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Theranostics 2019; 9(5):1417-1425. doi:10.7150/thno.28745 This issue Cite

Research Paper

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

Jose I. Varillas^1,2*, Jinling Zhang^2*, Kangfu Chen², Isis I. Barnes³, Chen Liu⁴, Thomas J. George^3✉, Z. Hugh Fan^1,2,5✉

1. J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, P.O. Box 116131, Gainesville, FL 32611, USA.
2. Interdisciplinary Microsystems Group, Department of Mechanical and Aerospace Engineering, University of Florida, PO Box 116250, Gainesville, Florida 32611, USA.
3. Department of Medicine, University of Florida, P.O. Box 100278, Gainesville, FL 32611, USA.
4. Department of Pathology, Immunology and Laboratory Medicine, University of Florida, P.O. Box 100275, Gainesville, FL, 32610, USA.
5. Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611, USA.
*These authors contributed equally to this work.

✉ Corresponding authors: Z. Hugh Fan (hfanedu) and Thomas J. George (thom.georgeufl.edu)

Abstract

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.

Keywords: circulating tumor cells, cancer stem-like cells, pancreatic cancer, microfluidics

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