Identification and isolation pipeline for single cell transcriptomic of magnetically enriched tumor cells
Peng Liu,
Sitian He,
Mette Ø. Agerbæk,
Ali Salanti,
Leon W.M.M. Terstappen,
Pascal Jonkheijm,
Michiel Stevens
Affiliations
Peng Liu
Personalized Diagnosis & Therapeutics, Department of Bioengineering Technologies, Technical Medical Center, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, the Netherlands; Department of Medical Cell Biophysics, TechMed Center, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, the Netherlands; Department of Molecules and Materials, Laboratory of Biointerface Chemistry and the TechMed Centre, University of Twente, Enschede, the Netherlands
Sitian He
Department of Medical Cell Biophysics, TechMed Center, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, the Netherlands; Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), 570311 Haikou, China
Mette Ø. Agerbæk
Centre for translational Medicine and Parasitology at Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen and Department of Infectious Diseases, Copenhagen University Hospital, 2200 Copenhagen, Denmark; VarCT Diagnostics, 2000 Frederiksberg, Denmark
Ali Salanti
Centre for translational Medicine and Parasitology at Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen and Department of Infectious Diseases, Copenhagen University Hospital, 2200 Copenhagen, Denmark
Leon W.M.M. Terstappen
Department of Medical Cell Biophysics, TechMed Center, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, the Netherlands; Department of General, Visceral and Pediatric Surgery, Heinrich-Heine University, University Hospital Düsseldorf, 40225 Düsseldorf, Germany
Pascal Jonkheijm
Department of Molecules and Materials, Laboratory of Biointerface Chemistry and the TechMed Centre, University of Twente, Enschede, the Netherlands; Corresponding authors.
Michiel Stevens
Department of Medical Cell Biophysics, TechMed Center, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, the Netherlands; FETCH BV, Deventer, the Netherlands; Corresponding authors.
Introduction: The transcriptomic analysis of circulating tumor cells provides valuable insights into cancer metastasis, heterogeneity and the identification of drugable targets. However, traditional methods for circulating tumor cel identification and isolation often compromise mRNA integrity, due to cell handling such as the cell permeabilization, required for intracellular staining. Here, we present a novel identification and isolation pipeline for single cell transcriptomics of magnetically enriched tumor by combining rVAR2 based identification cells with single-cell magnetic pickup. Methods: To test our method, we used tumor cells from four lung cancer cell lines spiked into blood. After magnetic enrichment using the CellSearch® Profile Kit, the resulting sample was divided for staining with either rVAR2 or CellSearch reagents. Single cells were isolated using a magnetic needle and analyzed for mRNA integrity using reverse transcription quantitative polymerase chain reaction (RT-qPCR) targeting the GAPDH and EpCAM genes. Results: The integration of single-cell magnetic pickup enabled precise single-cell separation, while the extracellular rVAR2 staining enables mRNA preservation, collectively facilitating the detection of low-expressed genes at the single-cell level. Compared to CellSearch staining, the rVAR2 staining resulted in our pipeline in a approximately 10 to 100 times higher mRNA yield from single magnetically enriched tumor cells. Conclusion: This mRNA-friendly method enhances our ability to study tumor heterogeneity at the single-cell level and supports the development of personalized cancer therapies, making it a valuable tool for circulating tumor cel research and clinical applications.
