD-4/3 diet resensitized Fn-induced resistant tumors to radiotherapy (Figures 7H–7L).

To determine whether the elimination of intratumoral Fn with antibiotics would resensitize tumors to radiotherapy, we treated the mice with doxycycline (Dox) to which intratumoral Fusobacterium is sensitive.²⁸ We found that Dox resensitized Fn-induced resistant tumors to radiotherapy, as shown by a reduction in tumor volume and weight (Figures 7J–7L). Notably, combining the LDD-4/3 diet and Dox achieves a better therapeutic effect than individual applications in Fn-positive NPC mice treated with radiotherapy (Figures 7J–7L). Further IHC analysis of tumor tissues from the aforementioned four groups revealed that the expression levels of ki67, SLC7A5, and p-S6K1 were significantly decreased in the Dox + LDD-4/3 diet group (Figures 7M and 7N). These results indicated that intratumoral Fn clearance and leucine intake control improve radiotherapy efficacy in the treatment of Fn-positive NPC.

Discussion

Herein, as a study focusing on intratumoral Fn in NPC and associated radioresistance, we revealed that Fn is prevalent and enriched in human NPC tissues. We demonstrated that tumor-resident intracellular Fn promotes NPC malignancy. Consistent with these findings, two recent studies have shown the presence of the genus Fusobacterium in NPC tissues via 16S rRNA sequencing.¹¹^,¹² Moreover, several studies have confirmed the significantly greater abundance of Fn in oral/head and neck tumors.²⁹ These studies suggested that Fn is widely present and is a key bacterium in oral/nasopharyngeal tumors. Remarkably, unlike other intratumoral bacteria, Fn can multiply inside tumor cells and promote the proliferation of several tumor-host cells.¹⁸^,³⁰ We also observed the simultaneous proliferation of Fn and its NPC host cells, suggesting that Fn-infected NPC cells share similar mechanisms of malignancy with other Fn-infected digestive tract cancer cells.

Recent preclinical mouse models and observational cohorts revealed that the intratumoral microbiome influences the tumor response to radiation and chemotherapy.³¹ Tumor-resident Lactobacillus iners was reported to confer resistance to chemoradiation treatment by inducing metabolic rewiring through lactate in patients with cervical cancer.³¹ Interestingly, our and Liu et al.’s studies also revealed that the genus Lactobacillus is enriched in NPC tissues, including corresponding and noncorresponding groups, suggesting that the metabolite lactate may promote NPC progression but not the key radioresistance factor in NPC.

Based on our clinical samples, we found that patients with NPC with a greater abundance of Fn exhibited poor radiotherapy efficacy. We observed that intracellular Fn inhibited irradiation-induced PANoptosis, diminishing the response of NPC host cells to radiotherapy. Typically, therapeutic ionizing radiation can induce DNA damage and kill tumor cells.³² Interestingly, Fn infection decreases the expression of γH2AX, which is a DNA damage marker,³³ suggesting that intracellular Fn can decrease oxidative DNA damage after irradiation exposure. Ionizing radiation-induced ROS are mediators of DNA damage.³⁴ Intriguingly, we found that intracellular ROS accumulation decreased after Fn treatment. NADPH oxidase is crucial for ROS generation. We confirmed that Fn infection decreases the expression of the NADPH oxidase subunits p47phox and gp91phox³⁵^,³⁶ in NPC cells treated with X-ray radiation. These findings indicate that intracellular Fn inhibits NADPH oxidase activity, thus decreasing ROS generation and preventing ionizing radiation-induced oxidative damage. This is a favorable intracellular environment for obligate anaerobic Fn to avoid lethal ROS toxicity.

Glycolysis is the most important metabolic pathway in tumor cells,³⁷ and its intermediates provide primary raw materials for OXPHOS in mitochondria.³⁸ Fn drives tumor glucose metabolism and oncogenesis in CRC.³⁹ Our data are consistent with a previous report showing that Fn facilitates glycolysis in NPC cells under nonradiation or radiation conditions. Recent studies have suggested that some tumor cells exhibit enhanced OXPHOS.⁴⁰^,⁴¹^,⁴² It is not clear whether aerobic glycolysis is fixed or reversible, especially under therapeutic stress conditions. Radiotherapy often leads to a reduction in OXPHOS in mitochondria due to cellular damage.⁴³ However, we observed increased mitochondrial protein levels and increased expression of key genes encoding respiratory complex subunits in Fn-infected NPC cells during X-ray radiation treatment. Herein, we describe a survival mechanism of Fn-mediated host cells triggered by X-ray radiation in NPC cells that substantially contributes to both acquired and intrinsic radioresistance. This evidence indicated that intracellular Fn maintains mitochondrial OXPHOS and a proper level of glycolysis to support cell proliferation in NPC.

