| BackgroundOncology is a hot field of biological science now.The rapid development of oncology biology,oncology immunology and oncology therapy depend on the research and exploration of specific tumor microenvironment and its regulatory mechanism.Squamous cell carcinoma is a common type of primary solid tumors.Because it occurs in the epidermis or its subsidiary cells,and can have different degrees of keratosis,it is also called epidermal cancer.It mostly occurs in the areas covered by squamous epithelium,such as oral cavity,pharyngeal,cervix,vulva,esophagus,lung and so on.The head and neck,as common parts of the disease,have attracted great attention.Head and neck cancer is the sixth most common cancer in the world.All malignancies of the upper digestive tract,from the vermilion border of the lips to the beginning of the esophagus,are known as head and neck cancers,of which oral,pharyngeal and lip cancers account for the vast majority.More than 90%of them are squamous cell carcinomas,namely head and neck HNSCC.Although in recent years,the treatment of head and neck squamous cell carcinoma(HNSCC)has been greatly improved by surgery combined with radiotherapy and chemotherapy,the prognosis of patients is still poor.Some of the head and neck squamous cell carcinomas are immunosensitive tumors.Therefore,it is an important premise and approach to improve the clinical therapeutic effect of head and neck squamous cell carcinoma to understand the immune cell infiltration and host inflammatory state of head and neck tumors,and to further explore the generation mechanism of different inflammatory states in tumor microenvironment.The cells and molecules in the tumor microenvironment are in a dynamic change process,which reflects the essence of the evolution of the tumor microenvironment,and the final result is that a large number of immune cells and a large number of inflammatory related factors aggregate in the tumor microenvironment,jointly promoting the occurrence and development of tumors.Therefore,starting with the concept of tumor microenvironment,this paper elaborates the composition and mechanism of specific tumor microenvironment,which is of great significance to the promotion of specific biological behavior,prognosis and prevention strategies of tumors.It has been proved that chronic inflammation is one uniform characteristic of the tumor microenvironment by epidemiological and experimental evidence.Extensive involvement of leukocytes in the microenvironment of the tumor may have a double effect on tumor progression and progression.Immune cells can directly clear tumor cells or participate in the induction of anti-tumor response,while tumor cells recruit and promote tumor growth and progress through appropriate training.The above immune cell infiltration included many participants,including macrophages and neutrophils.Neutrophils which are famous for their antimicrobial functions have been proved to be a critical factor in the development of tumor with the exception of macrophages.Nevertheless,tumor-associated neutrophils(TANs)have been has been demonstrated playing important role in the pathological processes of malignant tumor and in the modulation of the antitumor immunity.It has long been thought that neutrophils are end-differentiated effector cells that play an important role in the acute phase of inflammation and in resistance to microorganisms.But recent evidence challenges this limited view,suggesting that neutrophils can act on different cell populations and subsequently produce many cytokines and effector molecules.Therefore,neutrophils are involved in various inflammatory reactions,such as cancer immune response,which are combined with innate or adaptive immune response regulation to participate in cancer in this form.In recent years,there is also evidence that infiltration of neutrophils in tumors may be associated with poor prognosis of some tumors.However,there is no systematic study on the relationship between the infiltration of TAN and the clinical prognosis of HNSCC,especially OSCC.In recent years,the incidence of HNSCC,especially OSCC,is increasing,and more and more human papillomaviruses(HPV)are detected in OSCC.HPV positive HNSCC and HPV negative HNSCC are two different solid tumors in terms of biological characteristics,treatment response and patient prognosis.It has been reported that compared with HPV negative HNSCC patients,the patients with HPV have significantly better overall survival rate and lower recurrence rate.Scientists have found that HPV-related oropharyngeal squamous cell carcinoma is more radiosensitive than HPV-negative oropharyngeal squamous cell