Bioinformatics and immunohistochemistry analyses of expression levels and clinical significance of CXCL2 and TANs in an oral squamous cell carcinoma tumor microenvironment of Prophyromonas gingivalis infection
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- Published online on: January 6, 2021 https://doi.org/10.3892/ol.2021.12450
- Article Number: 189
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Copyright: © Guo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck (1), and is associated with high incidence, invasion and metastasis rates, as well as a poor prognosis. In the United States, ~50,000 individuals suffered from OSCC and 8,000 individuals died in 2013 (2). OSCC has gradually become a serious problem worldwide, despite the increase in basic research and the rapid development of clinical treatment in the past few decades. Furthermore, the 5-year survival rate of this disease has not significantly improved (3). With the identification of a causal link between Helicobacter pylori (H. pylori) infection and the occurrence of gastric tumors in the 1900s (4), an increased understanding has been achieved regarding the association between bacteria and tumors. Nevertheless, how oral microorganisms influence the tumor microenvironment (TME), and how they promote tumor development during tumor progression, remains unknown (1). It has been reported that a large number of pro-inflammatory cytokines secreted by microorganisms can transform tumor cells into more aggressive phenotypes by regulating oncogenes (5). Tumor growth and metastasis are not only affected by neoplastic cells, but also by the TME (6). Surgery combined with chemotherapy is an effective treatment for OSCC (1). However, chemotherapy is not a first-line therapy for OSCC, since most OSCC cases develop resistance to chemotherapeutic reagents (7). Periodontitis has been suggested to be associated with the TME of OSCC, which could be involved in the development of chemotherapy resistance in OSCC (7). Therefore, further investigations examining the regulatory mechanism of the TME and identifying novel biomarkers to optimize patient selection for this therapy have gained clinical significance and theoretical value.
Porphyromonas gingivalis (P. gingivalis) is a key pathogen in periodontal disease, and is known as an independent microorganism risk factor for increased tumor mortality (8). P. gingivalis is a special oral pathogen and a potentially independent microbiological factor that increases the risk of mortality in patients with OSCC (9). A previous study revealed the presence of the P. gingivalis antigen in the tissues of gingival squamous cell carcinoma (10). An abnormal increase in the number of P. gingivalis is an important factor in causing the imbalance of the local microecology of the oral cavity (7). The dominant bacterial pathogen in periodontitis is P. gingivalis, which affects the condition of the TME, increasing the likelihood of OSCC development (11).
Chemokines are a small molecular protein family that serve a role in chemical signaling during cell activation and differentiation, as well as in the process of movement. Furthermore, chemokines and their signaling receptors are important in the TME of OSCC (12). According to a previous study, P. gingivalis induces immune cells to secrete chemokines [including C-X-C motif chemokine ligand (CXCL)1, CXCL2, CXCL5 and CXCL8] and promotes tumor growth (13). CXCL2 is a type of small molecular pro-inflammatory chemokine and a member of four subfamilies (C, CC, CXC and CX3C), serving a role in coding protein secretion, immune regulation and the promotion of tumor angiogenesis (14). CXCL2 is highly expressed in breast, colon, prostate and liver tumors, and is closely associated with tumor growth, invasion and distant metastasis (15). However, the other biological functions of CXCL2 in the P. gingivalis-infected TME of OSCC remain unknown.
Polymorphonuclear neutrophils (PMNs) are the primary defense line of the immune system, and are the most abundant white blood cells in the peripheral blood circulation (16). Tumor-associated neutrophils (TANs) are infiltrates in the TME, and there are two polarized manifestations of TANs: Defined as the N1 phenotype, one has the ability of typical tumor inhibition, while another, defined as the N2 phenotype, has typical tumor promotion, the differentiation of which depends on the factors of the TME (17,18). In addition, CD66b+ has been assessed as a PMN marker in gastric tumors using immunohistochemistry (IHC), and CD66b+ has been revealed to be a reliable marker to identify the phenotype of TANs that promote tumor growth (19). It has also been revealed that TANs are primarily activated by CXC chemokines (20).
In order to analyze the immunoexpression and clinical significance of CXCL2 and TANs in the TME of P. gingivalis infection, the immunohistochemistry evaluation, bioinformatics analyses and statistical analysis were performed.