Additionally, ionizing radiation can cause significant changes in mitochondrial structure, mainly manifested as mitochondrial vacuolation, ridge breakage, and swelling.⁴⁴ The damaging effect on the OXPHOS process results in a decrease in mitochondrial respiration and the ATP synthesis rate, an increase in electron leakage, and ROS production.⁴⁴ Mitochondrial damage can be partially restored after ionizing radiation.⁴⁵ Recent results indicate that mitochondrial membrane fusion-mediated increases in OXPHOS and NADH/NAD+ metabolism contribute to tumor immortalization.⁴⁶ Our findings demonstrated that intracellular Fn moderates acute mitochondrial damage caused by X-ray radiation exposure by promoting mitochondrial fusion. These results suggested that Fn has evolved strategies to govern the mitochondrial structure and functions of host cells to evade killing and protect host cells, particularly under irradiation pressure conditions.

SLC7A5, an important amino acid transporter, is essential for the uptake of amino acids by tumor cells.⁴⁷ High SLC7A5 expression is associated with poor prognosis in several cancer types,⁴⁷ and SLC7A5 is expected to become an emerging therapeutic target for breast cancer.⁴⁸ Our study indicated that SLC7A5 expression was markedly increased in Fn-infected NPC cells during irradiation stress. SLC7A5 exports intracellular glutamine and promotes leucine uptake.⁴⁹ Leucine is an important regulator of protein turnover that stimulates mTORC1 activation.⁵⁰ The critical functions of mTORC1 include DNA double-strand break repair and mitochondrial function.⁵¹ We provide evidence that Fn promotes SLC7A5-mediated leucine influx and contributes to radioresistance via mTORC-induced OXPHOS reprogramming in NPC cells. Similar observations were observed in breast cancer cells, and increasing SLC7A5 levels in cultured estrogen receptor-positive MCF-7 cells effectively allowed the cells to acquire additional leucine, increasing their resistance to tamoxifen.⁵² Notably, a recent study showed that restricting leucine in the diet could be beneficial for patients with CRC.⁵³

Since rapamycin-based therapy suppresses immune function and may cause serious side effects,⁵⁴ compared with drug therapy, we believe a leucine-restricted diet is safe and convenient for the self-management of patients with Fn-positive NPC.

In summary, we demonstrated that Fn is enriched in the tumor tissue of patients with NPC and is associated with a poor response to radiotherapy. We showed that Fn promotes NPC radioresistance through the SLC7A5/leucine-mTORC1 axis to maintain energy metabolism. Our data suggested that Fn and its host cell form mutualistic symbioses to counter irradiation pressure. This finding raises an important clinical question: are conventional radiotherapeutic regimens suitable for treating patients with NPC in the Fn-positive subgroup? Alternatively, we suggest that Fn-positive patients with NPC be treated with radiotherapy in combination with the mitochondria-targeting antibiotic Dox and/or the dietary restriction of leucine. It is important to manage patients with NPC with different Fn levels differentially during radiotherapy.

Limitations of the study

Due to the complexity and ambiguity of the host-intratumoral microbiota interplay, it is not possible to definitively exclude the potential of other microbial taxa to enhance tumor radioresistance in NPC. Moreover, the current study is limited by a small sample size. Consequently, future research is essential for corroborating these findings across multiple centers.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ge Zhang (zhangge@mail.sysu.edu.cn).

Materials availability

This study did not generate new materials.

Data and code availability

•
This paper does not report the original code.
•
The 16S rRNA sequencing and RNA-seq data were uploaded to the NCBI Sequence Read Archive (SRA, PRJNA1060278; PRJNA1060782). Accession numbers are listed in the key resources table. The remaining data are shown in the manuscript and its supplemental information files.
•
Any additional information required to reanalyze the data presented in this paper is available upon request from the lead contact.

Acknowledgments

This work was financially supported by the Youth Foundation of the National Natural Science Foundation of China (no. 32300761), Guangdong Basic and Applied Basic Research Foundation (no. 2023A1515012256), Basic and Applied Basic Research Project of Guangzhou (no. 2024A04J4315), and Fostering Program for NSFC Young Applicants (Tulip Talent Training Program) of Sun Yat-sen University Cancer Center (no. 2023yfd17).

