In this study, we will develop a composite nanofibrous scaffold for cancer stem cell (CSC) enrichment.Chitosan and alginate will be coelectrospun using a dual-jet system to prepare nanofibrous matrix in differentratios, by which their physical, chemical, and mechanical properties can thus be delicately adjusted. Thesescaffolds will be applied to enrich CSCs from cancer cell lines, and the selected cells will be evaluated of theircapabilities of tumor spheroid formation, proliferation, invasion, and drug resistances. These cells will also beidentified by in vitro and in vivo systems. For in vitro experiments, the flow cytometry analysis will evaluatewhether the enriched cells show specific cell markers highly correlative to CSCs. Genomic and proteomicanalyses will also be performed to evaluate gene regulation and protein expression corresponding to cellstemness, angiogenesis, anti-apoptosis, extracellular matrix (ECM) invasion, drug resistances, epithelial tomesenchymal transition (EMT), and angiogenesis. Furthermore, the effects of ECM remodeling will beinvestigated by regulating the degradation of alginate fibers in composite scaffolds. This system can provideappropriate environmental cues because the remodeling of niches always occurs during tumor progression.Finally, the enriched cells will be implanted subcutaneously to athymic nude mice to validate their pathologicalproperties. After several-week implantation, the formed tumors will be harvest for sizing and weighting todetermine their tumor formation ability. Histological analysis and immunohistochemistry (IHC) staining willalso be performed to determine whether the implanted cells improve angiogenesis and exhibit invasion as wellas EMT which are direct evidences of metastasis and tumor recurrence. Through these assays we can determinethe extent to which the composite nanofibers promote CSCs enrichment. Furthermore, this system is highlyadjustable which can customize scaffold properties for screening cells derived from various tissues. Weanticipate that our dynamically controlled in vitro system can simulate in vivo tumor invasion, which can beapplied to high-throughput and cost-efficiently investigate the therapeutic effects of potential anti-cancercompounds on killing progressive tumors to reduce animal experiments and accelerate clinical cancer research.