The Protumoral Roles And Mechanisms Of Tumor Associated Inflammation Cells

Inflammation has emerged as a hallmark of cancer. The influences of inflammatory microenvironment on tumor have become a new research hotspot. Leukocyte infiltration, including mast cells, T cells, myeloid derived suppressor cells (MDSC), natural killer cells, tumor-associated macrophages (TAM) and tumor-associated neutrophils (TAN) are the key participants of the tumor microenvironment, where they exert a role of pro-tumor or anti-tumor.1. Lung adenocarcinoma, haemoptysis and inflammationLung cancer is the leading cause of cancer death worldwide. The most common symptoms of lung include cough, dyspnea, hemoptysis, etc. Though haemoptysis is one of the common clinical manifestations of lung cancer, few precise data have been reported about its incidence in patients before treatment, and it is not clear about the exact mechanisms of haemoptysis, especially for mild or moderate haemoptysis. The possible mechanisms involved in neovascularisation, vascular invasion, tumor necrosis, injuries due to invasive procedures or cough and so on. In our retrospective study, we found haemoptysis is an independent prognostic factor in lung adenocarcinoma; vascular invasion could be the most important mechanism of haemoptysis; and haemoptysis was associated with high white blood cell (WBC) level. To clarify the correlations between inflammation, WBC and the invasion ability of tumor, we performed further study.2. Tumor associated inflammation cells.Chronic inflammation is a consistent feature of tumor microenvironment. Cancer-related inflammation includes local inflammation and systemic inflammation. Macrophages and neutrophils are essential components of the inflammatory infiltrate of tumors. TAMs undergo classically activated (M1) and alternatively activated (M2) and exert antitumoral and protumoral functions. This plasticity has served as a paradigm for the functional polarization and a widely accepted hallmark for the monocyte-macrophage lineage which has been deeply explored. Like TAM, recent evidence finds that tumor-associated neutrophil (TAN) infiltrating in the tumor are also polarized toward antitumoral (N1) and protumoral (N2) phenotypes. TGF-β is the important effector in regulating the polarization of TAM and TAN.Based on the similar protumoral effects of macrophages and neutrophils, and lung adenocarcinoma with haemoptysis was associated with high WBC count, we analyzed the level of peripheral monocytes and neutrophils and found that neutrophils in haemoptysis group were significantly increased, but this was not observed in monocytes.3. TANCirculating neutrophils account for 70% of WBC. At the site of infection or tissue damage, they are firstly recruited to the lesion, engulfing pathogens, and releasing different factors. The tumor cells and mesenchymal cells in tumor microenvironment. secrete chemotactic factors which binding to the receptors CXCR1 and CXCR2 highly expressed on neutrophil surface and drawing circulating neutrophils towards tumor tissue, where they paly vital roles. TAN plays multiple roles at different stages of tumor development, invasiveness, and metastasis. However, the role of TAN in clinical studies remains controversial. We performed a systematic review and meta-analysis and concluded high levels of intratumoral TAN was associated with unfavorable outcome in head and neck cancer, non-small cell lung cancer (NSCLC), renal cell cancer, but not in gastric cancer. The effect and mechanism of TAN in the esophageal carcinoma (EC) remains unclear, we further investigated the roles of TAN in EC and lung adenocarcinoma with haemoptysis.4. TAN and Epithelial-mesenchymal transition (EMT)EMT describes the transition of cellular phenotype from an epithelial to mesenchymal state. It is an important biological process for cancer cells to obtain the migration and invasion ability, and also a key mechanism to obtain the malignant phenotype. EMT is regulated by many signaling pathways, including TGF-β, Ras, Wnt and etc. TGF-β signaling pathway, is one of the classical pathway. TGF-β signaling involves in EMT by canonical Smad dependent pathway and non-Smad dependent pathway.Researches show that TAN enhances invasion and metastasis of tumor cells, while little is known about the exact mechanisms. TANs induced EMT and promoted