Author contributions

S.G. performed most of the experiments and data analysis. Z.W., F.C., Q.L., and X.P. assisted in the in vivo or in vitro experiments. S.X. collected the clinical samples. S.G. and G.Z. wrote the original draft. F.C. contributed to experiment guidance. G.Z. and W.L. designed the project and revised and edited the manuscript.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Mice/rabbit anti-Fn polyclonal antibody	Homemade	N/A
α-Tubulin Rabbit mAb	Cell Signaling Technology	Cat#2125; RRID: AB_2619646
PARP antibody	Cell Signaling Technology	Cat# 9532; RRID: AB_659884
BCL2 antibody	Cell Signaling Technology	Cat# 4223;RRID: AB_1903909
BAX antibody	Cell Signaling Technology	Cat# 5023; RRID: AB_1055741
Caspase3 antibody	Cell Signaling Technology	Cat# 14220S; RRID: AB_2798429
Cleaved caspase3 antibody	Cell Signaling Technology	Cat# 9661; RRID: AB_2341188
GSDME antibody	Abcam	Cat# ab215191; RRID: AB_2737000
GSDMD antibody	Abcam	Cat# ab210070; RRID: AB_2893325
S6K1 antibody	Abcam	Cat# ab32529; RRID: AB_777800
p-S6K1 antibody	Abcam	Cat# ab60948; RRID: AB_944606
SLC7A5 antibody	Abcam	Cat# ab305251; RRID: NA
MT-CO1 antibody	Abcam	Cat# ab14705; RRID: AB_2084810
MT-ND1 antibody	Abcam	Cat# ab181848; RRID: AB_2687504
MT-ATP6 antibody	Abcam	Cat# ab190287; RRID: AB_2747745
GAPDH Monoclonal antibody	Proteintech	Cat# 10494-1-AP; RRID: AB_2263076
anti-rabbit IgG (DyLight 594) antibody	Abcam	Cat# ab96921; RRID: AB_10680407
anti-mouse IgG (DyLight 488)	Abcam	Cat#ab96879; RRID: AB_10687475
Anti-rabbit IgG, HRP-linked Antibody	Cell Signaling Technology	Cat# 7074; RRID: AB_2099233
Anti-mouse IgG, HRP-linked Antibody	Cell Signaling Technology	Cat# 7076; RRID: AB_330924

Chemicals, peptides, and recombinant proteins

RPMI 1640	Gibco	Cat# 12633012
Fetal bovine serum (FBS)	Gibco	Cat# 10100147
Lipofectamine 3000	Invitrogen	Cat# L3000008
Rapamycin	MedChemExpress	Cat# HY-10219; CAS: 53123-88-9
SLC7A5-specific siRNA oligo duplex	Origene	Cat#: SR305376
Neomycin	MedChemExpress	Cat# HY-150520; ACS: 1404-04-2
DAPI	Cell Signaling Technology	Cat# 4083
Ci [3 H]-L-leucine	Perkin Elmer	Cat# NET116600
XF 1.0 M Glucose Solution	Agilent Technology	Cat# 103577-100
Seahorse XF Base Medium	Agilent Technology	Cat#102353-100
XF DMEM Medium, pH 7.4	Agilent Technology	Cat#103575-100
XF 100 mM Pyruvate Solution	Agilent Technology	Cat#103578-100
XF 200 mM Glutamine Solution	Agilent Technology	Cat#103579-100

Critical commercial assays

TIANamp FFPE DNA Kit	TIANGEN	Cat# DP331-02
Evo M-MLV Reverse Transcription Kit	Accurate Biology	Cat# AG11711
SYBR® Green Pro Taq HS qPCR Kit	Accurate Biology	Cat# AG11702
Calcein/PI Cell Viability Assay Kit	Beyotime	Cat# C2015M
Cell Apoptosis Kit	Beyotime	Cat# C1062S
ROS Assay Kit	Beyotime	Cat# S0033S
LDH Cytotoxicity Assay Kit	Beyotime	Cat# C0016
Glucose Uptake-GloTM Assay Kit	Promega	Cat# J1341
ATP Assay Kit	Beyotime	Cat# S0026
Seahorse XFe96/XF Pro FluxPak mini	Agilent Technology	Cat# 103793-100
Seahorse XF Glycolysis Stress Test Kit	Agilent Technology	Cat#103020-100
Seahorse XF Cell Mito Stress Test Kit	Agilent Technology	Cat#103015-100

