Cancer Biology

Numerous oncogenes have been identified to cause leukemia. For example, chromosomal translocation generates various fusion genes of the mixed-lineage leukemia (MLL) gene and a partner gene in leukemia, whose gene products transform primary myeloid progenitors into an immortalized state. To characterize the transforming ability of leukemic oncogenes, researchers in the field have developed an ex vivo murine myeloid transformation assay using retroviral gene transduction and its protocol has been improved over the past 10 years. Here, we provide the detailed procedure for this assay.

Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation strategies must preserve their viability. The ability to isolate different populations of cells from the bulk of the tumor will continue to deepen our understanding of the biology of cancer stem cells. Here, we report the strategy combining mechanical tumor dissociation, enzymatic treatment and flow cytometry to isolate a pure population of epithelial cancer stem cells from their native microenvironment. This technique can be useful to further functionally profile the cancer stem cells (RNA sequencing and epigenetic analysis), grow them in culture or use them directly in transplantation assays.

The in vitro limiting dilution assay is used to determine the colorectal cancer initiating cell (CC-IC) frequency of a CC-IC enriched suspension culture, grown in growth factor enriched serum free media. The in vivo limiting dilution assay is used to determine the colorectal cancer initiating cell frequency of a primary colorectal cancer sample or an established suspension cell line using immunocompromised murine xenograft models. In vitro and in vivo limiting dilution assays (LDAs) can be used to determine the effect of a specific treatment or genetic knockdown strategy on the initiating cell frequency of a population of CC-ICs or colorectal cancer sample, respectively.