Materials and methods
Patient selection
The present study included 205 surgical specimens from patients [age range, 36–90 years; mean age, 63 years; 103 men (50.2%); 102 women (49.8%); 76 patients <60 years (37.1%); 129 patients ≥60 years (62.9%)] who underwent surgical treatment for primary OSCC at the Oncology Department of Oral and Maxillofacial Surgery of The First Affiliated Hospital of Xinjiang Medical University (Urumqi, China) between March 2007 and March 2019. Surgical resection was performed in all 205 patients with OSCC. The TNM and clinicopathological classification and staging of patients with OSCC were performed according to the American Joint Committee on Cancer (AJCC) guidelines (21). The inclusion criteria were as follows: i) patients with OSCC located in the tongue, buccal cavity, lip, floor of the mouth, or gingival and retromolar area as confirmed via biopsy; ii) patients who did not undergo any treatment before; and iii) patients with completed clinical data and follow-up. Clinical data, including age, sex, survival status, differentiation, tobacco and alcohol consumption, TNM stage, clinical stage, recurrence, periodontal condition, treatment (surgery, radiotherapy and/or chemotherapy, post-operative adjuvant radiotherapy or chemotherapy) and tumor size, were collected. In addition, the present study included 20 cases of benign tumor and non-tumor patients [age range, 23–48 years; mean age, 33 years; 10 men (50.0%); 10 women (50.0%)] who underwent surgical treatment at the Oncology Department of Oral and Maxillofacial Surgery of The First Affiliated Hospital of Xinjiang Medical University (Urumqi, China) between May 2017 and May 2019. The inclusion criteria were as follows: i) Patients with benign tumors located in the oral cavity; ii) patients with benign tumors located in the parotid gland, sublingual gland and submandibular gland; iii) patients with inflammatory lesions requiring surgical removal. The present study was approved by the ethics review board of the First Affiliated Hospital of Xinjiang Medical University (Urumqi, China) at the original time of data collection (approval no. IACUC20180411-13) and written consent was obtained at the original time of data collection.
Bioinformatics analyses
Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) is a public functional genomics data repository of high-accuracy gene expression data, chips and microarrays. A total of two gene expression datasets, GSE87539 (22) and GSE138206 (23), were downloaded from GEO (Affymetrix GPL570 platform; Affymetrix Human Genome U133 Plus 2.0 Array; Thermo Fisher Scientific, Inc.). The probes were converted into the corresponding gene symbols according to the annotation information in the platform. The GSE87539 dataset contained 3 samples of non-infected oral epithelial cells and 3 samples of P. gingivalis-infected oral epithelial cells. The GSE138206 dataset contained 6 OSCC samples and 6 non-cancerous samples.
The differentially expressed genes (DEGs) between GSE87539 and GSE138206 samples were screened using GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r). GEO2R is an interactive web tool that allows users to compare ≥2 datasets in a GEO series to identify DEGs across the experimental conditions. Hierarchical clustering of hub genes was performed using UCSU Cancer Genomics Browser (http://genome-cancer.ucsc.edu) (24). The adjusted P-values (adj. P) and Benjamini and Hochberg false discovery rate were applied to provide a balance between the discovery of statistically significant genes and limitations of false-positives. The probe sets without corresponding gene symbols or genes with >1 probe set were removed or averaged, respectively. Log fold-change >1 and adj. P<0.01 were considered to be statistically significant.
IHC evaluation
A total of 205 OSCC samples from patients were immersed in 10% neutral buffered formalin for fixation for 3 days at room temperature. A total of 205 paraffin-embedded OSCC samples from patients and consecutive 4-µm-thick sections cut from paraffin blocks were used for IHC evaluation. The expression levels of P. gingivalis were evaluated, and 119 samples exhibited strong expression. These 119 samples were used in the evaluation of the expression levels of CXCL2 and TANs. Non-cancerous samples were obtained from patients who suffered from benign tumors or inflammatory disease at the Oncological Department of Oral and Maxillofacial Surgery, The Affiliated Stomatology Hospital of The First Affiliated Hospital of Xinjiang Medical University (Urumqi, China) and maintained at the Institute of Stomatology of Science and Technology Building of the First Affiliated Hospital of Xinjiang Medical University (Urumqi, China). The IHC procedure was as follows: Slides were heated at 65°C for 2 h. Sections that adhered to the slides were deparaffinized in xylene and rehydrated in a gradient ethanol (50% ethanol for 10 min, 70% ethanol for 10 min, 80% ethanol for 10 min, 95% ethanol for 10 min, 100% ethanol for 10 min three times), followed by submerging into EDTA antigenic retrieval buffer, treatment with 3% hydrogen peroxide for 15 min at room temperature and incubation with 1% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at 37°C. The samples were then incubated with anti-P. gingivalis (dilution, 1:200; Dia-An, lnc; this antibody is not a commercial product, and it was prepared at the time), anti-CXCL2 (dilution, 1:500; cat. no. bs-1162R; BIOSS) and anti-TANs (CD66b+; dilution, 1:500; cat. no. ab197678; Abcam) antibodies overnight at 4°C. After being washed with PBS with 1% Tween, the slides were incubated with secondary antibodies [Goat Anti-Mouse IgG H & L (HRP); dilution, 1:2,000; cat. no. ab205719; Abcam; and Goat Anti-Rabbit IgG H & L (HRP); dilution, 1:2,000; cat. no. ab205718; Abcam] for 1 h at 37°C, followed by incubation with streptavidin horseradish peroxidase complex (Thermo Fisher Scientific, Inc.). The sample sections were then immersed in 3,3′-diaminobenzidine for 5 min at room temperature, counterstained with 10% Mayer's hematoxylin for 1 min at room temperature, dehydrated and mounted.
