In this study, we extracted exosomal miRNA from three-dimensional organoid culture and elucidated profiles of exosomal miRNAs from CRA organoids. Reportedly, the expression of exosomal miRNAs such as miR-21, miR-1246, miR-1290, miR-23a, miR-200c, miR-203a, miR-17, miR-19a and miR-7641 are increased in CRC [21–26]. Although expressions of miR-21, miR-1246, and miR-1290 are reportedly increased in CRC and CRA, no report has described the increased expression of miR-4323, miR-4284, miR-1268a, or miR-6766-3p in CRA. These miRNAs might be related to mutation of the APC gene, which causes adenoma initiation in the colonic epithelium. Results also suggest that expression of exosomal miRNAs is altered along with progress of stages of colorectal tumors.
To our knowledge, no study has examined exosomal miRNAs extracted from organoid culture as a candidate for liquid biopsy. The AUCs in CRA patients were 0.698 for the combination of exosomal miRNAs and 0.698 for the combination of serum miRNAs. Moreover, the AUCs in patients with larger CRA (approx. ≥12.6 cm2) were 0.834 for the combination of exosomal miRNAs and 0.834 for the combination of serum miRNAs. The AUC in FIT for advanced adenoma (those ≥ 10 mm size or with villous and/or high-grade dysplasia) is reportedly about 0.62 . The diagnostic capacity of liquid biopsy using miRNAs presented in this report is presumed to be higher. Furthermore, the results, demonstrating that better diagnostic capacity was obtained for larger tumors, suggests that the miRNAs used as candidates are secreted by tumors. Since it has been reported that the larger the size of adenoma, the more often complicated with cancer, it is expected that the candidate miRNAs especially useful as a screening for adenomas at high risk of cancer. However, the accuracy of liquid biopsy must be improved, particularly in consideration of the labor and costs of miRNA extraction and PCR testing. In this study, exosomes were extracted from the organoid culture supernatant by ultracentrifugation because few exosomes contained in the culture supernatant and required a large amount of medium. On the other hand, in a prospective study using patient sera, the polymer precipitation method was used. The available exosome kinds differ depending on the separation method [29, 30]. Presumably, miRNAs can be more effective biomarkers for CRA if it is possible to separate and extract from more tumor-derived exosomes. Result of our study showed no difference in miR-4283 or miR-4323 in serum miRNA levels: differences in expression were observed only for exosomal miRNAs. One influential factor might be that the change in expression was detected more sensitively because exosomes were extracted selectively.
In this prospective study, the expression of miRNAs in sera or serum exosomes of CRA patients were compared before and after endoscopic resection. Although differences in serum miRNAs before and after surgical resection in advanced CRC have been reported [9, 31], no report of the literature has described comparison of serum miRNA levels before and after endoscopic resection of adenoma. Because factors other than the tumor can be excluded, that method can be regarded as yielding more accurate study results. The expressions of serum exosomal miR-21, miR-29a, miR-92a have been reported for use of biomarkers for CRA patients . In this study, no difference was observed for miR-21 in the serum before and after treatment. Moreover, secretion of miR-29a or miR-92a from adenomatous organoids was not observed, possibly because of different extraction methods. Additionally, miR-29a and miR-92a might be secreted by cells other than tumor cells, such as fibroblasts and immune cells.
Reportedly, miR-4323 has been expressed less in patients of rectal cancer than in the normal colorectal epithelium , and it has also been expressed less in sera of patients with CRC than in healthy persons . One report describes that miR-4284 was enriched in the cyst fluid from pancreatic intraductal papillary mucinous neoplasm with low grade dysplasia, but it was depleted in the cyst fluids derived from invasive pancreatic carcinomas. . The expression of miR-4323 and miR-4284 might be lower in highly malignant tumor. Some reports have described that miR-1290 is expressed at a higher level in patients with CRC , and that it is related to epithelial mesenchymal transition , with miR-1246 promotes development of CRC via the expression of TGF-β  and as a diagnostically useful biomarker of CRC and recurrence [9, 20, 39, 40].
This study has several limitations. First, the investigation was conducted in a small number of patients and single center study. Second, studies of tumor lesions other than colorectal tumors are also need to clearly that candidate miRNAs are specific for patients with CRA. Finally, although serum at 6 months after endoscopic treatment was used as the control, the duration until the serum miRNA level returns to normal was not fully investigated.