Deposited data

16S rRNA-Seq data	This paper	NCBI Sequence Read Archive (SRA): PRJNA1060278
RNA-seq data	This paper	NCBI Sequence Read Archive (SRA): PRJNA1060782

Biological samples

Nasopharyngeal carcinoma tissue chip	Shanghai core Biological Technology Co., Ltd.	Number: HNasN110Su01
Nasopharyngeal carcinoma tissue samples	Cancer Center of Sun Yat-sen University	No: G2023-257-01

Experimental models: Cell lines

5-8F cell line	Gift from Musheng Zeng, SYSUCC	N/A
CNE2 cell line	Gift from Musheng Zeng, SYSUCC	N/A

Bacterial and virus strains

E. coli DH5α	TIANGEN	Cat# CB101-02
Fusobacterium nucleatum ATCC 25586	Guangdong Microbial Culture Collection Center	GDMCC NO.: 1.1290

Experimental models: Organisms/strains

BALB/c nude mice	Experimental Animal Center of Sun Yat-sen University	N/A

Oligonucleotides

FITC-labeled F. nucleatum probe	Synthesized by Servicebio, Co., Ltd	5′-CTT GTA GTT CCG C(C/T) TAC CTC-3′
Primer: Fusobacterium nucleatum	Synthesized by Sangon Biotech, Co., Ltd	Forward: 5′-AAGCGCGTCTAGGTGGTTATGT-3′ Reverse: 5′-TGTAGTTCCGCTTACCTCTCCAG-3′

Software and algorithms

GraphPad Prism 8	GraphPad	https://www.graphpad.com/
ImageJ	National Institute of Health	National Institute of Health
EndNote X9	EndNote	https://endnote.com/

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Experimental model and subject details

Clinical samples

Cohort 1

An NPC tissue chip (n = 110) was purchased from Shanghai Core Biological Technology Co., Ltd. (number HNasN110Su01). Approval for the tissue chips was granted by the Ethics Committee of Shanghai Outdo Biotech Co., Ltd. (SHY JS-CP-1704009). Data for 29 patients were excluded because the dots were off the chips during the experiment. In total, data for 81 patients with NPC were included in the final analysis. The clinical characteristics of the study participants are shown in Table S1.

Cohort 2

Seventeen paired paraffin-embedded NPC specimens of tumor and normal adjacent (normal adj.) tissue were obtained from Sun Yat-sen University Cancer Center (SYSUCC). The clinical characteristics of the study participants are shown in Table S2.

Cohort 3

Formalin-fixed paraffin-embedded (FFPE) samples were procured from a cohort of 15 patients with radiotherapy-resistant NPC and 15 patients with radiotherapy-sensitive NPC at SYSUCC. The clinical characteristics of the study participants are shown in Table S3.

Patients who exhibited disease stability or progression at three months after radiotherapy and recurrence within six months were classified as radioresistant; conversely, patients who achieved complete or partial remission at three months after radiotherapy were classified as radiosensitive. Samples were obtained before the administration of radiotherapy. For cohorts 2 and 3, patient samples were obtained with the approval of the Ethics Committee of SYSUCC (No: G2023-257-01).

Bacterial strains and culture conditions

F. nucleatum (ATCC 25586) was grown anaerobically by using AnaeroPack (Mitsubishi Gas Chemical Co., Japan) at 37°C on blood agar plates (Huankai, China) for 2–3 days. E. coli (DH5α; Tiangen, China) was aerobically cultured on LB agar plates at 37°C for 24 h. Subsequently, the bacteria were suspended in RPMI-1640 medium (Gibco, USA) at a final concentration of 1× 10⁸ colony forming units (CFU) mL-1 for use during infection experiments.

Cell lines and cell culture

The human NPC cell lines 5-8F and CNE2 were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). The cells were incubated at 37°C with 5% CO₂. All cell lines used in this study are free of mycoplasma contamination and have been authenticated by STR profiling.

Mouse xenograft experiments

Female BALB/c nude mice (4–6 weeks) were procured from the Experimental Animal Center of Sun Yat-sen University and housed in a controlled specific pathogen-free environment. All animal care and experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Guangzhou, China), NO: SYSU-IACUC-2024-000199.

For the subcutaneous tumor model, 5.0×10⁶ 5-8F cells were subcutaneously injected into each mouse. In the lung metastasis model, 1.0×10⁶ 5-8F cells were intravenously injected through the tail vein. Following a three-day interval, the mice were subsequently intravenously injected with Fn, K-Fn (both 10⁶ CFU/mouse), or PBS (control) every other day for a total of three administrations.