invasion and metastasis in pancreatic ductal adenocarcinoma. Co-culture peripheral blood mononuclear cells and gastric cancer cells promote tumor cells to release more TGF-β1. However, no further studies were done about the relevant signaling pathways and biological effects. Whether TGF-β is affected during the interactions of neutrophils and tumor cells has not been reported. Because of the important role of TGF-β in EMT and neutrophil plasticity, and the similarity in polarization of neutrophil and monocyte, we hypothesized neutrophils could promote tumor cells to generate more TGF-β1, and induce EMT via TGF-β signaling pathway.Due to the protumoral roles of tumor associated inflammation cells, tumor associated inflammation biomarkers have become new prognostic factors. Peripheral neutrophil-lymphocyte ratio (NLR) has been demonstrated to be an important prognostic factor in many solid tumors. While few study demonstrated the prognostic role for the peripheral lymphocyte-monocyte ratio (LMR) at diagnosis in solid cancers. Neutrophils and macrophages infiltrated in tumor microenvironment are important components of local inflammation. The prognostic value of LMR in lung cancer has not been reported. We further investigate the prognostic role of baseline LMR in lung cancer and demonstrate its prognostic value for the first time.This study will start from the hemoptysis, a common clinical manifestation, to explore its prognostic value and the related mechanisms, further investigate why hemoptysis was related to high WBC, and investigate the role and mechanisms of TAN in EC and lung adenocarcinoma in local inflammation. In addition, the important prognostic value of LMR in lung cancer is confirmed in systemic inflammation response level. This will provide more credible evidence to elucidate the protumoral role of tumor associated inflammatory cells in cancer. Section I Haemoptysis as a prognostic factor in lung adenocarcinoma after curative resection Objectives:1. To identify the prognostic value of hemoptysis in lung adenocarcinoma;2. To explore the possible mechanisms of hemoptysis. Materials and methods:1. Primary lung adenocarcinoma patients who underwent radical resection were retrospectively enrolled in this study. The prognostic value of hemoptysis with respect to overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) was analyzed;2. In order to explore the possible mechanisms of hemoptysis, vascular endothelial growth factor (VEGF) expression, tumor necrosis, vascular invasion and extratumoral microvessel density (MVD) were evaluated in randomly selected patients;3. Correlations between haemoptysis and clinicopathological data were analyzed. Results:1. Hemoptysis predicted poor OS, DSS and DFS in operable lung adenocarcinoma (all p＜0.001).2. Hemoptysis group showed more severe vascular invasion compared to non-hemoptysis group, but no statistically significant differences of VEGF expression, tumor necrosis, and extratumoral MVD were observed in hemoptysis and non-hemoptysis groups;3. Hemoptysis was associated with high WBC count (p= 0.032), high fibrinogen (Fib) (p＜0.001), high tumor greatest dimension (p＜0.001), severe vascular invasion (p=0.002) and central tumor location (p＜0.001). Conclusions:1. Hemoptysis predicts poor OS, DSS and DFS in lung adenocarcinoma after curative resection.2. Vascular invasion rather than angiogenesis or tumor necrosis could be the most important mechanism of hemoptysis in lung adenocarcinoma. Section II TAN contributes to EMT in lung adenocarcinoma cells Objectives:1. To clarify the relationship between haemoptysis and the subgroups of WBC: neutrophil count and monocyte count;2. To explore the role of TAN infiltration in tumor. Materials and methods:1. Mann-Whitney U-test was used to compare the distribution of neutrophil count and monocyte count in haemoptysis and non-haemoptysis groups;2. Immunohistochemistry (IHC) was used to detect CD66b+TAN and E-cadherin expression in tumor tissue;3. The expressions of EMT markers were assessed by western blot by using co-culture system of tumor cells and neutrophils;4. TGF-β1 concentrations in supernatant were measured by ELISA, and the source of TGF-β1 was detected by RT-PCR;5. The migration activities of tumor cells were performed by transwell migration assays. Results:1. Haemoptysis was associated with high neutrophil count, while