The Modulation And Related Mechanism Of PD-L1 Expression In Non-small-cell Lung Cancer

Background and objectivesLung cancer occupies the most part of solid cancer around the world, while among which, Non-small-cell lung cancer(NSCLC) is the most common histological type. In spite of a rapid development of traditional operation, platinum-based chemotherapy and radiotherapy, the advanced and metastatic NSCLC patients still have a poor prognosis. Therefore, novel and more effective strategies are urgently required.Currently, the targeted therapy of EGFR-tyrosine kinase inhibitors(TKIs), gefitinib and erlotinib, has come to be the first-line therapy in EGFR mutant NSCLC patients. Meanwhile, immunotherapies targeting on immune “check points” such as PD-1, PD-L1 and CTLA-4, have also been reported to obviously prolong the median progression-free survival of patients suffering tumors. However, what is the reasonable strategy and how to rationally combine the two novel strategies remain unknown.Recently, it is reported that NSCLC cell lines harboring EGFR mutations expressed a higher level of PD-L1 than those with other mutations, but the potential mechanism remains unexplored.This project aimed to find the potential correlation between EGFR mutation status and PD-L1 expression in different NSCLC cell lines and investigate the influence of EGFR-TKIs on PD-L1 expression in both EGFR-TKIs sensitive and acquired-resistant NSCLC in vitro and in vivo as well as their related underlying mechanisms. Consequently, this work may provide a novel strategy for improvement of anti-tumor immunotherapy.Methods1. The correlation between status of EGFR mutation and expression of PD-L1 in different NSCLC cell linesTotal DNA of NSCLC cell lines was extracted and EGFR mutations was identified by the peptide nucleic acid(PNA)–locked nucleic acid(LNA) polymerase chain reaction(PCR) clamp method. Levels of PD-L1 was tested through flow cytometry. Human recombinant epidermal growth factor(rh EGF) was added in culture environment of NSCLC cell lines and corresponding PD-L1 expression was test through flow cytometry and RT-PCR.2. The influence of EGFR-TKIs on PD-L1 expression of in both EGFR-TKIs sensitive and acquired-resistant NSCLC cell linesIn vitro experiments, different doses of gefitinib(0,2.5,5,10,100nM) was added in culture environment of NSCSC cell lines for a certain time(48h) or a certain dose of gefitinib(100nM) was added for different periods of times(0,6,12,24,48h). PD-L1 expression was tested through flow cytometry and immunofluorescence. In vivo experiments, NSCLC cell line was injected subcutaneously in nude mice and when tumor grew to a certain extent gefitinib was treated intragastricly. Tumor volume and weight were measured and PD-L1 expression in tumor tissue was tested through flow cytometry and immunofluorescence.3. Determine the mechanism involved in EGFR-mediated PD-L1 enhancement in NSCLC cell linesIn vitro experiments, NF-κB p65 expression in cytoplasmic and nuclear extracts of different NSCLC cell lines was tested via Western Blotting. Different doses of NF-κB inhibitor PDTC(0,50,100μM) was added in culture environment of NSCSC cell lines for a certain time, PD-L1 expression was tested through flow cytometry. Then different doses of gefitinib(0,2.5,5,10,100 n M) was added in culture environment of NSCSC cell lines for a certain time, NF-κB p65 expression in cytoplasmic and nuclear extracts was tested respectively by Western Blotting. In vivo experiments, in gefitinib-treated tumor-burdened nude mice model mentioned above, NF-κB p65 expression in tumor tissue was tested by immunofluorescence.Results1. EGFR activation was associated with PD-L1 expression in NSCLC cell linesPNA-LNA PCR showed that NSCLC cell lines PC-9, HCC827 and H1650 harbored mutant EGFR on exon 19, while H460, H1299 and SPC-1A harbored wild-type EGFR. Flow cytometry demonstrated that PD-L1 was much higher in EGFR mutant cell lines than that in EGFR wild-type cell lines. Treatment of rh EGR on EGFR wild-type cell lines could effectively upregulate their PD-L1 expression tested by flow cytometry and RT-PCR.2. Gefitinib could reversibly reduce the expression of PD-L1 in EGFR mutant NSCLC cell lines in a concentration and time dependent mannerFlow cytometry and immunofluorescence showed that different concentrations of gefitinib stimulated PC-9 and HCC827 cells for a certain time or a certain dose of gefitinib stimulated PC-9 and HCC827 cells for different periods of times could all attenuated their expression of PD-L1 and the attenuation degrees were positively correlated with treated concentrations and times. Besides, removal of gefitinib or rhEGF treatment could restore the expression of PD-L1 of gefitinib pre-treated PC-9 cells tested by flow cytometry.3. Gefitinib could reduce the PD-L1 expression in EGFR-TKI acquired-resistant NSCLC in a concentration and time dependent mannerGefitinib could also decrease PD-L1 expression of gefitinib acquired –resistant PC-9 cells tested by flow cytometry and attenuation degrees were positively correlated with treated concentrations and times.4. Geftinib could reduce the tumor growth and PD-L1 expression of tumor tissue in PC-9-xenografted mice model.Gefitinib treatment could effectively decrease the volume and weight of PC-9-formed tumor. Besides, flow cytometry and immunofluorescence displayed a lesser expression of PD-L1 in tumor tissue from mice of gefitinib-treated group than that from mice of control group.5. NF-κB pathway was involved in reduction of PD-L1 by EGFR-TKINF-κB p65 was expressed higher in cytoplasmic extracts and lower in nuclear extracts from EGFR wild-type cell lines than their expression from EGFR mutant cell lines tested by Western Blotting. Flow cytometry showed that NF-κB inhibitor PDTC treatment on PC-9 cells could reduce the expression of their PD-L1 in a dose dependent manner. Different concentrations of gefitinib stimulation could increase cytoplasmic expression and decrease nuclear expression of NF-κB p65 in PC-9 cells while not H1299 cells in a concentration dependent manner tested by Western Blotting. Intragastric gefitinib treatment could also attenuate NF-κB p65 nuclear expression in tumor tissue from PC-9-xenografted mice observed by immunofluorescence.Conclusions1. PD-L1 is highly expressed in EGFR mutant NSCSC cell lines and its expression is regulated by EGFR signaling in NSCLC wild-type cell lines.2. EGFR-TKI can both effectively decrease the expression of PD-L1 in EGFR mutant TKI sensitive and TKI acquired-resistant NSCSC cell lines in a concentration and time dependent manner.3. EGFR-TKI treatment alleviates tumor growth in xenografted mice model and decreases PD-L1 expression in tumor tissue.4. EGFR signaling sustains PD-L1 expression via NF-κB signaling pathways.