To assess the abundance of Fn within tumor tissue, 5.0×10⁶ DiD-labelled Fn was administered intravenously to mice through the tail vein. After three days, the mice were imaged using the NightOWL LB983 bioluminescence imaging system (Berthold, Germany).

To assess the impact of intratumoral Fn on NPC radiotherapy, 5.0×10⁶ 5-8F cells were subcutaneously injected into each mouse. After a three-day interval, the mice were intravenously injected with Fn (10⁶ CFU/mouse) or PBS (control) every other day for three administrations. Once the tumor size reached approximately 200 mm³, the nude mice were randomly assigned to either the non-IR group or IR group. In the IR group, the mice underwent local irradiation of the xenograft with a single 20 Gy dose of X-rays (Rs 2000, Rad Source Technologies, USA) at a dose rate of 0.883 Gy/min.

To assess the impact of SLC7A5 on the development of tumors and resistance to radiation therapy, 5-8F cells (5×10⁶/100 μL) were subcutaneously administered to nude mice with or without SLC7A5 overexpression (oe-SLC7A5). Using a random allocation method, the nude mice were then divided into two groups: non-IR group and IR group.

For the leucine-deficient diet (LDD) experiment, following the initiation of modeling on day 0 randomly assigned to four groups (n = 5). Each group was treated as follows: (1) the control group, mice were fed a standard diet; (2) the LDD-4/3 group, mice were fed on a cyclic schedule (4 days of the standard diet followed by 3 days of the LDD). Importantly, both the LDD and standard diet were comparable in terms of energy and nitrogen content and were procured from Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd; (3) doxycycline (Dox) group, Mice were administered with 0.2 mg/mL of Dox (Sangon Biotech) through drinking water; (4) LDD-4/3+Dox group, mice were fed with LDD-4/3 diet and administered Dox (0.2 mg/mL) orally via drinking water.

Tumor volumes were calculated using the following formula: volume (mm³) = length (mm) × width (mm) × width (mm)/2. Subsequently, the tumors were embedded in paraffin for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). As previously described,²⁰ bacterial DNA was extracted from tumor tissues for Fn detection via qPCR. The primers used are listed in the key resources table.

Method details

Fluorescence in situ hybridization (FISH)

FISH analysis was conducted to examine the abundance of Fn using a specific probe. All the experiments were performed according to manuals provided by Servicebio (Wuhan, China). The F. nucleatum probes used are listed in the key resources table.

DNA extraction and 16S rRNA sequencing

DNA was extracted from sections of paraffin-embedded NPC tissues using the TIANamp FFPE DNA Kit (TIANGEN, China) according to the manufacturer’s instructions. The 16S rRNA sequencing was performed by LC-Bio Co. Ltd. (Hangzhou, China). A five-region (5R) amplification method was utilized for sample analysis, as previously detailed.⁵⁵ The libraries were sequenced on the Illumina NovaSeq6000 system. Reads were demultiplexed per sample, filtered and aligned to each of the five amplified regions based on the primers’ sequences. The Short Multiple Regions Framework (SMURF) method was employed to merge read counts from the five regions into a cohesive profiling result by solving a maximum likelihood problem.⁵⁶ The GreenGenes database was used as a reference. This method can also quantify the relative abundance of microbe with the expectation maximization algorithm. The reliability of the data analysis can be affected by low quality, primer splice sequences etc. Therefore, a series of filters were applied to detect and remove contamination as described.⁵⁵

Quantitative PCR (qPCR)

Analysis of intratumoral bacteria was performed using an SYBR Green Pro Taq HS qPCR Kit (Accurate Biology, China) on a Bio-Rad CFX96TM (Bio-Rad, USA) qPCR system. E. coli DNA was used to plot a standard curve to calculate bacterial DNA concentration in the sample. The sequences of the primers used in the qPCR analysis are listed in the key resources table. Raw threshold cycle (Ct) values were normalized according to a bacterial standard curve produced with E. coli DNA.

Bacterial co-culture with cell and irradiation treatment

For in vitro co-culture experiments, cells were washed with PBS and then incubated with Fn, heat-killed Fn, or E. coli at the indicated multiplicity of infection (MOI) in antibiotic-free RPMI-1640 medium supplemented with 10% FBS at 37°C under 5% CO₂. The cells were irradiated using a Rad Source Rs 2000 X-ray Irradiator (Rad Source Technologies, USA) at a dose rate of 300 cGy/min (dose: 0, 2, 4, or 8 Gy) at room temperature.

