Identification and isolation pipeline for single cell transcriptomic of magnetically enriched tumor cells

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.