Microfluidic Isolation of Circulating Tumor Cells and Cancer Stem-Like Cells from Patients with Pancreatic Ductal Adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDAC) requires multimodal therapeutic approaches and disease monitoring for effective treatment. Liquid biopsy biomarkers, including circulating tumor cells (CTCs) and cancer stem-like cells (CSCs), hold promise for evaluating treatment response promptly and guiding therapeutic modifications. Methods: From 24 patients with metastatic PDAC (stage IV, M1) undergoing active systemic treatment, we collected 78 blood samples at different time points for CTC and CSC isolation using a microfluidic platform functionalized with antibodies against a CTC biomarker, epithelial cell adhesion molecule (EpCAM), or a CSC biomarker, CD133. These isolated cells were further verified, via fluorescent staining and imaging, using cytokeratin (CK), CD45, and nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI). Results: The majority (84.4%) of patient blood samples were positive for CTCs (EpCAM + CK + CD45 -DAPI + ) and 70.8% of patient blood samples were positive for CSCs (CD133 + CK + CD45 -DAPI + ), using the highest baseline value of healthy samples as threshold. The CTC subtypes (EpCAM + CK + CD45 -DAPI + CD133 + and EpCAM + CK + CD45 -DAPI + CD133 -) and CSC subtypes (CD133 + CK + CD45 -DAPI + EpCAM + and CD133 + CK + CD45 -DAPI + EpCAM -) were also analyzed using immunochemical methods. In several cases, CSCs exhibited cytokeratin expression that did not express EpCAM, indicating that they will not be detected using EpCAM-based isolation.