Bacteroidetes promotes esophageal squamous carcinoma invasion and metastasis through LPS-mediated TLR4/Myd88/NF-κB pathway and inflammatory changes

Gut microbiota plays a crucial role in gastrointestinal tumors. Additionally, gut microbes influence the progression of esophageal cancer. However, the major bacterial genera that affect the invasion and metastasis of esophageal cancer remain unknown, and the underlying mechanisms remain unclear. Here, we investigated the gut flora and metabolites of patients with esophageal squamous cell carcinoma and found abundant Bacteroides and increased secretion and entry of the surface antigen lipopolysaccharide (LPS) into the blood, causing inflammatory changes in the body. We confirmed these results in a mouse model of 4NQO-induced esophageal carcinoma in situ and further identified epithelial–mesenchymal transition (EMT) occurrence and TLR4/Myd88/NF-κB pathway activation in mouse esophageal tumors. Additionally, in vitro experiments revealed that LPS from Bacteroides fragile promoted esophageal cancer cell proliferation, migration, and invasion, and induced EMT by activating the TLR4/Myd88/NF-κB pathway. These results reveal that Bacteroides are closely associated with esophageal cancer progression through a higher inflammatory response level and signaling pathway activation that are both common to inflammation and tumors induced by LPS, providing a new biological target for esophageal cancer prevention or treatment.


Bacteroides induce inflammatory changes via LPS in patients with ESCC
We revealed that the alphadiversity (species richness) of gut flora species in the two populations demonstrated a reasonable progressive amount of sample sequencing data, and the betadiversity (microbial composition) exhibited a large difference in gut microbial structure between the two populations (Fig. S1A-B).We used the LEfSe analysis of effect size method to determine differentially enriched species and identified Family Bacteroidaceae, Order Bacteroidales, Class Bacteroidia, and Order Lactobacillales in the fecal flora of patients with ESCC compared to healthy controls.Additionally, Family Peptostreptococcaceaeg and Order Peptostreptococcales Tissierellaleh increased in abundance, whereas Family Ruminococcaceae and Order Veillonellales Selenomonadales decreased in abundance, with a clear dominance of gram-negative Bacteroides (Fig. 1A).More specifically, we observed an increased abundance of Bacteroides with ESCC progression (Fig. 1B).Moreover, we detected LPS levels in the serum of clinical subjects using enzyme-linked immunosorbent assay (ELISA) and revealed significantly elevated LPS in the serum of patients with esophageal cancer (Fig. 1C).We further analyzed the data using Spearman's correlation analysis to elucidate the relationship between Bacteroides and LPS and found that LPS was significantly and positively correlated with Bacteroides spp.(R = 0.4599, P < 0.001) (Fig. 1D).
Meanwhile, changes in the levels of endogenous bioactive cytokines, including IL-6, IL-10, TNF-α and TGF-β, which are usually diagnosed as indicators of cancer progression, were further assessed by ELISA of serum IL-6, IL-10, TNF-α, and TGF-β levels in clinical subjects, and we found significant differences in serum inflammatory cytokines in patients with ESCC compared with healthy controls (Fig. 1E).www.nature.com/scientificreports/

Inflammatory changes induced by Bacteroides via LPS in mouse ESCC models
We developed a 4NQO-inducible mouse model of ESCC to further investigate the association between fecal microorganisms and serum inflammatory factors.Figure 2A shows esophageal carcinoma in situ and invasive carcinoma in mice at week 32.The fecal flora of experimental mice was investigated using 16S rRNA technology.Alphadiversity (species richness) of mouse fecal flora species demonstrated a progressively reasonable amount of sequencing data from the samples, and betadiversity (microbial composition) exhibited large differences in the structure of gut microorganisms between the two groups of mice (Fig. S2A-B).Among them, Family Bacteroidaceaeb, Family Prevotellaceaec, Family Lachnospiraceaeh, Order Lachnospiralesi, Family Oscillospiraceaej, Family Ruminococcaceaek, Order Oscillospiralesl, Class Clostridium, Family Enterobacteriaceae with increased abundance of Order Enterobacterales, Family Erysipelotrichaceaed, Order Erysipelotrichalese, Family Clostridiaceaef, and Order Clostridialesg decreased in abundance.Interestingly, Bacteroides abundance was significantly higher in the resolving flora of the model mice than in the control group, which is similar to the findings of the clinical trial (Fig. 2B-C).Meanwhile, the serum LPS level of mice in the model group was significantly increased (Fig. 2D), and Spearman's correlation analysis revealed that LPS was significantly and positively correlated with Bacteroides (R = 0.8977, P < 0.0001) (Fig. 2E).Moreover, compared with the normal controls, significant changes in serum IL-6, IL-10, TNF-α, and TGF-β levels were observed in the ESCC mouse model and were consistent with the changes in inflammatory cytokines in patients with ESCC (Fig. 2F).

