Background: Despite the advances in overall cancer outcomes, pancreatic ductal adenocarcinoma (PDAC) remains among the deadliest cancers with a 5-year survival rate of less than 11%. As most cases of metastatic PDAC are resistant to conventional therapies, immunotherapy offers hope for improving PDAC outcomes and survival. To this end, an unbiased study of human leukocyte antigen (HLA)-bound peptides can facilitate future plans for T Cell-based immunotherapy. The goal of this study is to start with model cell lines and expand the pursuit to clinical specimens in the future.
Methods: Panc-1 model cell line was cultured and treated with IFN-γ (50 units/ml) in a 72h time course with increments of 24h. Snap-frozen pellets of size 5e7 and 5e6 were collected for HLA-peptidomics and proteomics analysis, respectively. Induction of HLA class I molecules was assessed using flow cytometry as a readout for IFN-γ effects. HLA-peptidomics pellets were lysed and HLA-peptide complexes were isolated using the anti-pan-HLA class I antibody W6/32. Bound peptides were prepared for mass spectrometry according to established protocols. The proteomics pellets were lysed and processed based on the S-TRAP method. HLA-peptidome data was acquired on a Bruker TimsTOF Pro in a data-dependent mode and proteomics data was acquired on Sciex 7600 ZenoTOF in the ZenoSWATH mode. Datasets were searched with a 1% FDR at the peptide and protein level.
Results: Panc-1 cells clearly show increased HLA class I expression upon IFN-γ treatment in a time-dependent fashion. Around ~25000 HLA-bound peptides in total were identified, with a time-dependent increase in peptide numbers post-IFN-γ treatment. A small subset of these peptides (0.4%) was considered cancer-specific; including known tumor-associated antigens (TAA) such as kinetochore protein Nuf5 and MAGEA1. A proportion of these peptides (0.11%) have been shown previously to act as immunogenic T Cell targets. Proteomics data shows a clear time dependence in proteome remodeling. Mainly, the members of the antigen processing pathway are upregulated earlier in the time course, while some proteins associated with cytoskeleton and metabolic remodeling are differentially expressed in the later time points.
Conclusion: The established workflow is sensitive to identifying up to 25000 peptides from as little as 5e7 cells from a PDAC cell line that is usually known for low HLA expression. Additionally, detecting known T Cell epitopes and potential targets derived from TAA proteins shows potential for using the workflow on patient-derived material to detect peptides with potential for the possibility of (semi-)personalized immunotherapy based on biopsies.