Detection and Characterization of Circulating Tumor Cells in Colorectal Cancer Patients via Epithelial-Mesenchymal Transition Markers

Abstract

Background/Objectives: Liquid biopsy methods have gained prominence as minimally invasive tools to improve cancer treatment outcomes. Circulating tumor cells (CTCs) offer valuable insights into both primary and metastatic lesions. However, validating the CTC test results requires confirmation that the detected cells originate from cancer tissue. While studies have identified CTCs in colorectal cancer (CRC) patients using molecular markers, simultaneous validation of their cancer tissue origin remains unexplored. Methods: This study introduces a simple approach to detect adenomatous polyposis coli (APC) gene abnormalities alongside established CTC markers using a molecular imaging flow cytometer (MI-FCM). Given that APC gene abnormalities occur in 60-70% of CRC patients, their detection serves as strong evidence of cancer origin. Results: Our method achieved 92% concordance with DNA sequence analysis of tumor-derived cells. In a proof-of-concept study using 5 mL of whole blood from CRC patients, we observed a high frequency of cells exhibiting APC abnormalities, cytokeratin (CK), and vimentin (Vim) expression. Extending the study to 80 CRC patients across pathological stages I-IV confirmed CK and Vim as valid CTC markers. Three distinct cell populations were identified in blood: CK+/Vim-, CK+/Vim+, and CK-/Vim+. CTC number and frequency increased progressively with cancer stage. Conclusions: This is the first report demonstrating CK and Vim as effective markers for direct CTC detection in CRC patients. Our findings provide evidence-based validation of CTC markers, offering new insights and advancing approaches for patient care.

Keywords: adenomatous polyposis coli; circulating tumor cells; colorectal cancer; liquid biopsy.

Figure 1

Study design. The study followed these stages sequentially: (1) development of the detection method using cell lines, (2) validation of Cytokeratin (CK) and Vimentin (Vim) as circulating tumor cell (CTC) markers using isolated tumor-derived cancer cells (iCCs), and (3) characterization of CTCs from individual patients.

Figure 2

Development of APC mutation, CK, and Vim detection methods. (A) Image of the mutated APC detection method. (B) Western blot image of wild-type APC and mutant APC using A549, HCT116, and DLD1 cell lines. (C) Intensity of the APC-C/APC-N signal. Each cell line was immunostained and measured using imaging flow cytometer (MI-FCM). The cutoff was determined to encompass 95% of DLD1 cells exhibiting confirmed detection of mutant APC (dAPC) expression through Western blotting. (D,E) Expression of CK and Vim in HCC827 and transforming growth factor-beta (TGF-β)-stimulated HCC827. Each cell line was immunostained and measured using MI-FCM. The uncropped blots are shown in Figure S3.

Figure 3

Validity assessment of the detection method for APC mutation using iCCs. (A) Intensity of APC-C/APC-N signals in iCCs obtained from 13 CRC patients. iCCs were immunostained and analyzed using MI-FCM. The sequencing results of the APC gene are listed alongside the corresponding donor numbers. (B) Immunostaining images of APC-mutated and APC-wild-type iCCs. APC-N (green) and APC-C (red) were detected using specific antibodies, and nuclei were counterstained with Hoechst 33342 (violet).

Figure 4

Evaluation of CK and Vim as CTC markers. Whole blood samples from nine patients with APC gene mutations detected in their iCCs were analyzed using the CTC detection system. (A) Frequency of APC mutations in CTCs. (B) Frequency of CK and Vim expression in CTCs. (C) Immunostaining image of APC-mutated CTCs, with APC-N (N-terminal of APC, green), APC-C (C-terminal of APC, red), CK (orange), and Vim (yellow) stained using specific antibodies. Nuclei were counterstained with Hoechst 33342 (violet).

Figure 5

Analysis of CTCs from 74 patients with CRC. A 5 mL whole blood sample from each of the 74 patients with CRC was analyzed using the CTC detection system. Statistical comparisons were performed using the Mann–Whitney U-test. (A) Total number of CTCs expressing CK+/Vim−, CK+/Vim+, and CK−/Vim+ CTCs markers, grouped by pathological stage. (B) Number of CK−/Vim+ CTCs by pathological stage. (C) Frequency of CK and Vim expression in circulating tumor microemboli (CTMs). (D) Immunostaining image of CTMs, showing CK (orange) and Vim (yellow) staining with specific antibodies, and nuclei stained with Hoechst 33342 (violet).