Gut barrier damage increases circulating LPS
Immunohistochemistry (IHC) was used to detect mouse intestinal barrier-associated proteins, zonula occludens 1 (ZO-1) and occludin.The results revealed a significant decrease in ZO-1 and occludin-positive staining in the model group compared to that in the control group (Fig. 3).This indicates a damaged intestinal barrier in mice, increased permeability, and elevated LPS entry into the blood through the intestinal mucosal barrier.

Increased tumor proliferation and EMT and activation of TLR4/Myd88/NF-κB signaling pathway in model mice
IHC revealed that the proliferation marker protein Ki67, invasion and metastasis marker proteins N-cadherin, and snail of ESCC tumor tissue were significantly increased in the model group (Fig. 4A).Additionally, IHC detected the expression and distribution of TLR4, Myd88, and NF-κB.The three indicators demonstrated increased expression in esophageal epithelial tissue compared to that in the control group (Fig. 4B).This indicates that EMT progression in mouse esophageal cancer may be associated with the TLR4/Myd88/NF-κB signaling pathway.

LPS promotes KYSE-150 cell proliferation and migration invasion
Studies have revealed that Pseudomonas fragilis is the most pathogenic of the genus Pseudomonas 8 .We obtained LPS for in vitro experiments by culturing P. fragilis.We used different LPS concentrations (0, 125, 250, and 500 ng/mL) to coculture with KYSE-150 cells for 6, 12, and 24 h.The proliferative ability of KYSE-150 cells was significantly improved after LPS treatment dose-and time-dependent manner (Fig. 5A).Additionally, LPS of 500 ng/mL was used to treat KYSE-150 cells for 12 and 24 h to observe their migration and invasion abilities.The results revealed significantly improved migration and invasion abilities of KYSE-150 cells in the LPS intervention group compared with the control group (Fig. 5B-E).

LPS promotes EMT in KYSE-150 cells and activates the TLR4/Myd88/NF-κB pathway
We investigated the expression of key EMT proteins in LPS-treated KYSE-150 cells and found that the epithelial marker E-cadherin was significantly downregulated, and the mesenchymal markers N-cadherin and snail were significantly upregulated at both 12 and 24 h (Fig. 6A, C).We explored the molecular mechanisms by which LPS promoted KYSE-150 cell proliferation, migration, and invasion.We examined the expression of TLR4/Myd88/NF-κB pathway-related proteins after LPS treatment of KYSE-150 cells.Western blot analysis revealed significantly elevated TLR4 and myd88 at 12 versus 24 h, and p-NF-κBp65 was significantly increased at 24 h.However, NF-κBp65 exhibited no significant changes.This indicated that the TLR4/Myd88/NF-κB pathway was activated by LPS in the order of signal transduction (Fig. 6B, D).