Each tumor specimen was observed by light microscopy and examined under 10 microscopic fields with a digital camera (AxiocamMRc; Zeiss AG) attached to a microscope (magnification, ×200; Axioskop 2 Plus Zeiss AG) to evaluate the immunoexpression levels of P. gingivalis, CXCL2 and TANs. Two experienced pathologists evaluated the three immunoexpression profiles of cancerous tissues and non-cancerous tissues. The final score was determined by multiplying the immunostaining intensity by the percentage of positive immunostaining cells. Subsequently, the cancerous samples were classified into three groups: 0, absent immunostaining; 1, weak immunostaining; and 2, strong immunostaining. The proportion of positive cells was classified as follows: 0, 0; 1, 1–25; 2, 26–75; 3, 76–100%. The final staining score was calculated by multiplying the staining intensity score by extent of staining score. A final staining score of ≥3 was considered positive, and others were classified as low expression.
Statistical analysis
Statistical analysis was performed using SPSS statistical software (version 21.0; IBM Corp.). Data are presented as value (%) and the association of P. gingivalis, CXCL2 and TANs immunoexpression levels in neoplastic cells with the clinicopathological variables was determined using χ2 or Fisher's exact tests (two-sided). For these analyses, the absence and weak immunoexpression levels were grouped together, obtaining final groups of absent/weak tumor immunoexpression and strong neoplastic immunoexpression. The cumulative survival rate (CSR) probability in 10 years was estimated using the Kaplan-Meier method, and survival curves were compared via a log-rank test. The follow-up period considered for cumulative survival consisted of the time between the date of surgery and mortality or the date of the last information collection regarding the patient. Cox regression analysis of the survival data was performed to test the statistical significance of regression coefficients. P<0.05 was considered to indicate a statistically significant difference.
Results
Study population
Between March 2007 and March 2019, a total of 205 samples from patients who were newly diagnosed with OSCC were retrospectively analyzed. There were slightly more men than women (50.2%) and patients with an age of ≥60 years (62.9%). Alcohol (18.0%) and tobacco (28.3%) consumption were considered as partial risk factors in patients.
With regard to survival status, the majority of patients in the present study were alive at the time of retrospective analysis (71.7%). Furthermore, the patients were clinically classified as stage I–II (54.1%) and III–IV (45.9%), and the TNM stages were classified as T1-T2 (65.9%) and T3-T4 (34.1%), and N0 (70.7%) and N(+) (29.3%). There were no patients with distant metastases at the time of physical examination (data not shown).
Among the enrolled patients, 67.3 and 23.9% were classified as well and moderately differentiated, while 8.8% were classified as poorly differentiated, according to the histopathological grade of tumor malignancy as described by Bryne et al (25). Recurrence occurred in 14.6% of patients, and there were 101 patients (49.3%) with poor periodontal condition. Only 13 patients (6.3%) underwent no therapy, 90 patients (43.9%) underwent surgical therapy, 14 patients (6.8%) received radiotherapy and/or chemotherapy and 88 patients (42.9%) received comprehension therapy (post-operative adjuvant radiotherapy and/or chemotherapy; Table I).
IHC expression of P. gingivalis in TME of OSCC
IHC expression of P. gingivalis in OSCC was weakly positive in 86 samples, while the expression was strongly positive in 119 samples. P. gingivalis immunoexpression was predominant in the cytoplasm of neoplastic cells. The strong immunoexpression of P. gingivalis was detected in carcinoma nests, while negative immunoexpression was observed in non-cancerous samples (Fig. 1).
Clinical variables, including the survival status, differentiation, tobacco consumption, T stage, N stage, clinical stage, periodontal condition, tumor size and treatment methods. The result revealed that death, poor differentiation, tobacco consumption, advanced T stage, N stage and clinical stage, poor periodontal condition, large size of tumor and no treatment exhibited significant associations with strong immunoexpression of P. gingivalis (Table II).
 | Table II.Immunohistochemical expression of P. gingivalis in samples from 205 patients with oral squamous cell carcinoma according to clinical data and follow-up. |
Identification of CXCL2 as a DEG in the TME of OSCC infected with P. gingivalis by bioinformatics analyses
After standardizing the microarray results, the DEGs (26,469 in GSE87539 and 9,443 in GSE138206) were identified (data not shown). The overlap between the two datasets containing 89 genes is presented in the Venn diagram (Fig. 2A), and included 67 upregulated genes and 22 downregulated genes.
Hierarchical clustering demonstrated that CXCL2 was a DEG between GSE87539 (P. gingivalis-infected oral epithelial samples compared with non-infected oral epithelial samples) and GSE138206 (cancerous samples compared with non-cancerous samples; Fig. 2B and C).
