Research On Molecular Mechanism Of CCL22 In Malignant Pleural Effusions

Background and objectivesMalignant pleural effusion(MPE)is a common complication of advanced malignant tumors and is significantly associated with tumor morbidity and mortality.It is estimated that there are about 150,000 people in the United States,and more than 100,000 people in Europe diagnosed with malignant pleural effusions each year.According to reports,most malignant tumors can cause malignant pleural effusions,of which lung cancer,breast cancer,and lymphoma are the most common causes.Although cytology is the gold standard for the diagnosis of malignant pleural effusions,the diagnostic sensitivity of repeated thoracentesis for malignant pleural effusions is usually only 50%-70%.Also,in cytology-negative cases,only 7%.-13% can be confirmed by pleural biopsy.Malignant pleural effusion is often regarded as markers of advanced cancer and usually indicate poor prognosis,with median survival ranging from 1 month to 12 months.Although malignant pleural effusions has been recognized for a long time,the current progress in treatment is slow,and palliative treatment is still the mainstay.The interaction between tumor cells and tumor stromal cells forms a tumor-specific microenvironment that promotes tumor invasion and progression.Malignant pleural effusion accumulating at the tumor site provides a unique opportunity to study the specific interactions between the tumor and its microenvironment and the immune system.Although studies on malignant pleural effusion are more and more common,the immune mechanism involved in their occurrence and development is still not very clear.Leukocytes,fibroblasts,and vascular endothelial cells constitute the tumor microenvironment,with immune cells accounting for the major component.These immune cells interact with tumor cells and affect the occurrence,development,and metastasis of tumors.Among them,Tumor-associated macrophages(TAMs)one of the prominent immune cells that coordinate various factors in the tumor microenvironment.However,the mechanism by which the interaction between immune cells of various groups in the tumor microenvironment promote tumor progression remains unclear.This study mainly clarified the role of tumor-associated macrophages in malignant pleural effusion and its effect on other immunosuppressive cells,providing a theoretical basis for the diagnosis and treatment of malignant pleural effusion.MethodsPleural effusion from untreated patients with non-small cell lung cancer(n=40)and non-tumor patients(n=40)were collected.Multiple chemokines with distinct pleural effusions were screened using multi-factor detection techniques and then validated by ELISA and real-time fluorescence quantitative PCR.Real-time fluorescence quantitative PCR was used to detect the expression of CCL22 in different immune cells.Cellular immunofluorescence was used to verify the expression of macrophage CCL22;THP-1 was induced by PMA and IL-4.The cells differentiated into M2 macrophages.The morphological changes of the cells at different time points were observed under inverted microscope.The expression of surface markers CD14 and CD163 of M2 macrophages after induction was detected by flow cytometry.Real-time fluorescence quantitative PCR was used to verify M2 macrophages.Expression of cell function molecules;analysis of C-FOS,a transcription factor that regulates CCL22 upregulation,using PCR Array technology;ELISA verification of CCL22 expression after knockdown of transcription factor C-FOS by small interfering RNA;luciferase reporter system verification of the binding relationship between transcription factor C-FOS and CCL22 promoter region was further verified by chromatin co-precipitation method.Results1.Multi-factor detection,real-time fluorescence quantitative PCR,and ELISA showed that CCL22 in malignant pleural effusion was significantly higher than that in infectious pleural effusion.2.Real-time fluorescence quantitative PCR results: In malignant pleural effusions,CCL22 is mainly derived from tumor-associated macrophages.3.Cellular immunofluorescence also confirmed that CCL22 is mainly derived from tumor-associated macrophages.4.Flow cytometry and real-time fluorescence quantitative PCR results showed that: PMA,IL-4 can induce THP-1 cells to M2 macrophage differentiation.5.Flow cytometry and real-time fluorescence quantitative PCR results showed that: PMA,IL-4 can induce THP-1 cells to M2 macrophage differentiation.6.ELISA results showed that the expression of macrophage CCL22 was significantly down-regulated after small interfering RNA knockdown of transcription factor C-FOS.7.The luciferase reporter system and chromatin co-precipitation results confirm that the transcription factor C-FOS can bind to the CCL22 promoter region and regulate its transcription.Conclusion1.Malignant pleural effusion CCL22 was significantly higher than infectious pleural effusion and the expression level of CCL22 was positively correlated with the survival of lung cancer patients and could be used as a potential diagnostic indicator of malignant pleural effusion.2.Malignant pleural effusion CCL22 is mainly derived from tumor-associated macrophages.The high expression of tumor-associated macrophage CCL22 in malignant pleural effusion is mainly mediated by C-FOS.