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

ABSTRACT Esophageal squamous cell carcinoma (ESCC) is a malignant upper digestive tract cancer, and its pathogenesis and etiology are poorly understood. Because gut microbes commonly impact progression, metastasis, and immunotherapy responses in colorectal cancer (CRC), the roles of the esophageal microbiota in ESCC have gradually drawn attention. As reported previously, Fusobacterium nucleatum (Fn), the notable “culprit” of CRC, can also influence the prognosis of ESCC in clinical studies. However, thus far, the underlying mechanism is unclear. In this study, 73 Chinese ESCC samples were collected. In those clinical samples, the abundance of Fn was found to be higher in tumors than in adjacent normal tissues, and a high abundance of Fn was correlated with shorter survival. Furthermore, using in vitro experiments, we demonstrated that Fn can invade ESCC cells, enhancing their proliferation capacity. The mechanism study revealed that Fn can produce high levels of putrescine after invasion, which disturbs polyamine metabolism and promotes the malignant proliferation of ESCC cells. In conclusion, Fn infection was found in Chinese ESCC tumor tissue samples and may promote ESCC progression by disturbing the polyamine metabolism pathway. IMPORTANCE Nowadays, the complex and varied interactions between microbes and human body are known to be crucial for maintaining the health of the human body. However, knowledge concerning the influence of esophageal microbes on the progression of esophageal squamous cell carcinoma is limited. Here, in our study, we confirmed that F. nucleatum can invade ESCC cells and consequently promote their proliferation, suggesting that esophageal microbes likely influence the progression of ESCC in clinical settings. Because the esophagus connects the oral cavity and stomach, acting as a canal for transporting foods, its special physical location makes it easily exposed to microorganisms. Thus, it is necessary to explore the interaction between esophageal microbes and ESCC.


Reviewer #3 (Comments for the Author):
Through a series of in vitro experiments, Ding and colleagues found that Fn could invade ESCC cells and further enhance the proliferation ability of ESCC cells. In addition, the expression levels of CXCL1, IL-6 and IL-1β in ESCC cells were significantly increased after Fn infection. I hope the authors may find some of the following suggestions useful: Major points: 1.There is no conclusion description in the abstract.
2.There is no validation and molecular biological rescue experiments For RNA-seq results regulated by Fn. 3.What is the specific pathway of Fn invasion into cells? It should be discussed or validated accordingly 4.For the change of tumor cell function caused by Fn, it is not enough to only observe the proliferation ability. Does it affect the invasion, migration and apoptosis of cells? 5. The abundance of Fn in clinical patients should be supplemented. Is there a correlation between Fn level and clinicopathological parameters? Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.

Introduction
Esophageal cancer is the seventh most diagnosed and the sixth leading cause of cancer deaths worldwide (1). More than 252500 new cases and 193900 annual deaths occurring in China (2). Majority of these cases are diagnosed as Esophageal squamous cell carcinoma (ESCC), one of the subtypes of EC (3). Although, alcohol consumption, cigarette smoking, poor oral hygiene, intake hot foods and pickled vegetables, and genetic mutation have been associated with increased risk of ESCC(4-6), the underlying etiology and pathology still not clear.
Billions of bacterium are co-existing in/on human body (7). Among them gut microbiota has been studied most thoroughly. Robust studies provided the important role of gut microbiota in cancer development (8), progression(9, 10), metastasis (11)(12)(13), and even immunotherapy response (14,15). Same as the gut, esophageal connects oral cavity and stomach, acts as a canal for transporting foods. Its special physical location makes it easy exposing to microorganism. Indeed, diverse bacteria can be detected in clinical esophageal samples by 16s-rDNA sequencing or shot-gun sequencing, including Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria and Fusobacterium (16,17). However, little was known about the impacts of those local bacterium on ESCC progression.
Fusobacterium nucleatum (Fn) is one of the Gram-negative anaerobic bacteria commonly detected in the dental plaque and intestinal tract of human body (18). Fn is frequently considered as an etiological agent of colorectal cancer (CRC) via multiple functional pathways (such as secreting bioactive metabolites, inducing DNA damage and disturbing immune system) (14,(19)(20)(21). As demonstrated, Fn can promote chemoresistance in CRC by targeting TLR4 and MYD88 innate immune signaling and downregulation miR-18a* and miR-4802 subsequently activation autophagy (22).
Fn also can targets lncRNA EN01-IT1 to promote glycolysis and oncogenesis in CRC (19). As for ESCC, clinical studies reported Fn can also be detected in the clinical esophageal samples (23)(24)(25)(26). Those Fn positive patients are associated with shorter survival and poorer response to neoadjuvant chemotherapy, but the underlying mechanism are still not clear.
In the present work, we found that Fn can penetrate and promoted the proliferation of ESCC cells. Along with Fn invading, there were 700 genes expressed differentially, among them there were 493 genes with increased expression levels and 207 genes with decreased expression levels. Those differentially expressed genes (DEGs) were mainly enriched in immunity relative signaling pathways, including the TNF signaling pathway, the IL-17 signaling pathway, the cytokine-cytokine receptor interaction pathway, and the chemokine signaling pathway. Concordantly, the dramatically elevated expression of cytokines were verified by Real-time PCR, such as IL-6, IL-1β and CXCL1. Additionally, the expression of ESCC progression relative genes, such as CCND1, NFE2L2, HAS3, DKK1 were changed significantly. Taken together, this study demonstrates Fn can invade into ESCC cells and promote the proliferation of ESCC cells along with elevated expression of cytokines (CXCL1, IL-6 and IL-1β ) and disturbed expression of some ESCC progression associated genes (DKK1, HAS3, NFE2L2 and CCND1).