IHC expression of CXCL2 and TANs in P. gingivalis-infected TME of OSCC
A total of 119 samples with high expression levels of P. gingivalis were selected for IHC examination of CXCL2 and TANs. In 119 samples with high P. gingivalis expression, CXCL2 and TANs were weakly positively expressed in 30 and 38 samples, respectively, but were strongly positively expressed in 89 and 81 samples, respectively. CXCL2 was strongly positively expressed in the OSCC samples, but was negatively expressed in the non-cancerous samples (Fig. 3).
CD66b+ TANs are associated with a poor prognosis of patients with cervical cancer (26). In the present study, CD66b+ TANs exhibited strong positive expression in OSCC samples and negative expression in non-cancerous samples (Fig. 3).
Clinical variables, including age, survival status, T stage, tumor size and treatment. The result revealed that high age, death, advanced T stage, large size of tumor and no treatment exhibited significant associations with strong CXCL2 expression.
In the present study, clinical variables, including advanced T stage and clinical stage, were statistically associated with strong expression of TANs (Table III). The present results indicated that the expression levels of P. gingivalis were positively associated with the expression levels of CXCL2 and TANs (Table IV).
| Table III.Immunohistochemical expression of CXCL2 and TANs in 119 samples with strong staining for Porphyromonas gingivalis from patients with oral squamous cell carcinoma according to clinical data and follow-up. |
| Table IV.Association between P. gingivalis immunohistochemical expression and CXCL2 and TANs in 205 patients. |
Cumulative survival analysis
In univariate analysis of the 10-year CSR, the following parameters exhibited an association with the survival rate: Age, tumor size, body mass index, alcohol consumption, recurrence and neck dissection. Furthermore, the immunoexpression levels of P. gingivalis, CXCL2 and TANs were significantly associated with the 10-year CSR. Patients with a high immunoexpression of P. gingivalis had an increased risk with a lower 10-year CSR [hazard ratio (HR), 0.260; 95% CI, 0.135–0.503; P<0.001], and those with high immunoexpression levels of CXCL2 (HR, 0.283; 95% CI, 0.156–0.514; P<0.001) and TANs (HR, 0.494; 95% CI, 0.283–0.862; P=0.013) also had an enhanced risk with a lower 10-year CSR compared with patients with low immunoexpression levels of P. gingivalis, CXCL2 and TANs (Table V).
| Table V.Univariate analysis of Cox risk ratio model regression in 205 patients with oral squamous cell carcinoma. |
In the multivariate survival analysis of the 10-year CSR, the results demonstrated that recurrence, BMI, CXCL2 expression, P. gingivalis levels, TANs expression and alcohol consumption were independent risk factors for the prognosis of patients with OSCC (Table VI; Fig. 4).
| Table VI.Multivariate analysis using Cox risk ratio regression model in 205 patients with oral squamous cell carcinoma. |
Discussion
As aforementioned, there are a number of factors, including tobacco and alcohol use, as well as microbiological agents, that serve important roles in the progression of OSCC (20). Microorganisms are recognized as the primary risk factors for OSCC carcinogenesis (27), and the role of microorganisms in the promotion of OSCC has gradually become a novel area of research. An epidemiological study has demonstrated that 16–18% of carcinomas occur due to inflammation (28). Previous studies have also demonstrated that microbial pathogens, including Epstein-Barr virus, and hepatitis B and C virus, serve a role in tumor development (29–31). Furthermore, bacterial pathogens are associated with carcinogenesis with H. pylori infection, which is a causative factor in chronic gastritis, and aids in the development of gastric cancer (4).
It has been reported that P. gingivalis is associated with the progression and metastasis of OSCC (32). P. gingivalis invades and exists in the neoplastic cells, reproduces and survives in the cytoplasm of infected cells, and spreads to the neighboring cells (33). In addition, following invasion, P. gingivalis evades the immune clearance mechanism of the host, meaning it survives, reproduces and affects the biological functions of immune cells (34). When P. gingivalis invades OSCC tissues, it is able to recruit myeloid-derived suppressor cells (MDSCs) by expressing factors, such as CXCL2 and IL-6 (35). Simultaneously, P. gingivalis promotes tumor progression by recruiting MDSCs by increasing the secretion of IL-6 and CXCL2 from infected oral dysplastic keratinocytes (35). Entry of microbial metabolites into the TME promotes tumor progression by eliciting tumor-potentiating immune cell responses (35).
In the present study, staining of P. gingivalis in OSCC samples was observed, and this was present to a lesser extent in the non-carcinoma samples. Furthermore, it was identified that staining was mainly localized in the cytoplasm of malignant cells. These findings indicated that, at the histological site, the bacteria have the ability to invade neoplastic cells in vivo. In addition, stronger P. gingivalis staining was identified to be associated with a poor prognosis.
