Fusobacterium nucleatum Infection Induces Malignant Proliferation of Esophageal Squamous Cell Carcinoma Cell by Putrescine Production

F. nucleatum was more abundant in ESCC tumor tissues.

Although it has been demonstrated that Fusobacterium nucleatum (Fn) can be detected in ESCC clinical samples and it was correlated with shorter survival in a Japanese cohort (23), whether Fn could be detected in a Chinese cohort and the clinical signature of Fn-positive patients were still unclear. To determine these issues, we detected the relative amount of Fn by qPCR assay in 73 ESCC clinical samples (with paired tumor [T] and adjacent normal tissue [N]) from Shanxi Province, China. Among the 73 clinical samples with paired N&T samples, the relative amount of Fn was significantly higher in tumor tissue than in adjacent normal tissue (Fig. 1A). These 73 Fn-positive samples were further divided into Fn-High and Fn-Low groups according to the relative Fn abundance in tumors. The Fn-High group exhibited significantly shorter survival than the Fn-Low group (Fig. 2B). In conclusion, Fn was significantly elevated in the tumor tissue of Chinese ESCC patients, and this increased Fn infection might have influenced the prognosis of ESCC patients.

Fn infection induces ESCC cell malignant proliferation and migration.

Because Fn could be detected in ESCC clinical samples and was correlated with shorter survival, we initially intended to determine the influence of Fn infection on the progression of ESCC patients using a Fn and ESCC cell coculture system. The ESCC cell line KYSE70 was incubated with Fn (ATCC 25586) at 400 multiplicities of infection (MOI) for 24 h, and the proliferation and migration ability were analyzed. As shown by CCK8 assay, Fn infection effectively promoted cell viability, whereas heat-killed Fn and another anaerobe, Bacteroides acidifaciens (Ba), had little effect on ESCC cell proliferation (Fig. 2A). Similarly, the cell colony-formation experiments also revealed that Fn infection significantly improved the colony formation ability of KYSE70 (Fig. 2B). In addition, using the live-cell imaging system, it was obvious that KYSE70 cells proliferate faster after Fn infection compared to cells without Fn infection (Movies S1 and S2). As in the transmission electron microscopy (TEM) results, Fn at the bottom of the petri dish disappeared along with the movement and division of KYSE70 cells (Movie S2), indicating that Fn invaded/adhered/was swallowed into KYSE70 cells. Moreover, the migration ability, another indicator of cancer cell malignance, was also significantly enhanced by Fn infection (Fig. 2C). In conclusion, Fn can promote ESCC cell proliferation and migration.

Fn can invade ESCC cells.

Because Fn can promote the proliferation of ESCC cells, we further determined whether Fn can invade living ESCC cells. Fn and KYSE70, one of the ESCC cell lines, were cocultured at 400 MOI for 24 h, and then cells were collected for TEM analysis or differential immunofluorescence experiments. In the TEM results, spindle-shaped Fn were found in the cytoplasm of KYSE70 cells (Fig. 3A). As in the TEM results, in the differential immunofluorescence experiment, some intracellular Fn, labeled with green fluorescence in the merged picture, could be detected (Fig. 3B), indicating that Fn can penetrate ESCC cells.

Fn promotes ESCC cell proliferation by putrescine production which can disturb polyamine metabolism.

To further delineate the potential mechanisms underlying Fn infection-induced malignant cell proliferation, KYSE70 and KYSE70-Fn were collected simultaneously for RNA-sequencing. Spermidine/spermine N1-acetyltransferase 1 (SAT1) was increased significantly after Fn invasion (Fig. 4A). Meanwhile, the relative expression level of SAT1 increased gradually over time after Fn invasion (Fig. 4B). SAT1 encodes a rate-limiting enzyme in the catabolic pathway of polyamine metabolism, predominantly acetylating the N¹ position of spermidine and spermine (27). We speculated that the polyamine metabolism pathway was disturbed during Fn infection. Therefore, we determined the polyamine concentrations in ESCC cells with and without Fn infection. Except for spermine, the arginine, ornithine, putrescine, spermidine, N¹-acetylspermidine, and N¹-acetylspermine concentrations significantly increased (Fig. 4D). Most noteworthy is that the putrescine concentration increased sharply, by more than 6-fold, after Fn infection, indicating that putrescine may play a crucial role in the Fn infection-induced disturbed polyamine metabolism. In addition, by analyzing polyamine concentrations in the Fn microbes themselves, we found that Fn can produce much higher levels of putrescine than other polyamines (Fig. 4E; putrescine, 16.66 nmol/billion Fn; arginine, 6.24 nmol/billion Fn; ornithine, 0.16 nmol/billion Fn; spermidine, 0.34 nmol/billion Fn; spermine, 0.0016 nmol/billion Fn). Meanwhile, the expression level of ornithine decarboxylase (ODC) stabilized in 6 h after Fn infection and decreased significantly at 24 h after Fn infection (Fig. 4F). The ODC gene encodes an enzyme involved in putrescine synthesis, and the decreased ODC expression level indicated that putrescine synthesis in ESCC cells was not active after Fn infection. Thus, the dramatically increased putrescine levels in Fn-infected ESCC cells were produced by Fn.