Fn infection induces ESCC cell malignant proliferation
To further figure out the influence of Fn infection in ESCC cells, KYSE70 and TE-5 cells were incubated with Fn at MOI400 for 24 hrs. As shown by CCK8 assay, Fn infection effectively promoted cell viability, whereas the heat killed Fn and another anaerobe Bacteroides acidifaciens (Ba) have 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 in KYSE70 (Fig. 2B). In addition, with the living cell imaging system, it is obvious that KYSE70 cells are proliferating faster after Fn infection compared to cells without Fn infection (Movie S1, S2). Same as the TEM results, Fn that at the bottom of the petri dish were disappeared along with the movement and division of KYSE70 cells (Movie S2), which indicated that Fn invaded/adhered/swallowed into KYSE70 cells. Taken together，Fn can not only invade into ESCC cells but also promote cell proliferation.

RNA-seq reveals a pro-inflammation signature associated with Fn infection
To further delineate the potential mechanisms underlying Fn infection induced cell proliferation changing, KYSE70 and KYSE70-Fn were collected simultaneously for RNA-sequencing. 700 DEGs were identified with more than two-fold expression change (FDR<0.05). Among them, there were 207 genes with decreased expression levels, and 493 genes with increased expression levels (Fig. 3A). Those DEGs significantly enrichened in the pathways related to immunity, such as TNF signaling pathway, IL-17 signaling pathway, cytokine-cytokine receptor interaction pathway and chemokine signaling pathway by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. 3B). The up-regulation expression levels of proinflammatory cytokines IL-1β, CXCL1 and IL-6 were verified by Real-time PCR (Fig.   3C). Indicating that Fn invasion provoke a pro-inflammation signature in the ESCC cells. Otherwise, the expression levels of ESCC progression relative genes, including CCND1, DKK1 (27), NFE2L2, HAS3 (28) were also verified by Real-time PCR (Fig.   3D). All these changes might be conducive to the rapid proliferation of ESCC cells. Numerous and variety microbiota are existed in/on human body with complicated physiological effects and can form different ecological communities in response to different lifestyle, location, and health condition of the host (7,32,33). Gut microbiota, the most well-known microbiota ecosystem in human body, can play roles in the development(8), progression(9, 10), metastasis (11)(12)(13), and immune therapy (14) of CRC and even some distal carcinoma(15, 34). Otherwise, tumor tissues which were used to be considered as sterile (such as breast cancer and pancreatic cancer) were also colonized by bacteria (13,35). Esophageal connects oral cavity and stomach, acts as a canal for transporting foods. Its special physical location makes it easy exposing to microorganism (17). We supposed some of the microbiota can interact with tumor microenvironment and disturb the ESCC progression.  (27). The HAS3 is a member of the hyaluronan synthase family, the products of HAS3 is responsible for secreting the growing hyaluronan polymer into the extracellular. As reported, HAS3 is overexpressed in ESCC clinical samples, and tumor growth can be inhibited by HAS3 knock down in ESCC cells (28). Similarly, the CCND1 and NFE2L2 were also demonstrated can influence the progression of ESCC (6). Taken together, those changes may also contribute to the proliferation of ESCC cells after Fn infection.
It has been reported that Fn can function by activating the TLR4 and MYD88 pathway in CRC cells (22), but in our study the expression levels of TLR4 and MYD88 were not changed (data not shown). Thus, Fn might work through other pathways in ESCC cells, which need to be explored in the following project.

Supplemental legend
Movie S1. Proliferation of KYSE70 without Fn infection.

Materials and methods
Cell culture Human ESCC cell lines KYSE70 and TE5 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin Solution (PS). Cells were incubated in the cell incubators at 37°C and 5% CO2 condition. Colony formation assay 600 cells were seeded in 60mm Petri Dish (Corning) containing complete RPMI1640 on day 1. The medium was replaced with PS-free cell culture medium on day 2, then Fn or PBS were added into cell plate at MOI400 and kept in the cell incubator for 24h. The medium was replaced with complete RPMI1640 medium on day 3 and maintained for another 8 days. On day 11, cells were fixed with methanol for 10 mins and stained with 1% crystal violet. After washing with running water, the cell colonies with more than 1mm diameter were counted.
Living cell imaging 2×10 5 KYSE70 cells were seeded in 6-well plates 24 hrs in advance, and the logarithmic phase Fn were added into the plates at MOI400. Then the co-culture system was photographed by BioTek Cytation 1 for more than 48hrs.  Table 1. All reactions were performed in triplicate with an Applied Biosystems StepOnePlus system. Data were normalized to PGK1 expression (ΔΔCT analysis). Statistical Analysis Data are expressed as mean ± SEM. All the experiments (except the living cell imaging) were repeated three times independently. Statistical comparison between two groups were analyzed by unpaired t-test by GraphPad Prism 7.0. Statistical comparison conducted in more than two groups were analyzed by Twoway ANOVA. p<0.05 was considered to be statistically significant. In order to explore the mechanism of Fn on esophageal cancer, the ESCC cell line and Fn were co-cultured in this study. Two conclusions were obtained:

Author's contribution
1. Fn can invade ESCC cells, thus enhancing the proliferation of ESCC cells.