Associations between oral cancer and tooth loss or periodontal disease have been reported in a previous study (36). IL-6 and IL-8 have been identified as vital cytokines involved in periodontitis under P. gingivalis infection (37). IL-6 has been verified as a biomarker to illustrate the role of P. gingivalis infection in favor of OSCC initiation and progression (38). IL-8 is commonly secreted in the TME, which promotes tumor progression via the chemotaxis of MDSCs (39). P. gingivalis expresses multiple types of virulence factors that serve different roles in subverting the host immune response (40). Different secreted virulence factors of P. gingivalis may exert contrasting influences on the production of IL-8 via various mechanisms (41). CXCL2 has been reported to promote the generation of monocytic MDSCs (42). Furthermore, CXCL5 and CCL5 are associated with tumor progression (43). The present results suggested that CXCL2 and TANs were markedly increased in OSCC tissues and the TME that were infected by P. gingivalis, indicating that P. gingivalis and CXCL2 are involved in the recruitment of TANs, which contributes to the progression of OSCC. However, using IHC, the association between the immunoexpression levels of related proteins in the TME of P. gingivalis infection and clinical data remains unclear, despite increased research that suggests a close association between P. gingivalis and OSCC (44).
In the present study, two mRNA microarray datasets were analyzed to identify DEGs. A total of 89 DEGs were identified between the two datasets, which included 22 downregulated genes and 67 upregulated genes. Higher mRNA expression levels of CXCL2 were associated with the TME of OSCC infected by P. gingivalis. Cancer cells with high CXCL2 expression due to transcriptional hyperactivation are primed for survival in the metastatic sites (14). CXCL2 expression appears to be involved in OSCC-induced bone destruction and promotes tumor progression (12). Furthermore, increased CXCL1 expression has been observed in different OSCC cell lines and tumor specimens, and is associated with leukocyte infiltration and lymph node metastasis (12). CXCL2 attracts CD66b+ TANs into the tumor, producing chemokines that enhance cancer cell survival (45). Furthermore, the chemokine CXCL2 is the core cytokine that mediates lung metastasis and chemoresistance in breast cancer. When the CXCL2 receptor is blocked, the effects of chemotherapy against breast cancer and metastasis are effectively augmented (46). However, to the best of our knowledge, clinical research regarding the involvement of CXCL2 in promotion of cancer metastasis in OSCC has not been reported.
In recent years, the outlook on OSCC has changed and the tumor is no longer considered as a bulk of neoplastic cells, but rather as an entirety comprising a complex TME and neoplastic cells, and it has been suggested that tumor progression occurs via the TME and neoplastic cell interaction (47). The stromal component of the TME involves different cell types, such as TANs, cancer-associated fibroblasts and macrophages (47). In early tumors, TANs may be able to stimulate T cell responses (48), but in established tumors, TANs are immunosuppressive and associated with a more protumor phenotype with tumor progression (49). These subpopulation of cells interact with each other, as well as with neoplastic cells, via complex communication networks through secreted cytokines and chemokines (50). It has been reported that H. pylori is causally associated with the malignancies of gastric epithelia (51). H. pylori causes inflammation of the gastric mucosa by inducing gastric epithelial cells to secrete IL-8, resulting in the recruitment of inflammatory cells at the site of infection (52). Furthermore, IL-8, other chemokines and their receptors have been implicated in tumor development and metastatic progression. It has also been revealed that IL-8 enhances the generation of CD163+ M2 macrophages, and CD163+ macrophages exhibit a significant association with poor overall survival in patients with OSCC (53).
The present results suggested that, in OSCC, the TME infected with P. gingivalis exhibits increased CXCL2 secretion, which recruits TANs to the site of neoplastic cells and further promotes tumor development. It was demonstrated that the immunoexpression levels of CXCL2 were increased in patients with high expression levels of P. gingivalis. Another study with similar findings to the present study reported that P. gingivalis contributed to the accelerated secretion of CXCL8 and CCL2 (54). The number of CD66b+ TANs in the TME of OSCC infected by P. gingivalis proportionally increased with the secretion of CXCL2, and the expression levels of P. gingivalis were associated with the expression levels of CXCL2 and TANs. Furthermore, all three of these factors were associated with the poor prognosis of patients. However, studies regarding the association between the TME of OSCC and P. gingivalis are considered to be at early stages, and future investigations are warranted to determine the underlying molecular mechanism.
In conclusion, to the best of our knowledge, to gain an increased understanding of the participation of P. gingivalis in the TME of OSCC, the association between the immunoexpression of P. gingivalis in OSCC and clinicopathological features and patient prognosis was analyzed for the first time in the present study. Using bioinformatics analysis, it was identified that CXCL2 was a DEG in the TME of OSCC infected by P. gingivalis. In addition, the associations of the immunoexpression levels of CXCL2 and TANs in OSCC with clinicopathological features and patient prognosis were further analyzed. The present study suggested a mutual interaction between P. gingivalis and the TME of OSCC, and that the course of chronic periodontal inflammation may create a tumor-favorable microenvironment that promotes tumor development and progression. Furthermore, strong immunoexpression levels of P. gingivalis, CXCL2 and TANs may be associated with a poor prognosis in patients with OSCC. Therefore, it is necessary to develop prevention strategies for patients with OSCC with chronic oral infection.
