Single-Cell RNA Sequencing of Pancreatic Circulating Tumor Cells.

Pancreatic cancer is one of the most aggressive malignancies, characterized by poor prognosis and high metastatic potential. Circulating tumor cells (CTCs) are critical mediators of metastasis and hold promise as biomarkers for early detection and therapy monitoring. However, their extreme rarity and heterogeneity pose significant challenges for their enrichment and analysis. Single-cell RNA sequencing (scRNA-seq) has emerged as a transformative technology to address these challenges by providing high-resolution insights into individual CTC biology.This chapter presents a comprehensive workflow for isolating and analyzing pancreatic cancer-derived CTCs using scRNA-seq. The protocol integrates size-based microfluidic enrichment, followed by negative selection via fluorescence-activated cell sorting (FACS), and concludes with single-cell transcriptome sequencing combined with target mutation detection. This method addresses critical purity and cell recovery limitations, allowing for the robust characterization of rare CTC populations. The insights gained from this workflow deepen our understanding of pancreatic cancer progression and facilitate the identification of novel therapeutic targets. By providing a practical example, this chapter serves as a valuable guide for researchers seeking to optimize single-cell sequencing techniques in their CTC studies.