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. Conclusion: The microfluidic platform enabled the reliable isolation of CTCs and CSCs from PDAC patient samples, as well as their subtypes. Complementary assessment of both CTCs and CSCs appears advantageous to assess the profile of tumor progressing in some cases. This research has important implications for the application and interpretation of approved methods to detect CTCs.

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