Acknowledgements
Not applicable.
Funding
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
ZCGo conceived and designed the study. ZCGu conducted the experiments. SJ, SLJ, XYJ and LLH performed the statistical analysis. ZCGu wrote the manuscript. ZCGu reviewed and edited the manuscript. All authors agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved. ZCGo and ZCGu confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of the Affiliated Stomatological Hospital of Xinjiang Medical University (approval no. IACUC20180411-13; Urumqi, China). Written consent was obtained at the time of the initial data collection.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
P. gingivalis |
Porphyromonas gingivalis |
|
CXCL2 |
C-X-C motif chemokine ligand 2 |
|
TME |
tumor microenvironment |
|
IHC |
immunohistochemistry |
|
OSCC |
oral squamous cell carcinoma |
|
DEGs |
differentially expressed genes |
|
TANs |
tumor-associated neutrophils |
|
GEO |
Gene Expression Omnibus |
|
CSR |
cumulative survival rate |
References
|
Chi AC, Day TA and Neville BW: Oral cavity and oropharyngeal squamous cell carcinoma-an update. CA Cancer J Clin. 65:401–421. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Siegel R, Naishadham D and Jemal A: Cancer statistics, 2013. CA: A Cancer J Clin. 63:11–30. 2013. | |
|
Li P, Cao Q, Shao P, Cai H, Zhou H, Chen J, Qin C, Zhang Z, Ju X and Yin C: Genetic polymorphisms in HIF1A are associated with prostate cancer risk in a Chinese population. Asian J Androl. 14:864–869. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Kim SS, Ruiz VE, Carroll JD and Moss SF: Helicobacter pylori in the pathogenesis of gastric cancer and gastric lymphoma. Cancer Lett. 305:228–238. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Whitmore SE and Lamont RJ: Oral bacteria and cancer. PLos Pathog. 10:e10039332014. View Article : Google Scholar : PubMed/NCBI | |
|
Gershkovitz M, Fainsod-Levi T, Zelter T, Sionov RV and Granot Z: TRPM2 modulates neutrophil attraction to murine tumor cells by regulating CXCL2 expression. Cancer Immunol Immunother. 68:33–43. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Song JM, Woo BH, Lee JH, Yoon S, Cho Y, Kim YD and Park HR: Oral Administration of Porphyromonas gingivalis, a major pathogen of chronic periodontitis, promotes resistance to paclitaxel in mouse xenografts of oral squamous cell carcinoma. Int J Mol Sci. 20:24942019. View Article : Google Scholar | |
|
Gao JL, Kwan AH, Yammine A, Zhou X, Trewhella J, Hugrass BM, Collins D, Horne J, Ye P, Harty D, et al: Structural properties of a haemophore facilitate targeted elimination of the pathogen Porphyromonas gingivalis. Nat Commun. 9:40972018. View Article : Google Scholar : PubMed/NCBI | |
|
Inaba H, Sugita H, Kuboniwa M, Iwai S, Hamada M, Noda T, Morisaki I, Lamont RJ and Amano A: Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation. Cell Microbiol. 16:131–145. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Lanzós I, Herrera D, Santos S, O'Connor A, Peña C, Lanzós E and Sanz M: Microbiological effects of an antiseptic mouth rinse in irradiated cancer patients. Med Oral Patol Oral Cir Bucal. 16:e1036–e1042. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Wen L, Mu W, Lu H, Wang X, Fang J, Jia Y, Li Q, Wang D, Wen S, Guo J, et al: Porphyromonas gingivalis Promotes oral squamous cell carcinoma progression in an immune microenvironment. J Dent Res. 99:666–675. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
da Silva JM, Soave DF, Moreira Dos Santos TP, Batista AC, Russo RC, Teixeira MM and da Silva TA: Significance of chemokine and chemokine receptors in head and neck squamous cell carcinoma: A critical review. Oral Oncol. 56:8–16. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Vashishta A, Jimenez-Flores E, Klaes CK, Tian S, Miralda I, Lamont RJ and Uriarte SM: Putative periodontal pathogens, Filifactor alocis and Peptoanaerobacter Stomatis, induce differential cytokine and chemokine production by human neutrophils. Pathogens. 8:592019. View Article : Google Scholar | |
|
Hardaway AL, Herroon MK, Rajagurubandara E and Podgorski I: Marrow adipocyte-derived CXCL1 and CXCL2 contribute to osteolysis in metastatic prostate cancer. Clin Exp Metastasis. 32:353–368. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang H, Ye YL, Li MX, Ye SB, Huang WR, Cai TT, He J, Peng JY, Duan TH, Cui J, et al: CXCL2/MIF-CXCR2 signaling promotes the recruitment of myeloid-derived suppressor cells and is correlated with prognosis in bladder cancer. Oncogene. 36:2095–2104. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Fridlender ZG, Sun J, Mishalian I, Singhal S, Cheng G, Kapoor V, Horng W, Fridlender G, Bayuh R, Worthen GS and Albelda SM: Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils. PLoS One. 7:e315242012. View Article : Google Scholar : PubMed/NCBI | |
