Limiting dilution assay to quantify the self-renewal potential of cancer stem cells in hepatocellular carcinoma.

Cancer stem cells (CSCs) are a heterogeneous subpopulation of self-renewing cancer cells that sustain tumorigenesis in various types of human malignancies. Due to the diverse phenotypes of CSCs in distinct pathological conditions, it remains challenging to define CSCs by specific cell surface markers. Thus, it is essential to develop experimental protocols to quantify the self-renewal and tumorigenic potential of CSC for therapeutic purposes. To address these technical difficulties, we introduce the limiting dilution assay (LDA), a well-recognized experimental approach that accurately measures the self-renewal capacity and tumorigenicity of CSCs. Using hepatocellular carcinoma (HCC) cells as a model, we describe detailed experimental procedures for CSC culture in three-dimensional soft fibrin gel. Moreover, we provide an updated protocol for assessing CSC self-renewal in vitro and tumorigenicity in vivo in NOD/SCID IL-2Rγ (NSG) mice by LDA. Taken together, these protocols are readily applicable to laboratories with basic cell culture equipment and access to experimental animal facilities, providing a valuable toolbox to dissect mechanisms underlying HCC tumorigenesis and identify CSC-targeting drugs via high-throughput screening.