Cell transfection and inhibitors

The human SLC7A5-specific siRNA oligo duplex (TT320001, ID 8140) and the ineffective Trilencer-27 fluorescence-labeled transfection control siRNA duplex (SR30002) were obtained from Origene Technologies. Lipofectamine 3000 (Invitrogen) was used for siRNA transfection following the manufacturer’s instructions. To establish stable SLC7A5-overexpressing NPC cell lines, an SLC7A5 overexpression vector was constructed with pcDNA3.1 by SynbioTech (Suzhou, China). The plasmids were transfected into 5-8F and CNE2 cells, which were subsequently selected using neomycin (800 μg/mL) for four weeks. To inhibit mTORC1 activation, the cells were treated with the mTORC1 inhibitor rapamycin (10 μM, MedChemExpress).

Immunofluorescence staining

Immunofluorescence staining was conducted according to previously described methods.¹⁸ In brief, cells or sections were washed and fixed with a 4% paraformaldehyde solution. Subsequently, the cells or sections were permeabilized with 0.3% Triton X-100, blocked with 3% BSA, and then incubated overnight at 4°C with antibodies against Fn and α-tubulin. Then, the cells or sections were incubated with anti-rabbit IgG (DyLight 594) and anti-mouse IgG (DyLight 488) antibodies for 40 min and DAPI for 15 min. Images were captured using a fluorescence microscope (Olympus FV300, Japan). The details of the antibodies used are listed in the key resources table.

Intracellular survival assays

Intracellular survival assays were conducted following a previously described protocol.¹⁸ In this study, NPC cells were infected with live Fn at a 10:1 infection ratio (MOI) and incubated at 37°C with 5% CO₂ for 12 h. Subsequently, the cells infected with Fn were rinsed thrice with PBS and treated with gentamicin (100 μg/mL) for 2 h to eliminate any extracellular bacteria. Finally, the cells were lysed using 0.1% Triton X-100 lysis buffer supplemented with protease and phosphatase inhibitors. The presence of intracellular Fn was determined by cultivating the lysed cells on blood agar plates and quantifying the colony-forming units (CFUs).

Transcriptome RNA sequencing

Cell samples were sent to Wuhan Maiteville Biotechnology Co., Ltd., in Wuhan, China, for transcriptome sequencing. The experimental procedures, including library preparation and sequencing, followed the standard protocols provided by Illumina, a company based in the United States. Sequencing was performed using an Illumina HiSeq 3000 system. The resulting RNA-Seq data were normalized using quantile normalization and are presented as log2-fold changes (log2FCs). Genes exhibiting significant up- or downregulated expression (log2 FC ≥ |0.5|) under specified conditions were analyzed using the web-based functional analysis tool Ingenuity Pathway Analysis (IPA).

Western blotting

Protein extraction and Western blotting were conducted following previously described methods.¹⁷ Briefly, extracted proteins were separated via 10%–15% SDS‒PAGE and subsequently transferred onto PVDF membranes. The membranes were then incubated with primary antibodies and HRP-conjugated secondary antibodies. A GAPDH antibody was used as a control. Protein detection was accomplished using a chemiluminescence detection system (Tianneng, China). The grayscale values of the resulting bands were analyzed using ImageJ software. The greyscale of targeted bands was normalized to the greyscale of GAPDH, and the relative greyscale was analyzed using SPSS software. The details of the antibodies used are listed in the key resources table.

Colony formation and migration assays

NPC cells were infected with live or heat-killed Fn (MOI 1:10) for 12 h at 37°C in 5% CO₂. Cells were washed with PBS to remove any unbound bacteria and then incubated in a medium containing gentamicin (100 μg/mL) for 2 h to kill the remaining extracellular Fn. For colony formation assays, the infected cells were seeded into 6-well plates at a density of 700 cells per plate, followed by a two-week cultivation period. Subsequently, the cells were fixed using a 4% paraformaldehyde solution and stained with 1% crystal violet to facilitate the quantification of colonies.

The migratory ability of the cells was examined using the transwell migration assay. The infected cells were resuspended in 200 μL medium or Leucine-limitation medium (0.1mM leucine/isoleucine) without serum and were seeded into upper chambers of transwell chambers (Corning, USA) while lower chambers were filled with medium containing 10% FBS. The migrated cells were observed and photographed under an inverted microscope at 24 h. Each experiment was analyzed in triplicate.

Assessment of cell viability and apoptosis

The cell viability rate was measured using a Calcein/PI Cell Viability Assay Kit (Beyotime, China). Cells were mixed with 1× Assay Buffer and stained with 2 μM calcein-AM and 4.5 μM PI at 37°C for 30 min. Subsequently, images were captured utilizing a fluorescence microscope (Olympus FV300, Japan).

