Effects Of Tumor-derived Tissue Factor Aberrantly Activating Complement In Lung Tumor And Underlying Mechanism

BackgroundLung cancer is one of most malignant type of the solid tumors that has the highest mortality and morbidity in our nation. Among all the lung cancer cases, 80 percent of them belong to non-small cell lung cancer (NSCLC) whose pathogensis remains unclear. Recent studies indicate that chronic inflammation plays a pivotal role in the development and progression of lung cancer. Thus, illuminating the role of chronic inflammation in the development and progression of lung cancer would enrich our understanding of lung malignancy and provide novel strategy in lung cancer treatment.Massive complement activation can be detected in specimen of many lung cancer patients. In the tumor microenvironment, C5a produced through complement activation is reported facilitating tumor progression via creating immunosuppressive milieu, enhancing tumor cell proliferation and metastasis and promoting endothelial cell angiogenesis.Nevertheless, the activation pathway of complement in lung tumor remain incomplete.Understanding the precise mechanism of complement activation in lung tumor may reveal novel target in lung tumor treatmentThe aberrant expression of tissue factor (TF) on tumor cell confers tumor cells the ability of triggering coagulant cascade, which leads to the hypercoagulant state in patients suffering lung, pancreatic, gastric malignancy and the subsequent complication of venous thromboembolism. Recent studies provide evidence that products from activated coagulation system can directly cleave complement protein and trigger complement activation cascade. However, this aberrant complement activation and its influence has never been reported in the tumor microenvironment yet.This study intended to explore the relationship between the coagulation activation induced by tumor-derived TF and the complement system within tumor microenvironment and its potential influence on lung tumor progression. This work may enrich knowledge of complement activation in tumor microenvironment and confer novel strategy in the clinical practice of lung cancer management.Method1. Measurement of TF expression in tumor cell lines and its association with tumor's procoagulant activity.The expression of TF on human lung adenocarcinoma cell line A549, human breast carcinoma cell line T47D, human ovarian carcinoma cell line A2780, human gastric carcinoma cell line AGS and human large cell carcinoma H460 had been measured by polymerase chain reaction (PCR). Short hairpin RNA was used for set up A549-shTF cell line that have a relatively low TF expression while A549-vec cell was built as a control.The expression of TF on these cell lines was validated by PCR and western blot. Platelet poor plasma of health volunteers was used for detecting the procoagulant activity of A549,A549-shTF, A549-vec cells and prothrombin time was measured.2. Analysis of the biological characteristics of A549 cell after TF knockdownCell Counting Kit assay was used for measuring the proliferative potential of A549,A549-shTF, A549-vec cells in vitro. Flow cytometric analysis was used for detecting ki-67 expression and apoptosis marker Annexin V and Propidium Iodide (PI) staining on A549,A549-shTF, A549-vec cells.3. Evaluating the growth and coagulant state in xenograft derived from A549-shTF and A549-vec cells.A549-shTF and A549-vec cell lines were subcutaneously injected into right flank of nude mice and tumor growth was measured every other day. Coagulant state in tumor tissue was reflected by measuring the immunohistochemistry (IHC) staining of TF and fibrin.4. Exploring the mechanism of delayed tumor growth after TF knockdown.C3b/iC3b/C3c and C5b-9 deposition was evaluated by IHC staining. Tumor infiltrating myeloid-derived suppressor cells (MDSC) was measured by flow cytometric analysis. C5a receptor antagonist was used for blunting the recruitment of MDSC to tumor derived from A549-shTF and A549-vec cells.Result1. TF expression dominants the procoagulant activity of lung tumor cell.Result from PCR showed A549 cell had a relatively high TF expression. A549-shTF and A549-vec were successfully built by shRNA transfection and were validated by PCR and western blot. Prothrombin time of plasma induced by tumor cell showed the clotting of plasma incubated with A549-shTF was prolonged comparing to that with A549 and A549-vec cells.2. TF knockdown unaffected the proliferative potential and apoptosis ratio of lung tumor cell in vitro.CCK-8 assay showed the proliferative potential was comparable among A549,A549-vec and A549-shTF cells; flow cytometric analysis showed the expression of Ki-67 was equal among A549, A549-vec and A549-shTF cells and the percentage of Annexin V+PI- cell in each cell population remain indistinguishable.3. Tumor growth of lung tumor cell was delayed and less coagulant molecule was stained in tumor tissue after TF knockdown.Tumor volume and tumor weight were less in mice transplanted with A549-shTF cells comparing to those transplanted with A549-vec cells. IHC staining of both TF and fibrin were less in tumor slice derived from A549-shTF cell in comparison with that in tumor slice derived from A549-vec cell.4. Complement activated by TF enhance the recruitment of MDSC to tumor tissue and facilitated tumor growth in vivo.IHC staining of C3b/iC3b/C3c and C5b-9 were significantly reduced in tumor slice derived from A549-shTF cell in comparison with that in tumor slice derived from A549-vec cell. Flow cytometric analysis showed the percentage of MDSC was lower in tumor derived from A549-shTF cell. By administrating of C5a receptor antagonist, tumor volume on mice transplanted with A549-vec cells showed comparable with that of mice transplanted with A549-shTF cell and the percentage of MDSC in tumor was equal between two groups.Conclusion1. Lung tumor cells possess procoagulant activity, which is due to the expression of TF.2. Down-regulation of TF expression on lung tumor cell has no effect on its proliferative potential and apoptosis ratio in vitro3. Down-regulation of TF expression on lung tumor cell delay the growth of their xenograft in nude mice and reduce the coagulant state in tumor microenvironment.4. Reducing the TF expression in tumor tissue alleviates complement activation degree in tumor microenvironment and decreases the percentage of MDSCs in tumor tissue.Antagonism of C5a receptor reduces MDSC recruitment and subsequently blunts the pro-tumor effect of TF expressed on lung tumor cell.