Hsu‐Huan Chou, Ting-Fang Che, K. J. Lee, Shin‐Cheh Chen, Jia-Yang Chen, Yen-Jang Huang, Syer Choon Lim, Shih‐Chiang Huang, Chia-Lung Tsai, Chia-Lung Tsai, Ying Chang, Chi-Neu Tsai, Chi-Neu Tsai
PURPOSE: Tumor heterogeneity and drug resistance remain key challenges in breast cancer management. While organoid cultures can be established from resected tumor specimens, patients undergoing systematic treatment often lack accessible tissue for ex vivo drug testing. Circulating tumor cells (CTCs) offer a minimally invasive alternative. The purpose of this study was to establish a clinically feasible workflow that integrates CTC enumeration and CTC-derived spheroid drug screening to overcome the limitations of tissue-dependent organoid models and enable personalized treatment in breast cancer. METHODS: CTCs were isolated from 34 newly diagnosed breast cancer patients using a liposome-tethered supported lipid bilayer (LIPO-SLB) microfluidic platform functionalized with anti-EpCAM antibodies. Thirteen patients undergoing systemic therapy and relapse were further assessed using CTC-spheroid culture for ex vivo drug screening. Hormone receptor expression and genomic mutation profiles were integrated with drug testing. One relapsed case underwent pre- and post-treatment spatial transcriptomic analysis (Xenium in situ). RESULTS: CTC and CTC-cluster counts significantly declined post-treatment in responders (p=0.044 and p=0.0264, respectively), but not in non-responders. CTC-spheroids were successfully generated in all 13 cases, enabling ex vivo drug testing. Effective therapies were identified in 9 patients (69.2%), of whom 7 achieved partial responses and 1 achieved stable disease; one patient did not receive the suggested treatment. Ex vivo drug sensitivity results correlated with clinical outcomes. Integrating CTC-based drug testing, hormone receptor expression in CTCs, and genomic DNA mutation profile further improved prediction of radiological response. Spatial transcriptomics revealed therapy-induced clonal shifts, with resistant subpopulations characterized by over-expression of chemoresistant gene, EIF4EBP1. CONCLUSION: This study demonstrates that CTC-derived spheroid drug screening provides actionable therapeutic guidance when tissue is unavailable. Combined with hormone receptor and genomic profiling, this liquid biopsy-based platform enables personalized treatment, and dynamic monitoring of resistance in breast cancer.