To clarify the influence of elevated putrescine induced by Fn infection, we mimicked the increased cell putrescine level by adding putrescine into the cell culture medium and found that putrescine can increase cell proliferation in a dose-dependent manner (Fig. 4G). In conclusion, the sharply increased putrescine levels in ESCC cells were produced by Fn and can promote the proliferation of ESCC cells.

Patients.

A total of 73 patients form Shanxi Province diagnosed with ESCC who had received no prior treatment were recruited in this study. Informed consent was obtained from all subjects. This study was approved by the ethical committees of Shenzhen Bay Laboratory.

Fn detection by TaqMan qPCR.

Total gDNA from Fn DNA in clinical ESCC samples was extracted and the Fn DNA in those clinical samples of gDNA was subsequently detected by TaqMan qPCR, as previously described (23). Briefly, the amplifications were performed by Universal U+ probe Master Mix V2 (Vazyme Biotech, Q513-02) following the manufacturer’s instructions. The following primer and probe sequences were used: Fn forward primer, 5′-TGGTGTCATTCTTCCAAAAATATCA′; Fn reverse primer, 5′-AGATCAAGAAGGACAAGTTGCTGAA′; Fn FAM probe, 5′-ACTTTAACTCTACCATGTTCA′; PGT forward primer, 5′-ATCCCCAAAGCACCTGGTTT′; PGT reverse primer, 5′-AGAGGCCAAGATAGTCCTGGTAA′; PGT FAM probe, 5′-CCATCCATGTCCTCATCTC’. Amplification and detection was performed with the ABI StepOne Plus real-time PCR system under the following reaction conditions: 5 min at 95°C, followed by 40 cycles of denaturation at 95°C for 10 s and at 60°C for 30 s. The cycle threshold (C_T) values of Fn were normalized to the C_T value of PGT (44).

Cell culture.

Human ESCC cell line KYSE70 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (PS). Cells were incubated at 37°C and 5% CO₂.

Bacterium culture.

F. nucleatum (ATCC 25586) was obtained from the Guangdong Microbial Culture Collection Center (GDMCC) and incubated in tryptic soy broth at 37°C under anaerobic conditions. B. acidifaciens (DSMZ 15896) was obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH and incubated in brain-heart infusion broth at 37°C under anaerobic conditions.

Bacterium invasion.

KYSE70 ESCC cells were seeded in a 96-well polypropylene microplates with complete cell culture medium (1640 medium with 10% FBS and 1% PS). Before Fn invasion, cells were washed twice with phosphate-buffered saline (PBS) solution and antibiotic-free medium was added. Fn was collected during the logarithmic phase, washed twice with PBS, and added to the cells at a MOI of 400, and cells were incubated in the cell incubator for 24 h. After infection, cells were washed with PBS, fresh complete medium was added, and cells were incubated for the following experiments.

Differential immunofluorescence experiment of Fn.

Intracellular bacteria detection has been described previously (45). Briefly, the ESCC cells with/without Fn infection for 24 h were fixed with 4% paraformaldehyde fix solution for 15 min, and blocked by 5% donkey serum (Jackson, 017-000-121) for 1 h at room temperature. Next, the extracellular Fn were reacted with primary antibody (Diatheva, ANT0084) at 1:200 at for 1 h at room temperature; after PBS washes, the primary antibody was revealed by the secondary antibody Alexa-594 (Thermo Fisher Scientific, cat no. A32753) for 1 h. After PBS washes, cells were permeabilized for 20 min with 0.1% Triton X-100. A second round of primary antibody reaction were reacted as previously: after PBS washes, the primary antibody was revealed by secondary antibody Alexa-488 (Thermo Fisher Scientific, cat no. A32790) for 1 h. After washing, the coverslips were mounted onto slides with ProLong Gold Antifade Mountant (Thermo Fisher Scientific, cat no. P36935).