|
Hagerling C and Werb Z: Neutrophils: Critical components in experimental animal models of cancer. Semin Immunol. 28:197–204. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Powell DR and Huttenlocher A: Neutrophils in the tumor microenvironment. Trends Immunol. 37:41–52. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Li S, Cong X, Gao H, Lan X, Li Z, Wang W, Song S, Wang Y, Li C, Zhang H, et al: Tumor-associated neutrophils induce EMT by IL-17a to promote migration and invasion in gastric cancer cells. J Exp Clin Cancer Res. 38:62019. View Article : Google Scholar : PubMed/NCBI | |
|
Sahingur SE and Yeudall WA: Chemokine function in periodontal disease and oral cavity cancer. Front Immunol. 6:2142015. View Article : Google Scholar : PubMed/NCBI | |
|
Carey LA, Metzger R, Dees EC, Collichio F, Sartor CI, Ollila DW, Klauber-DeMore N, Halle J, Sawyer L, Moore DT and Graham ML: American Joint Committee on cancer tumor-node-metastasis stage after neoadjuvant chemotherapy and breast cancer outcome. J Natl Cancer Inst. 97:1137–1142. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Geng F, Liu J, Guo Y, Li C, Wang H, Wang H, Zhao H and Pan Y: Persistent exposure to Porphyromonas gingivalis promotes proliferative and invasion capabilities, and tumorigenic properties of human immortalized oral epithelial cells. Front Cell Infect Microbiol. 7:57–61. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Qiu X, Lei Z, Wang Z, Xu Y, Liu C, Li P, Wu H and Gong Z: Knockdown of LncRNARHPN1-AS1 inhibits cell migration, invasion and proliferation in head and neck squamous cell carcinoma. J Cancer. 10:4000–4008. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Li L, Lei Q, Zhang S, Kong L and Qin B: Screening and identification of key biomarkers in hepatocellular carcinoma: Evidence from bioinformatic analysis. Oncol Rep. 38:2607–2618. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Bryne M, Koppang HS, Lilleng R, Stene T, Bang G and Dabelsteen E: New malignancy grading is a better prognostic indicator than Broders' grading in oral squamous cell carcinomas. J Oral Pathol Med. 18:432–437. 1989. View Article : Google Scholar : PubMed/NCBI | |
|
Carus A, Ladekarl M, Hager H, Nedergaard BS and Donskov F: Tumour-associated CD66b+ neutrophil count is an independent prognostic factor for recurrence in localised cervical cancer. Br J Cancer. 108:2116–2122. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Akinkugbe AA, Garcia DT, Brickhouse TH and Mosavel M: Lifestyle risk factor related disparities in oral cancer examination in the U.S: A population-based cross-sectional study. BMC Public Health. 20:153–163. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Danaei G: Global burden of infection-related cancer revisited. Lancet Oncol. 13:564–565. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Iqbal J, McRae S, Banaudha K, Mai T and Waris G: Mechanism of hepatitis C virus (HCV)-induced osteopontin and its role in epithelial to mesenchymal transition of hepatocytes. J Biol Chem. 293:200102018. View Article : Google Scholar : PubMed/NCBI | |
|
Horikawa T, Yang J, Kondo S, Yoshizaki T, Joab I, Furukawa M and Pagano JS: Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma. Cancer Res. 67:1970–1978. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Chandrakesan P, Roy B, Jakkula LU, Ahmed I, Ramamoorthy P, Tawfik O, Papineni R, Houchen C, Anant S and Umar S: Utility of a bacterial infection model to study epithelial-mesenchymal transition, mesenchymal-epithelial transition or tumorigenesis. Oncogene. 33:2639–2654. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Parkin DM: The global health burden of infection-associated cancers in the year 2002. Int J Cancer. 118:3030–3044. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Lee J, Roberts JS, Atanasova KR, Chowdhury N, Han K and Yilmaz Ö: Human primary epithelial cells acquire an epithelial-mesenchymal-transition phenotype during long-term infection by the oral opportunistic pathogen, Porphyromonas gingivalis. Front Cell Infect Microbiol. 7:4932017. View Article : Google Scholar : PubMed/NCBI | |
|
