Epitranscripomic markers in microbiome-derived cell-free RNA from plasma reveal potential for colorectal cancer detection

Circulating cell-free RNA (cfRNA) in human plasma represents a potential new avenue for cancer detection. We report the Low-Input Multiple Methylation Sequencing (LIME-seq) to profile methylation patterns within cfRNA, detecting diverse tRNAs and small non-coding RNAs (ncRNAs) originating from both the human genome and microbiome. Unlike abundance profiles through cfRNA/cfDNA shaped by microbial turnover, we find that methylation patterns in microbiome-derived cfRNA accurately reflect microbiota activity within the host, offering a unique class of non-invasive biomarkers with highly promising early colorectal cancer (CRC) diagnosis potential. A total of 27 patients with colorectal cancer and a total of 36 sex, age matched non-cancer individuals were recruited from the Lutheran General Hospital and the University of Chicago Medical Center. Blood from patients with CRC was collected before surgical resection (CRC). Peripheral blood samples from all participants were collected using standardized venipuncture protocols in antecubital venipuncture regions, with skin surfaces disinfected with 70% ethanol prior to phlebotomy to ensure aseptic conditions. All specimens were visually inspected to confirm absence of hemolysis. Blood from non-cancer individuals was collected from those undergoing screening colonoscopies at the University of Chicago Medical Center, with strict adherence to the same pre-analytical quality control measures, and only included subjects with no tumor upon post-colonoscopy pathological analyses. We included two validation cohorts in this study. Validation cohort 1 consisted of 8 non-cancer individuals and 30 patients with CRC. These samples were obtained from stored specimens, which were collected and processed by a separate research group at the same institute. To ensure comparability, CRC patients at different disease stages were age- and sex-matched with the non-cancer individuals. Validation cohort 2 included samples from additional individuals, with 8 non-cancer individuals and 7 patients with CRC, who were recruited in a different phase of this study. These samples were collected using the same procedures as the training cohort and processed by the same research group. Plasma cell-free RNA (cfRNA) or small RNA (<200 nt) were used to build libraries with LIME-seq following the protocol.