In vitro response of non-small cell lung cancer cells to T-lymphocytes surveillance

Abstract

Cancer immunotherapy includes immune checkpoint blockade that works by blocking the potential communication between cancer cells and T-lymphocytes, which will stop the cytotoxic actions of T cells on tumour cells. The aim of this study was to identify the changes of genomic and protein expression in NSCLC cells after in vitro co-culture with CD8+ T cells. In this study, CD8+ cytotoxic T-lymphocytes and NSCLC cell lines in vitro co-culture model was established from Chinese origin. Flow cytometry analysis indicated the PD-L1 expression levels on a half of lung cancer cells increased after 48-hours of co-culture with CD8+ T cells from non-cancer donors or from lung cancer patients; the PD-1 expression levels on CD8+ T cells showed an enormous reduction in the same co-culture condition. Subsequently, cytotoxicity assay revealed that three of the four NSCLC cell lines, TS260, CL100 and FA161.2 showed significant decline in viability after co-culture with CD8+ T cells but there was no significant viability drop when normal bronchial epithelial cell lines were co-cultured with CD8+ T cells with the same co-culture condition. To further explore gene mutations which maybe potential neoantigen from the NSCLC cells, whole exome sequencing was performed on the four cancer cell lines alone. Gene functional classification results indicated that the 34 commonly found somatic mutations among the four cancer cell lines affected mainly the metabolic and particularly the catalytic activity of cancer cells and there were three genes which may be related to lung carcinogenesis, namely Odorant binding protein 2A genes (OBP2A), Mini-chromosome maintenance complex component 7 (MCM7) and Lysine demethylase 4C (KDM4C). Proteomic analysis was performed on the lung cancer cell lines after 24-hours of co-culture with CD8+ T cells. Relative expression levels of 61 proteins were found to be increased while 81 proteins were found to show relative decrease in expression levels. These differential protein expressions involved mainly the metabolic and catalytic activity of cancer cells. Among 81 proteins with decreased level of expression, protein phosphatase 2 catalytic subunit α (PPP2CA) and protein phosphatase 2 scaffold subunit alpha (PPP2R1A) may be involved in regulation of immune escape mechanisms related to PD-L1 protein expression. To further validate our results on clinical specimens, immunohistochemical (IHC) staining was performed on 80 NSCLC tumour tissue sections. The results showed that PD-L1 expression was correlated with TNM lymph node stage; nuclear factor erythroid 2-related factor 2 (NRF2) expression is correlated with smoking history, gender and pathological types of NSCLC; while Phosphoinositide 3-kinase (PI3K) expression is correlated with smoking history and gender. In summary, these co-culture experiments provided evidence to support the immunosuppressive effects of cancer cells on T-lymphocytes; it also confirmed the different effects of T-lymphocytes on cancer cells and normal epithelial cell viability as reported in literature. Whole exome sequencing and proteomic analysis suggested that the metabolic activity of cancer cells might contribute the immune-tolerance of cancer cells. Finally, IHC data further confirmed the potential association of biomarkers PD-L1, NRF2 and PI3K protein expressions with clinic-pathological features of lung cancer.