Transcriptional levels of immune activation genes were altered in the tumor post circRNACRT-E7E6 treatment

Immunization with circRNACRT-E7E6 vaccines have proved delayed tumors growth in mice and promoted a strong infiltration of antigen-specific T cells into the tumor site. We therefore sought to verify whether these efficacies were corroborated at the transcriptomic level within bulk tumor samples. Accordingly, RNA sequencing (RNA-seq) was conducted on TC-1 tumor samples from mice administered circRNACRT-E7E6 and from empty LNP controls. Gene Ontology (GO) analysis and Gene set enrichment analysis (GSEA) suggested enhanced immune activation and anti-tumor T cell response in circRNACRT-E7E6 administrated mice. Overall design: C57BL/6 mice were subcutaneously inoculated with 2.5×10^5 TC-1 tumor cells. Twelve days post-inoculation, when average tumor volume reached approximately 100 mm³, mice were randomized into two groups (n=5 per group). The negative control group received intramuscular (IM) injections of empty LNP on Days 0, 3, and 7. The experimental group received IM injections of circRNACRT-E7E6 (2 µg) on the same schedule. Five days after the final treatment, tumor samples were collected, processed according to controlled standard operating procedures, and subsequently submitted for RNA sequencing (RNA-seq) to determine whole tumor transcriptomes.