Evaluation of Organoid-Derived Exosomal microRNA as Liquid Biopsy for Colorectal Cancer: A Multicenter Cross-Sectional Study

Abstract

Exosomal microRNAs (miRNAs) are candidates for liquid biopsies. Organoid culture systems enable long-term expansion of the colon epithelium. This study evaluated exosomal miRNAs from colorectal cancer organoids for liquid biopsy. Organoids were established from normal colon and colorectal cancer tissues. Exosomes were isolated from conditioned media. miRNAs were extracted from exosomes and compared using microarray analysis. Exosomal miRNAs expression levels in the sera of healthy patients and patients with colorectal cancer were compared at a single institution. The multicenter study was validated using miRNAs upregulated in the serum of colorectal cancer patients, along with exosomal miRNAs reported to be upregulated in colorectal adenoma organoids and sera. A total of 44 exosomal miRNAs were commonly expressed in both normal colorectal epithelial cells and colorectal cancer organoids, whereas 59 were exclusively expressed in colorectal cancer organoids. In a single-center cohort study, two exosomal miRNAs (miR-4284 and miR-5100) were upregulated in the serum of colorectal cancer patients. In a multicenter study, four exosomal miRNAs (miR-4284, miR-5100, miR-1246, and miR-1290) were upregulated in the serum of patients with colorectal cancer. The combination of these four exosomal miRNAs had comparable diagnostic performance to carcinoembryonic antigen, with an area under the curve of 0.75 (95% confidence interval: 0.65-0.83) versus 0.79 (95% confidence interval: 0.70-0.87). Combining the four miRNAs with carcinoembryonic antigen improved diagnostic accuracy, with an area under the curve of 0.82 (95% confidence interval: 0.74-0.89). Exosomal miRNAs derived from colorectal cancer organoids can serve as diagnostic biomarkers for colorectal cancer.

Keywords: carcinoembryonic antigen; miR‐1246; miR‐1290; miR‐4284; miR‐5100.

FIGURE 1

Study design and workflow. This study followed a three‐phase workflow to identify and validate exosomal miRNAs as biomarkers for CRC. CRC, colorectal cancer; miRNA, microRNA; RT‐PRC, real‐time polymerase chain reaction, MIRAI study; liquid biopsy using miRNAs associated with intestinal disease. The study was approved by the ethics committee in October 2021.

FIGURE 2

Expression levels of exosomal miRNAs and CEA in patients with CRC compared with healthy individuals in a single‐center cohort (phase 2). Statistical analysis was performed using unpaired t‐tests. *p < 0.05. miRNA, microRNA; CEA, carcinoembryonic antigen; CRC, colorectal cancer; NS, not significant.

FIGURE 3

Expression levels of exosomal miRNAs and CEA in patients with CRC compared with controls in a multicenter study (phase 3). Statistical analysis was performed using the t‐test, with false discovery rate adjustment applied using the Benjamini–Hochberg method. *p < 0.05. miRNA, microRNA; CEA, carcinoembryonic antigen; CRC, colorectal cancer; NS, not significant.

FIGURE 4

ROC curve analyses comparing the diagnostic performance of exosomal miRNAs and CEA in patients with CRC. (A) ROC curves comparing individual exosomal miRNAs, their combination, CEA, and the combination of four exosomal miRNAs with CEA. (B) ROC curves stratified by age (≥ 65 vs. < 65 years) and smoking status (smokers vs. non‐smokers) compared with the combination of four exosomal miRNAs and CEA. A significant difference was observed in smokers (p < 0.05). ROC, receiver operating characteristic; miRNA, microRNA; CEA, carcinoembryonic antigen; CRC, colorectal.