Capture of Epithelial-Mesenchymal Transitioning Circulating Tumor Cells using A/inity Based N-
Cadherin System

Introduction: 

Epithelial-to-mesenchymal transition (EMT) enables tumour cells to lose epithelial characteristics, acquire enhanced motility and aggressive invasiveness, and activate metastatic dissemination. Circulating tumour cells (CTCs) undergoing true EMT possess greater metastatic potential than purely epithelial CTCs because they exhibit increased survival in the bloodstream, immune evasion, and the ability to colonise distant organs. Thus, detection and characterization of EMT-CTCs provide clinically valuable insights into metastatic risk, disease progression, and treatment resistance beyond conventional epithelial CTC enumeration. Here, we report an N-Cadherin affinity-based ligand CTC-capture platform for capturing CTCs demonstrating varied EMT-CTC phenotypes.

Methods: 

Retrospectively, we analyzed blood samples from 30 cancer patients, including colorectal cancer stages I–IV (n=9), breast/TNBC (n=7), advanced lung adenocarcinoma (n=2), pancreatic cancer, anaplastic thyroid cancer, endometrial cancer, head and neck cancer, melanoma, and others. The cohort comprised 18 female and 12 male patients aged 19–84 years. We first validated Vimentin-positive MCF-7 cells using the OncoMetastat platform containing N-Cadherin glass beads for cellular localization. CTCs were captured using N-Cadherin glass beads from 1.5 mL of blood and subsequently confirmed based on Vimentin+, DAPI+, N-Cadherin+, and CD45− expression using a Nikon Ti-2 fluorescence microscope.

Results: 

Vimentin-positive CTCs were detected in 27/30 (90%) cancer patient blood samples. A total of 42 CTCs were captured, with a mean distribution of approximately 1.4 CTCs per sample and a range of 1–3 CTCs. Among the isolated CTCs, 13/42 (30.9%) expressed cellular N-Cadherin. Importantly, N-Cadherin expression was observed in 5/10 (50%) colorectal cancer (CRC) samples and 3/7 (42.8%) breast cancer samples. In six patients, intra-CTC heterogeneity was observed, with both N-Cadherin-positive and N-Cadherin-negative CTCs. The MCF-7 cell study demonstrated >90% cell capture using the N-Cadherin affinity platform, with Vimentin localization.

Conclusion: 

We demonstrate the capture of true epithelial-to-mesenchymal transitioning CTCs using an affinity-based N-Cadherin system in cancer patients. Further studies are required to investigate comparative CTC detection using anti-EpCAM antibodies and N-Cadherin, as well as their association with clinical outcomes.