carcinoma,and has better efficacy after adjuvant chemotherapy.Differential immune infiltration in tumors seems to be very important for the success of treatment because many studies on tumor immunity have shown that there are significant differences in tumor infiltrating immune cells between HPV-positive and HPV-negative tumors.In some types of head and neck tumors,HPV-positive head and neck squamous cell carcinomas(HPV HNSCC)have more B and T cell infiltration and fewer neutrophils than HPV-negative head and neck squamous cell carcinomas(HPV-HNSCC).CD8 T cell infiltration,NK cell infiltration and PD1 positive T cell infiltration were described as the reasons for the good prognosis of HPV positive oropharyngeal squamous cell carcinoma.These findings suggest that further understanding of the effect of HPV on OSCC immune cell infiltration and its possible mechanism is of great significance to the development of OSCC treatment strategies and the improvement of its prognosis.However,there is no systematic study on the effect of HPV on immune cells in OSCC,and there is no systematic study on the changes and effects of HPV status on neutrophil infiltration in specific tumor immune microenvironment caused by HPV infection.In addition,the neutrophil to lymphocyte ratio(NLR)in peripheral blood before operation,i.e.the ratio of specific circulating cell count in routine blood examination before operation,has become a cheap and widely used indicator.It has been reported that NLR is a prognostic factor for many kinds of tumors.Local neutrophil infiltration reflects the inflammatory state of tumor immune microenvironment at the microscopic and molecular levels,while NLR reflects the overall immune inflammatory state of the host.This parameter is now recognized in the literature as a reliable prognostic marker for HNSCC and predictive for occult metastases.However,there was also a lack of research on the correlation between NLR parameters and local tumor-related neutrophil infiltration,and the impact of HPV infection on NLR in patients with OSCC.From what has been discussed above,HPV is increasingly detected in HNSCC and HPV-related tumor also presents unique immune microenvironment,then the TAN was a potential target of immunotherapy combined with traditional therapy for patients with HNSCC,all of them are of great significance for HNSCC.We set up a project idea based on the above background and causes.In this study,OSCC was selected as the research object,and based on the existing research,through in vivo and in vitro experiments and database analysis,to explore whether the density of infiltrating neutrophils and HPV infection status can be used to determine the high-risk categories of OSCC patients.We further explored the infiltration of neutrophils and the effect of HPV on it,and analyzed the possible correlation between them by combining the neutrophil marker NLR in the overall inflammatory state of the host.We explored the effects of HPV and neutrophils on the clinicopathological characteristics of OSCC and their possible mechanisms at the level of micro-inflammation and overall-inflammation,which were expected to provide new ideas for the treatment risk assessment,efficacy prediction and individual scheme formulation of HNSCC.Section IInfiltration of neutrophils in oral squamous cell carcinoma and their relationship with HPV expressionObjectives1.To detect the infiltration of CD 15 neutrophils in oral squamous carcinoma cells.2.To detect the human papillomavirus expression in oral squamous carcinoma cells.3.To explore the correlation between CD15+ neutrophils and human papillomavirus expression.Methods1.PatientsIn this study,we retrospectively studied a series of 81 patients with OSCC who underwent primary and curative resection at Qilu Hospital of Shandong University,and through the medical record system and the form such as visiting,we collected the basic information of the patients,including age,sex,smoking history,drinking history,clinical stage,histological grade,lymph node metastasis,tumor radiotherapy and chemotherapy,etc.2.ImmunohistochemistryImmunohistochemistry was used to analyze the expression of CD 15+neutrophils and P16(HPV)in OSCC,and to explore the correlation with the clinicopathological factors and prognosis of patients with OSCC.3.Statistical AnalysisSurvival rate was analyzed by Kaplan-Meier curve and logarithmic rank test was performed.Cox proportional hazards model was used for multivariate analysis to assess the prognostic factors associated with neutrophil infiltration,HPV infection and patient survival.Correlations of infiltration of neutrophils and HPV expression were analyzed by Spearman's rho coefficient test.Results1.Immunohistochemical results showed that there were significantly higher number of CD15 neutrophils infiltration in OSCC.Human papillomavirus(HPV)was detected in some patients with OSCC.Higher numbers of CD 15 neutrophils infiltration was related to stage ?,?