Cell proliferation detection.

The proliferation of ESCC cell lines was measured by CCK8 assay. Briefly, ESCC cells with or without Fn invasion were washed with PBS before detection. Next, 100 μL of fresh cell culture medium containing 10% CCK8 solution was added, and cells were kept in the incubator for 1 h. Cell viability was then determined by measuring the absorbance at a wavelength of 450 nm.

Colony formation assay.

For the colony formation assay, 600 cells were seeded in a 60-mm petri dish containing complete RPMI 1640 on day 1. The medium was replaced with PS-free cell culture medium on day 2, then Fn or PBS was added into cell plate at 400 MOI and cells were kept in the incubator for 24 h. The medium was replaced with complete RPMI 1640 medium on day 3 and maintained for another 8 days. On day 11, cells were fixed with methanol for 10 min and stained with 1% crystal violet. After washing with running water, cell colonies with a diameter of more than 1 mm were counted.

Cell migration detection.

The migration ability of ESCC cells with/without Fn infection was measured by transwell assay. Briefly, 2 × 10⁵ ESCC cells with or without Fn infection for 6 h were resuspended in 2% FBS medium and seeded into the upper chambers of a 6.5 mm × 8.0 μm transwell (Corning, cat no. 3422). The bottom chambers contained 600 μL 20% FBS medium. After 40 h, the transwell chambers were sequentially washed with PBS, fixed with methanol, and stained with 0.1% crystal violet. After staining, the upper chambers were wiped gently with cotton swabs. Microscopy was used to image cells which migrated to the underside of the transwell membrane. Four fields were randomly selected, and the migrated cells were counted from those fields.

Live-cell imaging.

For live-cell imaging, 2 × 10⁵ KYSE70 cells were seeded in 6-well plates 24 h in advance, and logarithmic-phase Fn was added to the plates. Next, the coculture system was photographed by a BioTek Cytation 1 for more than 48 h.

Real-time PCR.

Real-time PCR was performed to measure the expression levels of differentially expressed genes of interest in ESCC cell lines. In brief, the extracted qualified total RNA was reversely transcribed into cDNA with commercialized kits (Vazyme, China), and then the prepared cDNA samples were subjected to real-time PCR using SYBR Green reagent (Vazyme, China) according to the manufacturer’s protocol. The primer sequences of targeted genes are listed in Table 1. All reactions were performed in triplicate with an Applied Biosystems StepOnePlus system. Data were normalized to PGK1 expression (ΔΔC_T analysis).

Polyamine detection by UHPLC-MS/MS.

The ESCC cells with/without Fn infection for 24 h were collected in 80% aqueous methanol. After ultrasonic treatment, the lysates were centrifuged, and the supernatant was dried and dissolved in 50 μL of 100 mM sodium carbonate. The chemical derivatization was initiated after adding 50 μL of 2% benzoyl chloride in acetonitrile. After derivatization, the sample was isometrically mixed with benzoyl-13C6 chloride-derivatized standards (as internal standards) prior to ultra-high-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) analysis. The UHPLC-MS/MS analysis was performed on an Agilent 1290 Infinity II UHPLC system coupled to a 6470A Triple Quadrupole mass spectrometer (Agilent, Santa Clara, United States).

The reagents used to prepare for standard solution, such as l-arginine, l-ornithine, putrescine, spermine, and spermidine, were purchased from Sigma-Aldrich. N1-acetylspermidine and N1-acetylspermine were purchased from Shanghai Yuanye Bio-Technology Co (Shanghai, China).

Statistical analysis.

Data are expressed as mean ± standard error of the mean. All experiments (except the live-cell imaging) were repeated three times independently. Statistical comparisons between two groups were analyzed by unpaired t test using GraphPad Prism 7.0. Statistical comparisons conducted in more than two groups were analyzed by two-way analysis of variance. P < 0.05 was considered to be statistically significant.

Data availability.

Data from this study have been deposited in Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA003868) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.

Other data and sources associated with this study are available from the corresponding author on reasonable request.