A Novel Microfluidic System for Immunomagnetic Isolation of Pancreatic Cancer CTCs: Plectin‑1 and EpCAM as Potential...

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide, with a five year survival rate below 10%. The high mortality rate of PDAC is primarily due to its aggressive metastatic nature, late diagnosis, and resistance to available therapies. A strategic research direction that demands significantly more attention is the identification of novel tumor markers and the development of tools for rapid, noninvasive diagnosis of this type of cancer. Circulating tumor cells (CTCs) are a promising target for early cancer detection via liquid biopsy, but their clinical application in PDAC is limited due to their rarity and technical challenges in reliable isolation and analysis. CTCs are typically isolated based on physical properties (size, density, deformability, charge, shape, hydrodynamics) or antigenic markers using specific antibodies. Although numerous cancer-specific antibodies have been identified, a marker uniquely distinguishing PDAC has yet to be found. In this work, we present the latest results concerning the development of a microfluidic platform that enables effective immunomagnetic isolation of PDAC cells from a complex biological sample using two potential PDAC markers: Plectin-1 and EpCAM. Here, we developed a low-cost, easy-to-manufacture, and user-friendly 3D-printed microfluidic platform that integrates a micromixer and a separation chamber for rapid cell isolation. Optimization tests showed that Plectin-1 at a concentration of 0.485 μg/mL captured 10² cells/ml with a recovery of 80% ± 13.3%, EpCAM at a concentration of 0.625 μg/mL captured 10³ cells/ml with a recovery of 47% ± 13.3%, and the mixture of EpCAM and Plectin-1 at concentrations of 0.3125 μg/mL and 0.242 μg/mL, respectively, achieved a recovery of 91.3% ± 4.4%. To demonstrate the applicability of the method in biological samples, blood with different anticoagulants was tested. The highest detection sensitivity was achieved in the blood sample with citrate: Plectin-1 at 0.485 μg/mL enabled detection at a concentration of 10² cells/ml (47 ± 9% recovery), and EpCAM at 1.25 μg/mL achieved a 92 ± 8% recovery. The entire isolation process from biological samples was performed in less than 1 min. Using this system combined with antibody-functionalized magnetic nanoparticles targeting EpCAM and Plectin-1, we demonstrated the efficient capture of PDAC cells from biological samples, highlighting Plectin-1 as a promising PDAC marker and the potential of immunomagnetic microfluidic separation for improved diagnostics.