(P<0.001),poor grade(p<0.001)and lymph node metastasis(p=0.014),while there was no explicit relationship between CD 15+neutrophils infiltration and age,gender,smoking history,drinking history,tumor extension,radiotherapy,chemotherapy.2.Human papillomavirus(HPV)was detected in some patients with OSCC.The expression of P16 was significantly heterogeneous.HPV negative status was also associated with stage ?,IV(p=0.001),poor grade(p=0.002),lymph node metastasis(p=0.005)and radiotherapy(p=0.038),while there was no explicit relationship between CD 15 neutrophils infiltration and age,gender,smoking history,drinking history,tumor extension,chemotherapy.3.According to immunohistochemical and data analysis,it was found that CD 15+neutrophils density was negatively correlated with HPV(P16)expression(p<0.001).The neutrophil infiltration was less in HPV(P16)positive expression group,but more in HPV(P16)negative group.In the other words,high density of and neutrophils was correlated with HPV negative status in OSCC(p<0.001).4.Survival analysis showed that there were significant differences in cancer-related survival rates between different types of neutrophil infiltration and HPV expression.The prognosis of oral squamous cell carcinoma patients with high infiltration of CD 15+neutrophil was worse than that of low infiltration of CD15+ neutrophil group,while the prognosis of HPV positive expression group was better than that of HPV negative expression group.In addition,high density CD 15 neutrophils and lymph node metastasis were independent prognostic factors in patients with OSCC(p<0.001).Conclusions1.A large number of neutrophils infiltrate the OSCC,The expression of HPV in oral squamous cell carcinoma is heterogeneous.In OSCC,high density of neutrophils was correlated with HPV negative status.2.In patients with OSCC,the prognosis of those with high infiltration of CD15+neutrophils and negative expression of HPV was poorer.High density CD 15+neutrophils and lymph node metastasis are independent prognostic factors in patients with OSCC.SignificanceOral squamous cell carcinoma(OSCC)is a type of head and neck cancer.Infection with human papillomavirus(HPV)is one of the most important causes of OSCC.HPV plays an important role in the development of OSCC.HPV-positive oral squamous cell carcinoma and HPV-negative oral squamous cell carcinoma are two different kinds of solid tumors in biological characteristics,treatment response and prognosis,especially in the infiltrating immune cells between HPV-positive and HPV-negative tumors.In this study,oral squamous cell carcinoma(OSCC)was selected as the research object to detect the infiltration of CD 15 neutrophils in OSCC and the expression of HPV in OSCC,and to explore the correlation between CD 1 5 neutrophils and HPV expression in OSCC.It was found that the expression of HPV was negatively correlated with neutrophil infiltration in oral squamous cell carcinoma.The findings of this phenomenon provide new arguments and evidence for the prognostic impact of specific tumor immune microenvironment caused by HPV infection on head and neck squamous cell carcinomas,especially oral squamous cell carcinomas,and provide new inspiration for further study of the mechanism.Section ?Possible mechanism of the effect of HPV overexpression on neutrophil infiltration in oral squamous cell carcinomaObjectivesTo explore the difference of cytokines and chemokines secreted by HPV-positive oral squamous cell carcinoma and HPV-negative oral squamous cell carcinoma in order to influence the infiltration and expression of tumor-related neutrophils.Methods1.Cell cultureCal-27 cells were cultured in a constant temperature and humidity incubator at 37 C and 5%carbon dioxide,and DMEM medium containing 10%fetal bovine serum(FBS)was used as a conventional medium.2.Construction of the plasmid HPV18-E7HPV18-E7-HA-Flag full-length cDNA sequence was purchased from Addgene Company,USA.The target gene was inserted into the framework of PCDNA 3.1.The construction and packaging of the plasmid were completed by Boshang Company,Shanghai.The target plasmid was named E7 and the empty plasmid was named PC.3.Plasmid transfection to Cal-27 cellsTransfection of target plasmid and empty plasmid was carried out on Cal-27 cells,and control group was set up.After 24 and 48 hours,the transfection was