miRNA Biomarkers in Metastatic Colorectal Cancer Uncovered

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Colorectal cancer (CRC) is a leading global cause of cancer-related death. The spread of tumor cells through the bloodstream, known as circulating tumor cells (CTCs), is a key driver of metastasis. Accurately detecting CTCs remains clinically challenging. In our recent study, conducted at the Faculty of Medicine, Bursa Uludag University and published in the Journal of Clinical Laboratory Analysis, we investigated whether specific microRNA (miRNA) profiles could reliably predict the presence of CTCs in patients with metastatic CRC (mCRC).

Tackling an unmet need for monitoring prognosis and metastasis in CRC

mCRC poses a major clinical burden due to the difficulty in early detection and monitoring of metastasis. CTCs are shed into the bloodstream from primary tumors and play a critical role in the metastatic process. However, their detection remains technically challenging due to their rarity and heterogeneity. Small non-coding RNAs involved in gene regulation, miRNAs, are stable in blood and have shown promise as biomarkers in cancer diagnostics. Our study aimed to explore whether specific circulating miRNA profiles could distinguish between CTC-positive (CTC(+)) and CTC-negative (CTC(-)) mCRC patients. By combining immunomagnetic CTC detection with high-throughput miRNA profiling, we sought to identify miRNA signatures that could support non-invasive risk stratification and provide new insights into the molecular mechanisms of metastasis in CRC.

miRNAs reflect CTC status in mCRC

To explore whether miRNAs can act as surrogate biomarkers for CTC status in mCRC, blood samples from mCRC patients and healthy individuals (HIs) were analyzed. The CTC status of 48 mCRC patients was determined using AdnaTest ColonCancer, which isolates CTCs based on colon-specific surface markers. Patients were classified as CTC(+) and CTC(-) and distinct miRNA signatures were determined for eight patients from each group, along eight HIs, using miRNA microarrays. Candidate miRNAs were subsequently validated by quantitative polymerase chain reaction (qPCR). Statistical and bioinformatics analyses, including KEGG pathway enrichment and miRNA–target gene mapping, were conducted to explore the functional relevance of differentially transcribed miRNAs (Figure 1). The results were compared against The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) databases to assess clinical significance. Three miRNAs were further validated in a larger cohort and analyzed for diagnostic performance. This integrated approach aimed to identify novel miRNA signatures correlating with CTC presence and provide potential biomarkers for mCRC progression and liquid biopsy applications.

Figure 1: The workflow of the study. The study compares circulating miRNA profiles among mCRC patients that were CTC(+) (mCRC CTC(+)), CTC(-) (mCRC CTC(−)) and healthy individuals (HIs). Credit: Created by Doğan, B. (doi:1 0.1002/jcla.70013).

The key findings of this study were:

• 62% of mCRC patients were CTC(+) based on gene marker detection.
• A total of 11 miRNAs were identified as differentially transcribed based on CTC status.
• Three miRNAs (miR-199a-5p, miR-326 and miR-500b-5p) were validated as significantly downregulated in CTC(+) patients.
• Receiver operating characteristic (ROC) analysis confirmed the diagnostic utility of these miRNAs (area under the curve (AUC) > 0.7).
• Bioinformatics analysis linked these miRNAs to cancer-related pathways and regulatory networks, highlighting their translational potential.

Circulating miRNAs support liquid biopsy-based CTC assessment in mCRC

This study provides evidence that circulating miRNAs reflect the presence of CTCs in patients with mCRC. Using high-throughput miRNA microarrays followed by qPCR validation and integrative bioinformatics analyses, we suggest that specific miRNA signatures can distinguish CTC presence in mCRC patients. Notably, miR-199a-5p, miR-326 and miR-500b-5p were significantly downregulated in CTC(+) patients and showed high diagnostic accuracy (AUC > 0.7), indicating their utility as non-invasive biomarkers. These miRNAs are known tumor suppressors and are functionally involved in cancer-related pathways, with validated target genes such as GPD2, AKT3 and PLAG1 linked to CRC pathogenesis. Our findings are further supported by bioinformatic analysis that indicates their potential involvement in metastasis-related processes. Our results, based on miRNA patterns in blood samples, showed some differences compared to tissue-derived signatures from the TCGA dataset. However, the overall consistency between the two sources supports the reliability of these markers as potential tools for clinical use. These results pave the way for integrating miRNA signatures into clinical decision-making and surveillance strategies for mCRC.

Our findings contribute to the growing field of liquid biopsy by proposing circulating miRNAs as minimally invasive biomarkers for CTC detection in mCRC patients. This approach has the potential to improve metastasis monitoring, enable personalized risk assessment and guide treatment decisions without relying on complex or costly cell-based assays. The integration of miRNA-based diagnostics with clinical CTC evaluation could enhance early metastasis detection and support novel translational applications in CRC management. Ultimately, this may reduce reliance on tissue biopsies and provide real-time insights into disease progression and therapy response.

Despite the promising findings, it should be noted that our study has several limitations. First, only a subset of differentially transcribed miRNAs was validated through qPCR, and additional experimental validation is needed for the remaining candidates. Second, the CTC detection platform (AdnaTest) used in this study targets three epithelial markers (CEA, EGFR and EpCAM), and CTC heterogeneity may be explored by further comprehensive platforms. Third, non-metastatic CRC (nmCRC) patients were not included, preventing assessment of the diagnostic utility of suggested miRNAs across the entire disease spectrum. Fourth, some miRNAs identified in our study were not represented in public datasets such as TCGA, which limited external validation efforts. Lastly, while target genes such as GPD2 and RPS6KA3 were predicted to play functional roles, their regulatory mechanisms and downstream effects were not experimentally confirmed. Therefore, further in vitro and in vivo studies are needed to validate their regulatory mechanisms and clinical relevance. To advance these findings toward clinical application, large-scale, multicenter studies are essential to validate the diagnostic and prognostic potential of the identified miRNAs across diverse patient populations and sample types.

Toward clinical validation of miRNA-based metastasis tools

These three miRNAs emerge as promising liquid biopsy biomarkers for non-invasive prediction of CTC status in mCRC. Clinically, they could augment or, in some settings, replace existing CTC detection methods. Functionally, they offer insights into the molecular underpinnings of metastatic spread. Moving forward, we plan to:

1. Validate these markers in larger, independent patient cohorts
2. Develop integrated diagnostic panels combining miRNA markers with CTC assays
3. Explore therapeutic strategies targeting these miRNA networks to impede CRC metastasis

Our findings reveal a promising set of circulating miRNAs associated with CTC status in mCRC. These molecules may serve not only as diagnostic or prognostic markers but also as functional mediators of metastasis. Once further validated and clinically integrated, these biomarkers could support risk-based patient stratification and facilitate the development of novel miRNA-targeted therapies in CRC.

Reference: Doğan B, Pirim D, Işık Ö, Evrensel T. Candidate biomarkers associated with circulating tumor cell status in metastatic colorectal cancer. J Clin. Lab. Anal. 2025;39(7):e70013. doi:10.1002/jcla.70013