Single-cell transcriptomic profiling of platelet-adherent circulating tumor cells using a microcavity-gel manipulation platform.

Pancreatic cancer remains one of the most lethal malignancies, with a 5-year survival rate of less than 9 %, primarily due to its aggressive metastasis and drug resistance. Circulating tumor cells (CTCs), which are key mediators of metastasis, are critical for understanding these clinical challenges. In this study, we applied a single-cell transcriptome analysis platform combining a microcavity array (MCA) and gel-based cell manipulation (GCM) technique for marker-independent cell recovery to analyze CTCs from patients with metastatic pancreatic cancer. Using pancreatic cancer cell lines, we demonstrated that this microcavity-gel manipulation platform enables high-quality single-cell RNA sequencing while preserving intrinsic molecular characteristics. Furthermore, spiking experiments with cancer cells in blood confirmed that the process minimizes contamination from non-target blood cells. Application of this platform to patient-derived CTCs revealed that most CTCs exhibited epithelial-mesenchymal transition (EMT)-like features and high expression of platelet-related genes such as PF4 and PPBP, suggesting platelet-driven EMT activation. In addition, CTCs were consistently arrested in the G1 phase of the cell cycle, implying a potential survival mechanism under therapeutic stress. These findings highlight the utility of microcavity-gel manipulation platform for robust single-cell transcriptomic profiling and provided novel insights into the biology of pancreatic CTCs.