Circular RNA as a source of neoantigens for cancer vaccines.

BACKGROUND: The effectiveness of somatic neoantigen-based immunotherapy is often hindered by the limited number of mutations in tumors with low to moderate mutation burden. Focusing on microsatellite-stable colorectal cancer (CRC), this study investigates the potential of tumor-associated circular RNAs (circRNAs) as an alternative source of neoepitopes in CRC. METHODS: Tumor-associated circRNAs in CRC were identified using the MiOncoCirc database and ribo-depletion RNA sequencing of paired clinical normal and tumor samples. Candidate circRNA expression was validated by quantitative real-time PCR (RT-qPCR) using divergent primers. TransCirc database was used for translation prediction. Human leukocyte antigen binding affinity of open reading frames from potentially translatable circRNA was predicted using pVACtools. Strong binders from messenger RNA-encoded proteins were excluded using BlastP. The immunogenicity of the candidate antigens was functionally validated through stimulation of naïve CD8+ T cells against the predicted neoepitopes and subsequent analysis of the T cells through enzyme-linked immunospot (ELISpot) assay, intracellular cytokine staining (ICS) and granzyme B (GZMB) reporter. The cytotoxicity of T cells trained with antigen peptides was further tested using patient-derived organoids. RESULTS: We identified a neoepitope from circRAPGEF5 that is upregulated in CRC tumor samples from MiOncoCirc database, and two neoepitopes from circMYH9, which is upregulated across various tumor samples from our matched clinical samples. The translation potential of candidate peptides was supported by Clinical Proteomic Tumor Analysis Consortium database using PepQuery. The candidate peptides elicited antigen-specific T cells response and expansion, evidenced by various assays including ELISpot, ICS and GZMB reporter. Furthermore, T cells trained with circMYH9 peptides were able to specifically target and eliminate tumor-derived organoids but not match normal organoids. This observation underscores the potential of circRNAs as a source of immunogenic neoantigens. Lastly, circMYH9 was enriched in the liquid biopsies of patients with CRC, thus enabling a detection-to-vaccination treatment strategy for patients with CRC. CONCLUSIONS: Our findings underscore the feasibility of tumor-associated circRNAs as an alternative source of neoantigens for cancer vaccines targeting tumors with moderate mutation levels.

A functional personalised oncology approach against metastatic colorectal cancer in matched patient derived organoids.

Globally, colorectal cancer (CRC) is the third most frequently occurring cancer. Progression on to an advanced metastatic malignancy (metCRC) is often indicative of poor prognosis, as the 5-year survival rates of patients decline rapidly. Despite the availability of many systemic therapies for the management of metCRC, the long-term efficacies of these regimens are often hindered by the emergence of treatment resistance due to intratumoral and intertumoral heterogeneity. Furthermore, not all systemic therapies have associated biomarkers that can accurately predict patient responses. Hence, a functional personalised oncology (FPO) approach can enable the identification of patient-specific combinatorial vulnerabilities and synergistic combinations as effective treatment strategies. To this end, we established a panel of CRC patient-derived organoids (PDOs) as clinically relevant biological systems, of which three pairs of matched metCRC PDOs were derived from the primary sites (ptCRC) and metastatic lesions (mCRC). Histological and genomic characterisation of these PDOs demonstrated the preservation of histopathological and genetic features found in the parental tumours. Subsequent application of the phenotypic-analytical drug combination interrogation platform, Quadratic Phenotypic Optimisation Platform, in these pairs of PDOs identified patient-specific drug sensitivity profiles to epigenetic-based combination therapies. Most notably, matched PDOs from one patient exhibited differential sensitivity patterns to the rationally designed drug combinations despite being genetically similar. These findings collectively highlight the limitations of current genomic-driven precision medicine in guiding treatment strategies for metCRC patients. Instead, it suggests that epigenomic profiling and application of FPO could complement the identification of novel combinatorial vulnerabilities to target synchronous ptCRC and mCRC.