Immunopeptidomics of colorectal cancer organoids reveals a sparse HLA class I neoantigen landscape and no increase in neoantigens with interferon or MEK-inhibitor treatment

Abstract

BackgroundPatient derived organoids (PDOs) can be established from colorectal cancers (CRCs) as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFNγ) or MEK-inhibitor treatment.MethodsFour microsatellite stable PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analyzed by MS.ResultsWe identified an average of 9936 unique peptides per PDO which compares favorably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. In contrast, computational HLA binding prediction estimated that 304 mutations could generate neoantigens. One hundred ninety-six of these were located in expressed genes, still exceeding the number of MS-detected neoantigens 65-fold. Treatment of four PDOs with IFNγ upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFNγ-inducible genes. HLA class II presented peptides increased dramatically with IFNγ treatment. MEK-inhibitor treatment showed no consistent effect on HLA class I or II expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment.ConclusionsOnly 3 out of 612 non-silent mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

Integrative -omics and HLA-ligandomics analysis to identify novel drug targets for ccRCC immunotherapy

“…Today, HLA ligandomics is the only method for direct measuring of the HLA peptidome of a cell or tissue, as reliable in silico methods to predict peptide presentation from, for example, transcriptome or proteome data are missing [93,94]. A recent study demonstrates even feasibility of immunopeptidomics using colorectal cancer organoids, thereby enabling the identification of only cancer cell-specific peptides without impairment of peptides derived from intratumoural stroma or immune cells [95]. The application of similar technologies using organoids derived from renal cell carcinoma in the future may further refine characterization of ccRCC-specific peptides.…”

Prioritizing Gene Cascading Paths to Model Colorectal Cancer Through Engineered Organoids

“…Using functional enrichment, many cancer-associated functions, such as MAPK cascade, Ras signaling pathway, PI3K-Akt signaling pathway, positive regulation of cell migration and positive regulation of cell proliferation, were identified (Table S7). With the accumulation of published studies about CRC organoids and multidimensional omics data of organoids (Fumagalli et al, 2017;Newey et al, 2019;Ooft et al, 2019), our method could be used to identify more extensive gene paths and construct the landscape of molecular pathogenesis for CRC cancer. Sequential introduction of the mutations in gene paths may provide a new avenue for understanding the dynamic progression of CRC.…”

An innovative approach for HLA typing, molecular tumor testing and the validation of tumor exclusive antigens

“…In addition, it can be investigated whether the source protein was differentially processed in the tumor compared to the surrounding benign tissue. To date, semiquantitative analyses have usually considered the total amount of peptides as a comparative value 18,46 . However, the allotypic peptides might be a better reference as demonstrated in this study.…”