After Fn infection, gene expression in ESCC cells was changed, and gene
expression related to inflammation related signals and cancer signals was upregulated.
There are few studies on the correlation between Fn and esophageal cancer, and its corresponding mechanism is not clear. The topic of this study is very meaningful, and the language description of the manuscript is clear.
However, the mechanism of Fn and the proliferation of esophageal cancer has not been clearly proposed. It is suggested to supplement the corresponding data to better understand the pro-proliferative effect of Fn in esophageal cancer.
The suggested supplementary data are as follows: 1. Why is there a significant difference in the morphology of Fn in Fig. 1

Dear reviewers,
We are very grateful to your encouraging and thoughtful comments and suggestions regarding our original submission. In response to these comments, we have made a number of modifications to our manuscript and performed additional suggested experiments. Below we detail these specific modifications with the specific comments from the reviewers followed by our response. We hope the reviewers will find this manuscript improved following these changes and more suitable for publication in Microbiology Spectrum.

Reviewer #1 (Comments for the Author):
Fusobacterium In order to explore the mechanism of Fn on esophageal cancer, the ESCC cell line and Fn were co-cultured in this study. Two conclusions were obtained: 1. Fn can invade ESCC cells, thus enhancing the proliferation of ESCC cells. 2. After Fn infection, gene expression in ESCC cells was changed, and gene expression related to inflammation related signals and cancer signals was up regulated.
There are few studies on the correlation between Fn and esophageal cancer, and its corresponding mechanism is not clear. The topic of this study is very meaningful, and the language description of the manuscript is clear. However, the mechanism of Fn and the proliferation of esophageal cancer has not been clearly proposed. It is suggested to supplement the corresponding data to better understand the pro-proliferative effect of Fn in esophageal cancer.
The suggested supplementary data are as follows:

Q1.
Why is there a significant difference in the morphology of Fn in Fig. 1? How can you prove that the marker is a bacterium, not a cell structure or other components?
Response 1: Indeed, we can see different morphology of Fn in Fig.1 (Fig. 3A), because cell is a three-dimensional structure, the bacteria were random distribution in the cells, and the picture were photographed from one layer of the cell, thus we can see different morphology. And they are identified as Fn bacteria, because we can see the "cell wall" in light gray at the margin of those bacteria (Fig. S1). In order to better clarify that Fn can invade into ESCC cells, the classic "differential immunofluorescence experiment" were carried out (   Q2. In Fig2b, Does Bacteroides acidifaciens also enter the cell?

Response 2:
The cells were collected after co-culture with Bacteroides acidifaciens (Ba) for 24h, and the TEM result presented that Ba can't entry into ESCC cells. (Fig.  S2).

Fig. S2. Bacteroides acidifaciens (BA) cannot invasion into ESCC cells. BA-infected cells were detected by transmission electron microscopy. Scale bar = 20μm.
Q3. In this study, there is only co-culture model in vitro, and corresponding animal experiments should be supplemented to observe whether Fn promotes the proliferation of esophageal cancer in vivo; Response 3: To be honest, I quite agree with you that the in vivo experiments should be done to better understand the influence of Fn infection during tumor progression and metastasis. Unfortunately, due to Fn is a pathogenic bacterium, we have consulted 3 available animal experimental centers, Fn relative experiment were not permitted. And this limitation was discussed in the final part of Discussion. Hope you will understand this problem.

Q4.
To clarify the specific target and corresponding mechanism of Fn promoting the proliferation of esophageal cancer; In our study, we found that the SAT1 were highly expressed after Fn infection (Fig.  4A,) and the relative expression level of SAT1 were increased gradually with time extension after Fn invasion (Fig. 4B), indicating the SAT1 may take part in the Fn-induced cell proliferation. As the SAT1 encodes an rate-limiting enzyme in the

Fig. S3. Fn infection didn't impact the apoptosis of ESCC cells. The apoptosis of ESCC cells with/without Fn infection were collected and detected by flow cytometry. The rates of early apoptosis and late apoptosis cells were analyzed (right).
Q5. The abundance of Fn in clinical patients should be supplemented. Is there a correlation between Fn level and clinicopathological parameters?

Response 5:
To answer this question, we collected 73 ESCC samples with paired tumor and adjacent normal tissues from Shanxi province, China. We found Fn were more abundant in tumor tissues and were significantly corelated with shorter survival (New Fig 1A and B).