completed,the cell-free supernatants of three groups of cells were collected,and their proteins and RNA were extracted simultaneously.Western blot and real-time quantitative PCR were used to detect the plasmid-transfected cell lines.The expression of HPV18-E7 in oral squamous cell carcinoma was explored at the level of protein and RNA.Therefore,we can detect the efficiency of plasmid transfection.4.Morphological analysis,proliferation detection and apoptosis detection of cell lines after plasmid transfectionAfter successful plasmid transfection,morphological observation and analysis of the three groups of cells were carried out under optical microscope.MTT method was used to detect the difference of proliferation ability of the three groups of cells after transfection.And flow cytometry was used to detect the difference of apoptotic level of the three groups of cells after transfection.5.Detection of cytokines and chemokinesAfter 48 hours of transfection,the acellular supernatants of three groups of oral squamous cell carcinoma cell lines cultured under different conditions were collected,and the cytokines and chemokines were detected and analyzed.Real-time quantitative PCR was used to detect the cell supernatants of three groups under different culture conditions,and to verify whether cytokines or chemokines secreted differently at the level of mRNA secreted differently.6.The expression of CXCL8(IL-8)in oral squamous cell carcinoma was detected by immunohistochemical method,and the correlation between CXCL8(IL-8)and CD15+ neutrophils as well as P16(HPV)in oral squamous cell carcinoma was explored and verified.7.Statistical AnalysisThe correlations between neutrophil infiltration and IL-8 expression,as well as the correlation between HPV expression and IL-8 expression were analyzed by Spearman's rho coefficient test.8.Validated the results based upon mRNA expression data from The Cancer Genome Atlas(TOGA)(http://cancergenome.nih.gov/)database and Gene Expression Omnibus(GEO)(https://www.ncbi.nlm.nih.gov/gds/)database.In the GEO database(CSE3292 and GSE6791)and TCGA database of more than 500 cases of head and neck cancer,the data labeled HPV status were extracted and the differential expression of IL-8 was analyzed to verify the results of in vivo and in vitro experiments.Results1.HPV18-E7-HA-Flag targeted plasmid DNA vector was constructed.2.HPV18-E7-HA-Flag targeted plasmid DNA vector was introduced into oral squamous cell carcinoma cell lines to simulate the in vivo environment.The expression of HPV18-E7 in HPV18-E7 transfection group was significantly higher than that in empty vector transfection group and control group by qRT-PCR and Western blot analysis.3.There was no significant difference in cell morphology by optical microscopy after transfection.MTT assay showed that there was no significant difference in cell proliferation among the three groups of the cells,that is,HPV18-E7-HA-Flag had no significant effect on cell proliferation of Cal-27.Flow cytometry was used to detect the changes of apoptotic level in the three groups.The results showed that there was no significant difference in apoptotic level among the three groups,that is,HPV18-E7-HA-Flag had no significant effect on the apoptotic level of Cal-27 cells.4.The results of cytokine and chemokine analysis showed that the expression of IL-8 was down-regulated in oral squamous cell carcinoma cell lines with over-expression of HPV18-E7.qRT-PCR was used to detect the cell supernatants under different culture conditions,which confirmed that IL-8 secreted differently at the level of mRNA.In other words,the expression of IL-8 in oral squamous cell carcinoma cell lines with over-expression of HPV18-E7 was also significantly down-regulated at the level of mRNA.5.According to immunohistochemical,the density of CXCL8(IL-8)expression was detected to be positively correlated with CD15+neutrophils(p<0.001)and negatively correlated with HPV(P16)expression(p<0.001)by using Spearman's rho coefficient test analysis.6.In(GEO)database(GSE3292 and GSE6791 datasets),the expression of CXCL8(IL-8)was significantly different.In TCGA database,we found that 112 cases were labeled with HPV status.In 112 samples,CXCL8(IL-8)was significantly up-regulated-Conclusions1.HPV18-E7-HA-Flag targeted plasmid transfection can significantly increase the expression level of HPV 18-E7-HA-Flag in Cal-27 cells.Overexpression of HPV 18-E7-HA-Flag in Cal-27 cells can down-regulate the expression of IL-8 in cell supernatant,which is the chemokine of neutrophils.2.HPV may participate in depressing neutrophils infiltration to some extent through downregulating expression of