Simple SummaryCirculating tumor cells (CTCs) are important diagnostic and prognostic markers in cancer patients. However, current methods for isolating CTCs primarily focus on epithelial markers, such as EpCAM, leading to the underrepresentation of mesenchymal CTCs. This study proposes a novel method for continuous CTC isolation using magnetic nanoparticles coated with both EpCAM and vimentin antibodies, a mesenchymal marker. Using lateral magnetophoresis, the method demonstrated increased efficiency and reliability in isolating CTCs from patients with prostate (n= 17) and pancreatic (n= 5) cancer. Results indicated that using both EpCAM and vimentin antibodies significantly improved CTC isolation compared to using either marker alone, regardless of cancer stage.AbstractCirculating tumor cells (CTCs) display antigenic heterogeneity between epithelial and mesenchymal phenotypes. However, most current CTC isolation methods rely on EpCAM (epithelial cell adhesion molecule) antibodies. This study introduces a more efficient CTC isolation technique utilizing both EpCAM and vimentin (mesenchymal cell marker) antibodies, alongside a lateral magnetophoretic microseparator. The effectiveness of this approach was assessed by isolating CTCs from prostate (n= 17) and pancreatic (n= 5) cancer patients using EpCAM alone, vimentin alone, and both antibodies together. Prostate cancer patients showed an average of 13.29, 11.13, and 27.95 CTCs/mL isolated using EpCAM alone, vimentin alone, and both antibodies, respectively. For pancreatic cancer patients, the averages were 1.50, 3.44, and 10.82 CTCs/mL with EpCAM alone, vimentin alone, and both antibodies, respectively. Combining antibodies more than doubled CTC isolation compared to single antibodies. Interestingly, EpCAM antibodies were more effective for localized prostate cancer, while vimentin antibodies excelled in metastatic prostate cancer isolation. Moreover, vimentin antibodies outperformed EpCAM antibodies for all pancreatic cancer patients. These results highlight that using both epithelial and mesenchymal antibodies with the lateral magnetophoretic microseparator significantly enhances CTC isolation efficiency, and that antibody choice may vary depending on cancer type and stage.