Discussion
Gut microbiota plays an important role in cancer development and is frequently characterized by altered microbiota in cancer.Gut microbiota alterations play an important role not only in colorectal cancer 9 but also in extraintestinal tumors, including melanoma 10 , hepatocellular carcinoma 11 , and gastric cancer 12 .A related study on esophageal cancer by Natasha et al. revealed that a high-fat diet accelerates esophageal allopatric hyperplasia by altering the esophageal microenvironment and gut microbiome 5 .Additionally, Qunate et al. revealed that metabolites of intestinal microorganisms are important mediators in accelerating the progression of esophageal cancer 13 .
Previous studies revealed the involvement of microbiota changes in the progression of ESCC in patients with esophagitis and ESCC 14 .We performed microbiota analysis of fresh feces from patients with ESCC using the 16S rRNA technique to investigate the critical role of major strain changes and their metabolites in ESCC development, and the results revealed a significantly elevated abundance of gram-negative bacteria, Bacteroides, in all cases.Interestingly, both gastric and colorectal cancers demonstrated a significant increase in the abundance of Bacteroides 15,16 .Our step studies revealed a positive correlation between serum LPS and Bacteroides abundance in patients with ESCC and the ESCC mouse model.
Meanwhile, experiments revealed an increased inflammatory infiltration level in the rectal tissues of mice in the model group and decreased expression of tight junction proteins, occluding, and ZO-1.Occludin and ZO-1 are the most abundant components of intercellular tight junction formation 17 .This indicates intestinal barrier damage and increased intestinal permeability in mice 18,19 .LPS can not only damage the intestinal tract but also trigger bacterial translocation, impair the intestinal barrier, and lead to abnormal expression of intestinal tight junction proteins.This allows LPS to penetrate the intestinal wall 20 .The increase in LPS in the blood further activates the host immune response 21 , dysregulating the cancer-inflammatory microenvironment, which plays an important role in extraintestinal diseases 22,23 .Therefore, we hypothesized that the increased abundance of www.nature.com/scientificreports/Bacteroides in the intestinal flora plays an important role in ESCC development and that its surface antigen-LPS may be a key factor.
Our study revealed that elevated serum IL-6, TNF-α, and TGF-β and decreased IL-10 levels revealed that patients with ESCC and model mice demonstrated increased immune levels, and these inflammatory cytokines are considered important mediators associated with inflammation and cancer.Imbalances in pro-and antiinflammatory signaling factors promoted inflammatory cancer transformation and cancer progression 24 .IL-6 is considered a pleiotropic molecule that promotes tumor transformation and can, particularly, promote tumor cell invasion and metastasis 25 .IL-10 is involved in suppressing inflammatory responses, promoting tissue repair, and maintaining immune tolerance 26 .Under normal conditions, IL-10 is involved in maintaining immune tolerance in the gut microbiota 27 , which limits intestinal inflammation and tumor formation 28 .Many studies revealed that the effects of TNF-α and TGF-β include the promotion of EMT invasion and metastasis [29][30][31][32] .
Research revealed that LPS generated a broad inflammatory cascade response by binding to TLR4 33 .TLR4 is not only a key hub that connects innate and adaptive immunity but also a sensor for recognizing microbial ecological dysregulation, which reflects the dysregulation of the body's immunity as well as the microbiota and is closely related to tumors 34,35 .Sato et al. revealed that high TLR4 expression predicts poor prognosis in patients with advanced thoracic ESCC after esophagectomy 36 .Upon LPS stimulation, TLR4 uses MyD88 junction protein to transmit signals that activate NF-κB and induce inflammatory cytokines 37,38 .Recent studies revealed that TLR4/Myd88/NF-κB is a signaling pathway that improves IL-6, TNF-α, and TGF-β production [39][40][41] .The results of this study indicate significantly increased protein expression of key nodes of the TLR4/Myd88/NF-κB pathway in the esophagus of mice modeled for esophageal cancer in situ, and the same changes in protein expression were observed in esophageal cancer cells after LPS treatment in vitro.This indicates that the TLR4/Myd88/NF-κB signaling pathway is involved in esophageal cancer disease progression and is closely associated with LPS.www.nature.com/scientificreports/EMT is abnormally activated in human cancers, providing a special capacity for tumor cell movement that contributes to tumor cell invasion 42 .Some evidence indicates that the TLR4/Myd88/NF-κB signaling pathway, as well as inflammatory cytokines, influenced the EMT process [43][44][45][46] .We performed in vivo and in vitro experiments to further validate the effect of LPS on the EMT process in esophageal cancer and revealed the accelerated EMT process progression in ESCC, which provides invasive and migratory properties to cancer cells.
This study has several limitations.The human gut microbiota is a diverse ecosystem that is closely related to cancer; however, we only investigated the promotional effects and possible mechanisms of Bacteroides, which were the most significant differences in our study.And the roles of gut microorganisms in ESCC still need to be collected from more clinical samples and experimental data and investigated in depth.Additionally, due to ethical issues, we were unable to obtain intestinal tissue samples from esophageal cancer patients and normal volunteers to compare the extent of intestinal barrier disruption between the two groups.

Conclusions
Overall, the increased abundance of Bacteroidetes was associated with ESCC development.Additionally, Bacteroidetes may further initiate the EMT process through LPS-mediated alteration of the inflammatory environment and TLR4/Myd88/NF-κB signaling pathway, thereby promoting the invasion and metastasis of ESCC (Fig. 7).

Clinical samples and ethical statement
This study included 30 patients newly diagnosed with esophageal cancer (ESCC group) and 30 healthy controls from the Department of Physical Examination (control group) of the Fourth Hospital of Hebei Medical University in 2021.Inclusion criteria 47 were as follows: (1) patients aged > 18 years, (2) patients with primary esophageal cancer, (3) patients who had not received radiotherapy, chemotherapy, and/or surgery, and (4) all healthy controls with normal bowel habits.Exclusion criteria 48 were as follows: (1) patients with diabetes and depression, (2) patients who had taken antibiotics, H2 receptor antagonists, proton pump inhibitors, and probiotics within the past 1 month, (3) patients with other cancers, and 4) dental bacterial diseases.The study was conducted in strict accordance with the protocol approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University, and all subjects provided written informed consent (2022KY057).The ESCC group referred to the "Chinese Society of Clinical Oncology (CSCO) Esophageal Cancer Diagnosis and Treatment Guidelines-2020" developed by the CSCO, and diagnosed squamous cell carcinoma of the esophagus by histopathology.

Figure 3 .
Figure 3. Disruption of the intestinal barrier and increased permeability in esophageal cancer mice.Histopathological changes in the rectum were examined by H&E under a microscope, H&E: hematoxylin and eosin.ZO1 and Occludin staining of mouse rectum tissue, brown for positive staining; ZO1 and Occludin: Mouse intestinal barrier-associated proteins.Observed under a 40 × microscope.

Female C57BL/ 6
mice (6 weeks old) were purchased from Beijing Wei Tong Li Hua Laboratory Animal Technology (License number: SCXK [Beijing] 2016-0011).The mice were housed in the Experimental Animal Center of the Fourth Hospital of Hebei Medical University (SPF level) following national and international guidelines.The Institutional Laboratory Animal Care Guidelines of the Fourth Hospital of Hebei Medical University approved the study (IACUC-4th Hos Hebmu-2022,003).

Figure 7 .
Figure 7. Schematic representation of the mechanism by which Bacteroidetes promotes migratory invasion of esophageal cancer cells.

Table 1 .
Descriptive data of included subjects in the study.