IL-8.SignificanceTumor microenvironment is a complex framework,and bone marrow-derived cells,play an important role in the process of the tumor from initiation to metastasis.Immune cells are key players in tumor microenvironment,and they can promote or inhibit tumor formation and development.Immune cells can directly clear tumor cells or participate in the induction of anti-tumor response,while tumor cells recruit and promote tumor growth and progress through appropriate training.The above immune cell infiltration included many participants including neutrophils.Our study selected oral squamous cell carcinoma,a typical tumor microenvironment,to explore the influence of oncogenic HPV overexpression microenvironment on the biological behavior of specific cell populations such as neutrophils in tumors.It was found that in OSCC,HPV may inhibit the aggregation of neutrophils by down-regulating the expression of the chemokine IL-8 of neutrophils,so as to affect the prognosis of patients with OSCC.It is of great significance to understand the biological behavior and prognosis of head and neck squamous cell carcinoma,especially OSCC.HPV and TAN are potential targets,which may help to provide new ideas for the development of new therapeutic methods for head and neck squamous cell carcinoma.Section ?Analysis of correlation between HPV status and NLR in patients with OSCCObjectives1.To determine if NLR can be used to define a high-risk category of patients with OSCC.2.To explore the correlation between NLR and CD15+ neutrophils as well as HPV.Methods1.PatientsIn this study,we retrospectively studied a series of 81 patients who were diagnosed as OSCC at Qilu Hospital of Shandong University,and through the medical record system and the form such as visiting,we collected the basic information of the patients,including NLR.2.Statistical analysis According to the best critical value determined by ROC curve analysis and You den's index,NLR is divided into two categories.Survival rate was analyzed by Kaplan-Meier curve and logarithmic rank test was performed.The correlation between NLR and neutrophil immersion and HPV expression was analyzed by Spearman's rho coefficient test.Results1.The NLRs were analyzed in two-categories according to the optimal cutoff values determined using the receiver operating characteristic(ROC)curve analysis and You den's index(sensitivity+specificity-1):NLR?2.925 vs NLR>2.925.2.Survival analysis showed that NLR>2.925 lead to the worse OS and DFS in patients with OSCC.3.According to immunohistochemical and data analysis,it was found that the NLR was higher in the group with higher density of CD15+neutrophils and lower in the group with lower density of CD15+neutrophils.The NLR of patients was lower in the HPV(P16)positive expression group,while the NLR of patients was higher in the HPV(P16)negative group.Conclusions1.Compared with NLR?2.925,NLR>2.925 was significantly associated with worse DFS and OS in the patients with OSCC.2.In the OSCC,the density of locally infiltrated CD15+neutrophils was positively correlated with NLR.3.The infection of carcinogenic HPV in patients with OSCC was negatively correlated with NLR in the overall inflammatory state.SignificanceNeutrophils play an important role both in systemic and local inflammatory responses.It has been reported that the up-regulation of neutrophils can reflect the invasive characteristics of cancer cells,because it is mainly stimulated by hematopoietic cytokines of cancer cells.Neutrophils can produce the substances,such as reactive oxygen species,arginase and inhibit the function of cytotoxic lymphocytes.At the same time,lymphocytes have anti-cancer activity by inhibiting the growth or metastasis of tumors.By binding neutrophils to lymphocytes,high NLR level has been shown to be a promising indicator of poor prognosis for patients with cancer.Through in vivo and in vitro experiments and database validation,HPV might inhibit the aggregation of local neutrophils by down-regulating the expression of IL-8,thus changing the local inflammatory state in tumor microenvironment.Here,we added the NLR as a parameter to the study.We found that the infection of carcinogenic HPV in patients with OSCC was negatively correlated with NLR in the overall inflammatory state.We discussed the effect of HPV and neutrophils on the clinicopathological characteristics of OSCC not only from the level of microinflammation,but also from the level of overall inflammation,which provided new ideas for the treatment risk assessment,curative effect prediction and individual scheme fonnulation of HNSCC. |
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