High-throughput generation of patient-derived cancer stem cells for precision medicine using a microwell-chip platform.

Substantial evidence indicates that cancer stem cells (CSCs) drive breast cancer (BRC) initiation, progression, therapy resistance, recurrence and metastasis. Evaluating drug efficacy against patient-derived CSCs can advance precision oncology. However, high-throughput isolation of functional CSCs from small clinical samples remains challenging. Tumoursphere formation, arising from the clonal expansion of a single cancer cell, is a standard in vitro method for CSC assay and enrichment. Here we show a microwell cell-chip culture platform that enables rapid, high-throughput generation of uniform micro-tumourspheres (MTSs) from limited BRC specimens, such as puncture biopsies, facilitating functional CSC enrichment and personalized drug testing within 8 days. Single-cell transcriptomics and limiting dilution tumour initiation assay confirm that MTSs recapitulate CSC functionality, uniformity and heterogeneity. MTSs predicted clinical outcomes in 16 patients with BRC with ~93.8% accuracy, underscoring their translational potential for personalized drug testing. Overall, this platform provides a robust tool for CSC-targeted precision medicine.