Woo BH, Kim DJ, Choi JI, Kim SJ, Park BS, Song JM, Lee JH and Park HR: Oral cancer cells sustainedly infected with Porphyromonas gingivalis exhibit resistance to Taxol and have higher metastatic potential. Oncotarget. 8:46981–46992. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Wen L, Mu W, Lu H, Wang X, Fang J, Jia Y, Li Q, Wang D, Wen S, Guo J, et al: Porphyromonas gingivalis Promotes oral squamous cell carcinoma progression in an immune microenvironment. J Dent Res. 99:666–675. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Peres MA, Macpherson LMD, Weyant RJ, Daly B, Venturelli R, Mathur MR, Listl S, Celeste RK, Guarnizo-Herreño CC, Kearns C, et al: Oral diseases: A global public health challenge. Lancet. 394:249–260. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Yee M, Kim S, Sethi P, Duzgunes N and Konopka K: Porphyromonas gingivalis stimulates IL-6 and IL-8 secretion in GMSM-K, HSC-3 and H413 oral epithelial cells. Anaerobe. 28:62–67. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Geng F, Wang Q, Li C, Liu J, Zhang D, Zhang S and Pan Y: Identification of potential candidate genes of oral cancer in response to chronic infection with Porphyromonas gingivalis using bioinformatical analyses. Front Oncol. 9:912019. View Article : Google Scholar : PubMed/NCBI | |
|
Bunt SK, Yang L, Sinha P, Clements VK, Leips J and Ostrand-Rosenberg S: Reduced inflammation in the tumor microenvironment delays the accumulation of myeloid-derived suppressor cells and limits tumor progression. Cancer Res. 67:10019–10026. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Zenobia C and Hajishengallis G: Porphyromonas gingivalis virulence factors involved in subversion of leukocytes and microbial dysbiosis. Virulence. 6:236–243. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang Y and Li X: Lipopolysaccharide-regulated production of bone sialoprotein and interleukin-8 in human periodontal ligament fibroblasts: The role of toll-like receptors 2 and 4 and the MAPK pathway. J Periodontal Res. 50:141–151. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Shi H, Han X, Sun Y, Shang C, Wei M, Ba X and Zeng X: Chemokine (C-X-C motif) ligand 1 and CXCL2 produced by tumor promote the generation of monocytic myeloid-derived suppressor cells. Cancer Sci. 109:3826–3839. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Ban Y, Mai J, Li X, Mitchell-Flack M, Zhang T, Zhang L, Chouchane L, Ferrari M, Shen H and Ma X: Targeting autocrine CCL5-CCR5 axis reprograms immunosuppressive myeloid cells and reinvigorates antitumor immunity. Cancer Res. 77:2857–2868. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Lafuente Ibáñez de Mendoza I, Maritxalar Mendia X, García de la Fuente AM, Quindós Andrés G and Aguirre Urizar JM: Role of Porphyromonas gingivalis in oral squamous cell carcinoma development: A systematic review. J Periodontal Res. 55:13–22. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Ohms M, Moller S and Laskay T: An attempt to polarize human neutrophils toward N1 and N2 phenotypes in vitro. Front Immunol. 11:5322020. View Article : Google Scholar : PubMed/NCBI | |
|
Feliciano P: CXCL1 and CXCL2 link metastasis and chemoresistance. Nat Genet. 44:8402012. View Article : Google Scholar | |
|
Eckert AW, Wickenhauser C, Salins PC, Kappler M, Bukur J and Seliger B: Correction to: Clinical relevance of the tumor microenvironment and immune escape of oral squamous cell carcinoma. J Transl Med. 16:402018. View Article : Google Scholar : PubMed/NCBI | |
|
Eruslanov EB, Bhojnagarwala PS, Quatromoni JG, Stephen TL, Ranganathan A, Deshpande C, Akimova T, Vachani A, Litzky L, Hancock WW, et al: Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer. J Clin Invest. 124:5466–5480. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Wu P, Wu D, Ni C, Ye J, Chen W, Hu G, Wang Z, Wang C, Zhang Z, Xia W, et al: γδT17 cells promote the accumulation and expansion of myeloid-derived suppressor cells in human colorectal cancer. Immunity. 40:785–800. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Peltanova B, Raudenska M and Masarik M: Effect of tumor microenvironment on pathogenesis of the head and neck squamous cell carcinoma: A systematic review. Mol Cancer. 18:632019. View Article : Google Scholar : PubMed/NCBI | |
|
Yasunaga JI and Matsuoka M: Oncogenic spiral by infectious pathogens: Cooperation of multiple factors in cancer development. Cancer Sci. 109:24–32. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Wen J, Wang Y, Gao C, Zhang G, You Q, Zhang W, Zhang Z, Wang S, Peng G and Shen L: Helicobacter pylori infection promotes Aquaporin 3 expression via the ROS-HIF-1α-AQP3-ROS loop in stomach mucosa: A potential novel mechanism for cancer pathogenesis. Oncogene. 37:3549–3561. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Nagarsheth N, Wicha MS and Zou W: Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy. Nat Rev Immunol. 17:559–572. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Damgaard C, Kantarci A, Holmstrup P, Hasturk H, Nielsen CH and Van Dyke TE: Porphyromonas gingivalis-induced production of reactive oxygen species, tumor necrosis factor-α, interleukin-6, CXCL8 and CCL2 by neutrophils from localized aggressive periodontitis and healthy donors: Modulating actions of red blood cells and resolvin E1. J Periodontal Res. 52:246–254. 2017. View Article : Google Scholar : PubMed/NCBI |
