| Background:Malignant pleural effusion(MPE),a common disease of the chest,is caused by primary pleural tumors such as pleural mesothelioma,or by metastasis of lung cancer,breast cancer,lymphoma and other chest tumors.Non-small cell lung cancer(NSCLC)is the most common cause of MPE.Malignant tumor accompanied with MPE often indicates poor prognosis.Up to now,there is no uniform standard for the management of MPE.In addition to systemic treatment,mechanical intervention in the pleural cavity has become a mainstream view.After the programmed checkpoint molecule 1(PD-1)binds to its ligand PD-L1,it could activate downstream pathways,inhibit lymphocyte proliferation and activation,and form tumor-adaptive immune escape.Anti-PD1monoclonal antibodies represented by Nivolumab and Pembrolizumab have been widely used in clinical anticancer treatment.However,no clinical studies or case reports have so far used immune-checkpoint inhibitors to treat NSCLC patients with MPE,especially local injections in the pleural cavity.The therapeutic effect and mechanism of intrathoracic injection of Anti-PD1 on MPE need to be explored.Methods:1.MPE xenografts mice model was established.The amount of MPE in mice at different time points was observed by CT.Anti-PD1 monoclonal antibody was injected into the chest of MPE model mice for treatment,and the survival time was recorded.The amount of pleural effusion and the number of nodules in the two groups were observed to determine the therapeutic effect.During the treatment,the body weight of the mice was monitored,and biochemical indicators such as liver enzymes and creatinine were measured for safety evaluation.Different dosages, different administration methods and contralateral chest cavity were compared.2.The infiltration of CD8 in the thoracic cavity of mice was observed by IHC.In vitro LDH release assay was used to observe whether lymphocytes participate in killing tumor cells.Flow cytometry was used to detect the proportion of CD4~+T cells and CD8~+T cells in the tumor nodules and spleen of mice in different treatment groups.Flow cytometry was used to detect CD69,as well as the cytokines IFN-γand granzyme B.Established an animal model of"CD8 depletion",and the above experiments were repeated to clarify the importance of CTL.3.Tissue RNA-seq was performed,and differentially expressed genes were screened.The enrichment analysis of GO,KEGG,and Reactome pathways was used to identify the most obvious pathways.Real-time quantitative PCR was performed to measure the expression levels of chemokines and related immune genes.ELISA assay was used to determine the release of related cytokines.IHC and western blot experiments were performed to observe the changes of related pathway protein levels.4.Patients with advanced NSCLC with MPE who met the inclusion criteria were screened clinically and injected with human Anti-PD1 m Ab.Patients were evaluated for effusion drainage by CT and safety before,5 and 10 weeks after administration.The proportion of lymphocytes and expression of related molecules in pleural effusion and peripheral blood of patients before and after local treatment was determined to verify the conclusions of animal experiments.Results:1.In the animal model,it was proved that the local intrapleural injection of Anti-PD1 m Ab had therapeutic effect on MPE,which significantly reduced the amount of effusion and the number of cancerous nodules,and no obvious adverse reactions were observed.Local treatment with low-dose immune monoclonal antibodies could safely and effectively control the mass production of MPE,which could control the growth of distant cancer.2.The topical use of Anti-PD1 m Ab for MPE resulted in significant local CD8 infiltration.Lymphocytes were involved in killing tumor cells under Anti-PD1 treatment in vitro.The proportion of CD3~+CD4~+T cells in spleen and cancer was relatively stable.CD8~+T cells are important participating cells for local Anti-PD1 treatment.Intrathoracic injection of Anti-PD1 could improve local CTL activity and killing effect.3.A large number of immune-related genes including chemokine CCL20 and CXCR5 were significantly up-regulated after local Anti-PD1 treatment.GO enrichment analysis confirmed that local Anti-PD1 treatment has a greater impact on multiple immune-related pathways in the local immune microenvironment.Local Anti-PD1 treatment promoted the activation of CCL20/CCR6 pathway.The levels of Tcf7,Lef1,CXCR5,and BTLA related to lymphocyte activation significantly increased, and Tfh and Tfc cells may be affected.After local treatment,the release of inflammatory factors TNF-αand IL-1βin the pleural effusion of mice increased. PD-1/PD-L1 pathway was inhibited in cancer and its adjacent areas.4.Intra-thoracic injection of human Anti-PD1 m Ab could significantly reduce fluid accumulation in advanced NSCLC patients with MPE.The pleural atresia rate was higher at 5 weeks and fewer adverse reactions occurred.Local Anti-PD1 treatment could improve the activity and killing function of CTL in the chest cavity of patients.Conclusions:1.Intra-thoracic injection of Anti-PD1 m Ab in animal models had therapeutic effect and safety on MPE.Symptoms can be controlled with low-dose local treatment,and safety is better compared with systemic treatment.Local treatment could effectively control distant cancer.2.CD8~+T cells participated in local Anti-PD1 treatment of MPE.Local Anti-PD1 treatment may exert immune effects by increasing local CTL activity and enhancing CTL killing function.3.Local Anti-PD1 treatment promoted activation of the CCL20/CCR6 pathway in the local microenvironment.Tfh and Tfc cells in the local microenvironment may be affected.Anti-tumor response of CTL in the local thoracic cavity was enhanced. PD-1/PD-L1 pathways in cancer and adjacent areas were inhibited.4.Intrathoracic injection of Anti-PD1 m Ab had therapeutic effect on pleural effusion in clinical NSCLC patients with MPE,and the safety was acceptable.Anti-PD1 treatment of patients with MPE improved patients’local CTL activity and killing function. |
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