Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product

Circulating Tumor Cells (CTCs) in blood encompass DNA, RNA, and protein biomarkers, but clinical utility is limited by their rarity. To enable tumor epitope-agnostic interrogation of large blood volumes, we developed a high-throughput microfluidic device, depleting hematopoietic cells through high-flow channels and force-amplifying magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from seven patients with metastatic cancer. High CTC yields (mean 10,057 CTCs per patient; range 100 to 58,125) reveal considerable intra-patient heterogeneity. CTC size varies within patients, with 67% overlapping in diameter with WBCs. Paired single-cell DNA and RNA sequencing identifies subclonal patterns of aneuploidy and distinct signaling pathways within CTCs. In prostate cancers, a subpopulation of small aneuploid cells lacking epithelial markers is enriched for neuroendocrine signatures. Pooling of CNV-confirmed CTCs enables whole exome sequencing with high mutant allele fractions. High-throughput CTC enrichment thus enables cell-based liquid biopsy for comprehensive monitoring of cancer.