This project examines the CXCR4/ACKR3 signaling axis underlying cancer stem cell (CSC)-driven therapy resistance in colorectal cancer (CRC) using patient-derived organoids (PDOs). Doctoral researchers will establish CRC PDO biobank lines from primary tumors and metastases, engineer advanced co-culture assembloids incorporating CAFs and immune cells within organ-on-chip platforms, and perform longitudinal experiments tracking CSC enrichment (LGR5+, CD133+) and receptor dynamics under chemotherapy (5-FU, oxaliplatin) and targeted therapy pressure. Spatial transcriptomics, single-molecule imaging (e.g., CXCR4 internalization), single-cell proteomics, and CRISPR-based axis perturbation combined with high-throughput ligand screening will map bidirectional signaling networks promoting CSC maintenance, invasion, and immune evasion. Patient-correlated validation identifies predictive biomarkers and validates pharmacological ACKR3 antagonists or axis inhibitors, aiming to develop CSC-specific therapies that overcome CRC treatment resistance and improve progression-free survival.