Abstract:
Background
The non-immunogenic and immunosuppressive microenvironment of pancreatic ductal adenocarcinoma (PDAC) had been a major obstacle for immunotherapeutic interventions. Recent findings had shown that epigenetic changes play a critical role in altering immune contexture in PDAC. Therefore, this study aims to identify aberrant epigenetic changes that attribute in immunosuppressive state in PDAC, particularly focusing on tumour cell intrinsic factors that mediates PDAC cells resistant to cytotoxic T lymphocytes (CTL) mediated killing.
Methods
A total of 396065 CpG variables methylation data involving 102 patients derived from The Cancer Genome Atlas (TCGA) project were retrieved from International Cancer Genome Consortium (ICGC) portal. Student t-test was used to filter out the insignificant CpG variables. Differentially methylated CpG sites were subsequently identified through 3 independent supervised clustering (SVM, kNN, random tree) methods, mean-variance filtering and hierarchy clustering. The identified differentially methylated CpG sites were further annotated to their corresponding genes to obtain differentially methylated genes. Scatterplot was used to analyse
correlation of methylation with gene expression to determine functional relevance of methylation at varying regulatory region. REACTOME over-representation analysis tool was used to identify enriched biological processes. Venn diagram was used to identify common differentially methylated genes between TCGA primary tumour and PDAC cell line datasets.
Result
Out of 9 differentially methylated genes that were significantly correlated to gene expression, 3 of the aberrant genes were deteced in both primary tumour and cell line datasets. Based on pathway enrichment analysis, TP53 regulated death receptor signalling pathways were enriched with hypermethylated genes while RUNX1, PPARα and RhoGTPases signalling were enriched with hypomethylated genes. Moreover, demethylating agent was observed to enhance cytolytic killing in PDAC cytotoxic T lymphocytes (CTL)-resistant cell lines in the presence of activated CD8+T cells
Conclusion
Hypomethylated Rab17 and PLCG; hypermethylated RGS12 were identified in both primary tumour and cell line model suggesting the potential of these intrinsic factors in contributing to the low cytolytic state in PDAC. Aberrant methylation event is evident to attribute in CTL resistance in pancreatic cancer cell lines. The deployment of DNMT inhibitor had shown to enhance the T cell mediated cytolytic killing in CTL resistant cell lines.
