Non-coding regions are the main source of tumor-specific antigens [mouse]

Purpose: Tumor-specific antigens (TSAs) represent ideal targets for cancer immunotherapy, but very few of them have been identified. Therefore, the goal of this study was to develop a novel approach, combining RNA-Sequencing and mass spectrometry, to enlarge the landscape of actionable TSAs in two murine cell lines, namely EL4 and CT26 cells. Methods: We performed RNA-Sequencing on both tumor cell lines (unreplicated) as well as on the syngeneic mature medullary thymic epithelial cells (mTEChi, in triplicate), using either the Illumina HiSeq200 or the Illumina NextSeq 500. Using those RNA-Sequencing data to build a global cancer database for each tumor cell line, we performed a transcriptomic-informed mass spectrometry analysis of their MHC I-associated peptides to identify TSAs. Results: We identified a total of 21 TSAs, 90% of which derived from allegedly non-coding regions and would have been missed by standard approaches. Moreover, about 70% of these TSAs derived from non-mutated yet cancer-restricted transcripts (e.g., endogenous retroelements) that can be shared by multiple tumors. Conclusions: In conclusion, the strategy reported herein is readily applicable to human tumors and should considerably enlarge the landscape of actionable TSAs. Transcriptomic analysis of EL4 and CT26 tumor cell lines (unreplicated) using the Illumina HiSeq 2000. C57BL/6 mTEChi were extracted from the thymi of 2 female and 2 male 5-8 week-old C57BL/6 mice and sequenced in triplicate using the Illumina HiSeq2000. Finally, Balb/c mTEChi were extracted from the thymi of 2 female and 2 male 5-8 week-old Balb/c mice and sequenced (in triplicate) on an Illumina NextSeq 500.