Cell apoptosis was assessed using a Cell Apoptosis Kit (Beyotime, China). Briefly, 5 × 10⁵ cells were collected and washed twice with cold PBS. The cells were centrifuged at 500 × g for 5 min at 4°C. Subsequently, the cells were resuspended in 50 μL of 1× binding buffer and incubated with 2.5 μL of Annexin V-FITC and 2.5 μL of PI staining solution at room temperature for 10 min. Then, 250 μL of 1× binding buffer was added to the mixture. Finally, flow cytometry was used to quantify cell apoptosis.

Measurements of LDH release and ROS production

LDH assays were conducted utilizing an LDH Cytotoxicity Assay Kit (Biyuntian, China) according to the manufacturer’s instructions. Absorbance was measured at a wavelength of 490 nm using a microplate reader (Bio-Rad, USA).

ROS detection was conducted using a ROS Assay Kit (Biyuntian, China). The growth medium was discarded, and DCFH-DA (10 μM) was diluted with 1640 medium and added to the plate. The cells were then cultured for 20 min and then washed thrice to remove DCFH-DA from the plate. The ROS concentration was quantified using a CytoFLEX Flow Cytometer (Beckman Coulter, USA).

Oxidative phosphorylation and glycolysis assays

A Seahorse XF96 Extracellular Flux Analyser (Seahorse Bioscience) was used to measure the intact cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in real-time. A total of 2.0 × 10⁴ cells were seeded into 96-well Seahorse microplates containing 80 μL of growth medium. The cells were subsequently incubated at 37°C in a 5% CO₂ environment for 16 h. A calibrator plate was also allowed to reach equilibrium overnight in a non-CO₂ incubator. Before the experiment commenced, the cells were washed twice using assay running media. The media consisted of unbuffered DMEM supplemented with 25 mmol/L glucose, 1 mmol/L glutamine, and 1 mmol/L sodium pyruvate for OCR measurements and unbuffered DMEM supplemented with 1 mmol/L glutamine for ECAR measurements. After the washes, the cells were equilibrated in a non-CO₂ incubator. Once the probe calibration was completed, the probe plate was substituted with the cell plate. This refined protocol was developed to facilitate concurrent measurements.

For the OCR, the analyzer generated a plot of the measured values as the cells underwent sequential treatment with oligomycin (1 μmol/L), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP, 0.5 μmol/L), and antimycin A (1 μmol/L) in combination with rotenone (1 μmol/L). Similarly, for the extracellular acidification rate (ECAR), the analyzer produced a plot of the measured values as the cells were subjected to sequential treatment with glucose (10 mmol/L), oligomycin (1 μmol/L), and 2-deoxy-glucose (2-DG, 100 mmol/L).

Mitochondrial fusion assay and electron microscopy

The assay was performed as described previously.⁵⁷ In brief, Green FP-Mitochondrion-expressing cells were seeded into 6-well plates and cultured on coverslips overnight alongside an equal number of Red FP-Mitochondrion-expressing cells. The following morning, the cells were incubated for 60 s in 50% PEG1500 (Roche), washed extensively with 1× PBS, and cultured for 7 h in a medium supplemented with 20 μg/mL cycloheximide before fixation.

Transmission electron microscopy (TEM) was conducted by the Electron Microscopy Facility at Servicebio (Wuhan, China) utilizing a HITACHI HT780 instrument to capture images.

Measurement of glucose uptake and ATP production

Cells were initially seeded at 2 × 10⁴ cells per well in 96-well plates and subsequently exposed to 1 mM 2-deoxyglucose for 2 h. Following a rinse with PBS, the extent of glucose uptake by the cells was quantified utilizing a Glucose Uptake-GloTM Assay Kit (Promega, USA) following the manufacturer’s instructions. Relative fluorescence was assessed using a Synergy 2 Multi-Detection Microplate Reader (BioTek, USA).

To determine intracellular ATP production, 1 × 10⁶ cells were collected, and the concentration of intracellular ATP was measured using an ATP Assay Kit (Biyuntian, China) following the manufacturer’s guidelines.

