Microfluidic-based isolation of circulating tumor cells in pancreatic cancer patients

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with poor survival rate largely owing to its usual late diagnosis at an advanced metastatic stage. Circulating tumor cells (CTCs) originate from primary and metastatic tumor foci and circulate in the peripheral blood of PDAC patients. Liquid biopsies allow the acquisition of CTCs, which can be used as cellular biomarkers for assessing disease state, molecular phenotype, and genotype. For this thesis, we devised a workflow for the efficient detection and retrieval of CTCs from the peripheral blood of PDAC patients based on microfluidic isolation with Parsortix™ and immunostaining. We first confirmed the robustness of a novel α-Caprin-1 antibody to specifically bind to primary PDAC cells as assessed by flow-cytometry. Then, we established an immunostaining protocol and confirmed the antibody’s binding to primary PDAC cells fixed on microscopic slides or in microscopic chamber slides. We further corroborated this protocol on PDAC cells spiked into blood from healthy donors and isolated by Parsortix™. Finally, we isolated CTCs from the peripheral blood of various PDAC patients of various disease stages. Retrieved single CTCs were collected using the single-cell picking system CellCelector™ (ALS, Jena, Germany). Our results confirm the specificity of the α-Caprin-1 antibody for tumor cells. The developed workflow provides a promising method for the successful identification, enumeration, and isolation of CTCs from patient blood samples. The obtained CTCs are currently being used for downstream single-cell RNA-seq (scRNA-seq) to allow full transcriptome analysis. As such, our data provides an efficient method of the capture, identification and isolation of CTCs, a crucial step towards their analysis which could potentially validate the relevance of liquid biopsies as a simple and non-invasive diagnostic, prognostic, and predictive tool for PDAC.