no relationship was found in monocyte count;2. Haemoptysis significantly associated with neutrophil infiltration (OR=4.25, 95% CI 1.246-14.502);3. Intratumoral CD66b+TAN expression was negatively associated with E-cadherin.4. Neutrophils promoted EMT and enhanced the migration activity of tumor cells in vitro;5. TGF-β1 was up-regulated after co-culture and tumor cell was the main source of TGF-β1;6. Smad4 translocated into nucleus, indicating TGF-β/Smad signaling pathway was initiated during co-culture. Conclusions:1. Lung adenocarcinoma with haemoptysis was associated with more TAN infiltration;2. Neutrophils promoted EMT, and TGF-β/Smad signal pathway may be the key pathway;3. TAN may induce EMT and promote invasion and metastasis of lung adenocarcinoma, which provides a mechanistic reason for why haemoptysis is associated with poor outcome.Section III Intratumoral TAN Is Associated with Poor Prognosis in Squamous EC by Promoting EMTObjectives:1. To investigate the prognostic role of TAN in squamous EC;2. To explore the protumoral mechanisms of TAN in squamous EC.Materials and methods:1. IHC was used to detect the infiltration of CD66b+TAN, TGF-β1 and E-cadherin expression;2. The interactions of neutrophils and EC cells were investigated by using in vitro co-culture system;3. Western blot was performed to detect the expressions of EMT markers;4. Tanswell migration assays were performed to evaluate the migration activities of tumor cells;5. ELISA was used to measure TGF-β1 concentrations in supernatant;6. MTT assay was used to access cell proliferation;7. The prognostic value of TAN infiltration was evaluated by using Kaplan-Meier survival curves and Cox proportional hazard models.Results:1. A total of 159 squamous EC patients were enrolled in the study;2. Intratumoral TAN infiltration was positively correlated with distant metastasis occurred during follow-up (Spearman’s rho=0.429, p<0.001), TGF-β1 expression in tumor (Spearman’s rho=0.487, p<0.001), and circulating neutrophil count (Spearman’s rho=0.432, p<0.001), negatively correlated with E-cadherin expression (Spearman’s rho=-0.551, p<0.001);3. Multivariate Cox analysis demonstrated that intratumoral TAN infiltration was an independent predictor of poor outcome with HR= 2.453,95% CI,1.543-3.900;4. Co-culture with neutrophils induced EMT of EC cells with down-regulation of E-cadherin expression but up-regulation of N-cadherin and vimentin expression;5. Neutrophils enhanced the migration activity of tumor cells in vitro-,6. TGF-β1 level was increased in the supernatant of co-culture system, and TGF-β/Smad signaling pathway was initiated;7. MTT assays demonstrated co-culture promoted EC cell proliferation. Conclusions:1. Intratumoral TAN infiltration is an independent unfavorable predictor in squamous EC;2. PMN promotes cancer progression partly by its ability to induce EMT and promote proliferation. squamous EC;Section IV Prognostic Significance of Systemic Inflammation-Based LMR in Patient s with Lung Cancer:Based on a Large Cohort StudyObjectives:1. To explore the prognostic value of LMR in operable lung cancer;2. To investigate the prognostic role in subgroups of NSCLC and small cell lung cancer (SCLC).Materials and methods:1. Primary lung cancer patients who underwent complete resection at Shandong Provincial Hospital Affiliated to Shandong University from 2006 to 2011 were enrolled in this retrospective study;2. Lymphocyte count and monocyte count were collected from routinely performed preoperative blood tests, and the LMR was calculated;3. Survival analyses were calculated for OS and DFS by using Kaplan-Meier survival curves and Cox proportional hazard models.Results:1. A total of 1453 patients were enrolled in the study;2. The LMR was significantly associated with OS and DFS in multivariate analyses of the whole cohort (HR= 1.522,95% CI:1.275-1.816 for OS, and HR= 1.338,95% CI:1.152-1.556 for DFS);3. Univariate subgroup analyses disclosed that the prognostic value was limited to patients with NSCLC instead of SCLC (HR:1.718,95% CI:0.946-3.122). Multivariate analyses demonstrated that LMR was still an independent prognostic factor in NSCLC (HR=1.511,95% CI:1.256-1.819 for OS; and HR= 1.335,95% CI: 1.141-1.561 for DFS).Conclusions:LMR can be considered as a useful independent prognostic marker in patients with NSCLC after complete resection.