Cancer stem cells (CSCs) represent a rare yet critically important subpopulation of tumor cells that drive tumor initiation, progression, and therapy resistance through their unique capabilities of self-renewal and differentiation. Their role in mediating chemoresistance and radioresistance makes them a pivotal target for developing more effective cancer treatments. This review provides a comprehensive overview of current methodologies for studying CSCs, beginning with established techniques for their identification and isolation, including surface marker profiling and functional assays such as ALDH activity detection and side population analysis. We examine in vitro approaches including tumorsphere formation assays, colony formation assays for evaluating CSC tumorigenicity. The discussion then explores cutting-edge technologies that are transforming CSC research, including microfluidics platforms, laser capture microdissection for precise isolation, and advanced single-cell multi-omics approaches that reveal CSC heterogeneity at unprecedented resolution. Particular emphasis is placed on innovative in vitro models such as 3D spheroids and organoids that faithfully recapitulate the tumor microenvironment, enabling more accurate study of CSC behavior and drug resistance mechanisms. Additionally, we highlight the emerging role of miRNAs as promising CSC biomarkers and discuss how integrating these diverse methodologies can provide new insights into CSC biology and accelerate the development of targeted therapeutic strategies to overcome treatment resistance.
Farshbaf et al. (Mon,) studied this question.