High-throughput generation of tumor-initiating cell-derived micro-tumorspheres for robust personalized drug testing in breast cancer

Many technologies emerged to establish tumor organotypic models for precision medicine; however, inherent variability and complexity of these models limit their application in therapeutic decision-making. Here, we develop a microwell cell-chip culture platform capable of co-culturing approximately 100 dissociated tumor cells within individual microwells under defined low-attachment and Matrigel-free selective conditions. This platform facilitates high-throughput formation of uniform micro-tumor spheres (MTSs) derived from single stress-tolerant tumor-initiating cells (TICs) within a heterogeneous tumor microenvironment (TME), ensuring the recapitulation of TIC population heterogeneity within primary tumors as confirmed by single-cell transcriptomics analysis. Our platform enables the generation of 5,000-12,000 MTSs comprising exclusively TICs from small (~50 mg) breast cancer (BRC) samples and allows for personalized drug testing on puncturing biopsies with high reproducibility within 10 days, resulting in an impressive accuracy rate of 93.75% in predicting clinical outcomes for 16 BRC patients in drug testing using MTSs. Additionally, the MTSs can be co-cultured with CD8+ T cells to evaluate the efficacy of immune-oncology (IO) therapies such as PD-1 blockade antibody treatment. This platform offers a robust tool for isolating functional TICs from patient tumors and generating uniform TIC-derived MTS models in a simple and high-throughput manner, thereby facilitating personalized drug testing specifically targeting refractory TICs.