Leucine uptake assay

The [3H]-L-leucine uptake procedure was conducted following previously described methods.⁵⁸ Briefly, cells were cultured in 96-well plates in RPMI medium supplemented with or without 10 mM BCH for 1 h. Then, the cells were exposed to 0.3 μCi [3H]-L-leucine (200 nM; PerkinElmer) in leucine-free RPMI medium (Invitrogen) for 15 min at 37°C, with or without 10 mM BCH. Subsequently, the cells were harvested and transferred to filter paper using a 96-well plate harvester (Wallac PerkinElmer). After the completion of the drying process, scintillation fluid was added to the cells, and counts were quantified utilizing a liquid scintillation counter manufactured by PerkinElmer.

H&E staining and IHC

Paraffin-embedded sections (5 μm) were stained with H&E following a standard protocol. For IHC staining, paraffin-embedded sections were prepared accordingly. Endogenous peroxidase activity was inhibited using 3% H₂O₂, followed by blocking with serum. Subsequently, the sections were incubated with primary antibodies. Then, HRP-conjugated secondary antibodies were added, and the sections were incubated for 2 h at room temperature. Diaminobenzidine was used as the chromogen. The details of the antibodies used are listed in the key resources table.

Quantification and statistical analysis

Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad, USA). The data were bar graphs or dot plots (means ± SDs). A Student’s t test or ANOVA was used to compare multiple groups according to the data type. Differences were considered statistically significant at p < 0.05.

Published: October 1, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.xcrm.2024.101753.

Contributor Information

Wanli Liu, Email: liuwl@sysucc.org.cn.

Ge Zhang, Email: zhangge@mail.sysu.edu.cn.

Supplemental information

Document S1. Figures S1–S4 and Tables S1–S3

mmc1.pdf^{(836.9KB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(8MB, pdf)}

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S4 and Tables S1–S3

mmc1.pdf^{(836.9KB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(8MB, pdf)}

Data Availability Statement

•
This paper does not report the original code.
•
The 16S rRNA sequencing and RNA-seq data were uploaded to the NCBI Sequence Read Archive (SRA, PRJNA1060278; PRJNA1060782). Accession numbers are listed in the key resources table. The remaining data are shown in the manuscript and its supplemental information files.
•
Any additional information required to reanalyze the data presented in this paper is available upon request from the lead contact.

[bib1] 1.Chen Y.P., Chan A.T.C., Le Q.T., Blanchard P., Sun Y., Ma J. Nasopharyngeal carcinoma. Lancet. 2019;394:64–80. doi: 10.1016/S0140-6736(19)30956-0. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Leucine restriction ameliorates Fusobacterium nucleatum-driven malignant progression and radioresistance in nasopharyngeal carcinoma

Songhe Guo

Shan Xing

ZhenYu Wu

Fangfang Chen

Xiaoyun Pan

Qifan Li

Wanli Liu

Ge Zhang

Summary

Graphical abstract

Highlights

Introduction

Results

F. nucleatum is prevalent in human NPC tissues and is associated with poor radiotherapy efficacy

Figure 1.

F. nucleatum invades NPC cells and promotes tumor growth and metastasis in NPC mice

Figure 2.

F. nucleatum accumulates in mouse NPC tissues and promotes radioresistance

Intracellular F. nucleatum promotes radioresistance in NPC cells by suppressing host apoptosis and DNA damage

Figure 3.

Intracellular F. nucleatum promotes host mitochondrial fusion and inhibits ROS generation during irradiation stress

Figure 4.

Intracellular F. nucleatum maintains the host’s OXPHOS and reduces PANoptotic cells during irradiation stress

Figure 5.

Intracellular F. nucleatum promotes radioresistance via the SLC7A5/leucine-mTOR axis in NPC cells

Figure 6.

Intratumoral F. nucleatum clearance and isoleucine restriction improve NPC tumor radiotherapy sensitivity in vivo

Figure 7.

Discussion

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental model and subject details

Clinical samples

Cohort 1

Cohort 2

Cohort 3

Bacterial strains and culture conditions

Cell lines and cell culture

Mouse xenograft experiments

Method details

Fluorescence in situ hybridization (FISH)

DNA extraction and 16S rRNA sequencing

Quantitative PCR (qPCR)

Bacterial co-culture with cell and irradiation treatment

Cell transfection and inhibitors

Immunofluorescence staining

Intracellular survival assays

Transcriptome RNA sequencing

Western blotting

Colony formation and migration assays

Assessment of cell viability and apoptosis

Measurements of LDH release and ROS production

Oxidative phosphorylation and glycolysis assays

Mitochondrial fusion assay and electron microscopy

Measurement of glucose uptake and ATP production

Leucine uptake assay

H&E staining and IHC

Quantification and statistical analysis

Footnotes

Contributor Information

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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