This study investigates the potential of tumor-associated circular RNAs (circRNAs) as an alternative source of neoantigens for cancer vaccines, particularly in microsatellite-stable colorectal cancer (CRC). The researchers identified circRNAs upregulated in CRC tumors using the MiOncoCirc database and ribodepletion RNA sequencing of paired clinical normal and tumor samples. They validated the expression of candidate circRNAs using quantitative real-time PCR and predicted the translation potential of their open reading frames (ORFs) using the TransCirc database. The HLA binding affinity of the ORFs was predicted using pVACtools, and strong binders were further validated by BlastP to exclude peptides from linear mRNA-encoded proteins. The immunogenicity of the candidate antigens was functionally validated through stimulation of naive CD8+ T cells against predicted neoepitopes, with subsequent analysis of T cells using ELISpot, intracellular cytokine staining (ICS), and granzyme B (GZMB) reporter assays. Patient-derived organoids were also used to test the cytotoxicity of T cells trained with antigen peptides. The study identified a neoepitope from circRAPGEF5 and two neoepitopes from circMYH9, which were upregulated in CRC tumor samples. These neoepitopes elicited antigen-specific T cell responses and expansion, as evidenced by various assays. Additionally, T cells trained with circMYH9 peptides were able to specifically target and eliminate tumor-derived organoids but not normal organoids. The detection of circMYH9 in liquid biopsies suggests a potential detection-to-vaccination treatment strategy for CRC. The findings highlight the feasibility of tumor-associated circRNAs as an alternative source of neoantigens for cancer vaccines targeting tumors with moderate mutation levels.This study investigates the potential of tumor-associated circular RNAs (circRNAs) as an alternative source of neoantigens for cancer vaccines, particularly in microsatellite-stable colorectal cancer (CRC). The researchers identified circRNAs upregulated in CRC tumors using the MiOncoCirc database and ribodepletion RNA sequencing of paired clinical normal and tumor samples. They validated the expression of candidate circRNAs using quantitative real-time PCR and predicted the translation potential of their open reading frames (ORFs) using the TransCirc database. The HLA binding affinity of the ORFs was predicted using pVACtools, and strong binders were further validated by BlastP to exclude peptides from linear mRNA-encoded proteins. The immunogenicity of the candidate antigens was functionally validated through stimulation of naive CD8+ T cells against predicted neoepitopes, with subsequent analysis of T cells using ELISpot, intracellular cytokine staining (ICS), and granzyme B (GZMB) reporter assays. Patient-derived organoids were also used to test the cytotoxicity of T cells trained with antigen peptides. The study identified a neoepitope from circRAPGEF5 and two neoepitopes from circMYH9, which were upregulated in CRC tumor samples. These neoepitopes elicited antigen-specific T cell responses and expansion, as evidenced by various assays. Additionally, T cells trained with circMYH9 peptides were able to specifically target and eliminate tumor-derived organoids but not normal organoids. The detection of circMYH9 in liquid biopsies suggests a potential detection-to-vaccination treatment strategy for CRC. The findings highlight the feasibility of tumor-associated circRNAs as an alternative source of neoantigens for cancer vaccines targeting tumors with moderate mutation levels.