Intra-patient clonal diversity in HLA class I peptide ligand presentation

Tumor heterogeneity results from clonal diversity and evolutionary selection, and is a major cause of therapeutic resistance. Immunotherapy may exploit alternative vulnerabilities of drug resistant cells, and tumor-specific HLA peptide ligands on the cell surface are promising leads to invoke such targeted anti-tumor responses. In this work, we investigate the variability in HLA class I peptide presentation between tumor cells of the same patient, as differential ligand presentation could impact the efficacy of immunological interventions. By organoid amplification, we modeled single-cell level heterogeneity in a MSI- colorectal cancer patient, and directly correlated clonal tumor proteomes with the respective ligandomes. With deep comparative proteome profiling and sensitive detection of >7000 peptide ligands from each tumor organoid line, we found tumor-surface peptide presentation to be largely uncoupled from the steady-state protein abundance. Within the small minority of tumor-specific ligands (~3%), source proteins needed for DNA damage sensing and repair, and tumor suppression were prominently featured, leading us to speculate that such peptides may be consistently presented, as degradative byproducts of intended silencing. While clone-specific differences in peptide presentation were observed, inter-clone variability was also highly prevalent (15-25%). Collectively, these illustrate the heterogeneous peptide presentation landscape even within one individual, and hint that a multi-peptide vaccination approach against highly conserved tumor suppressors may be a viable option in patients with low tumor-mutational burden.