Molecular characterization of quiescent melanoma cells

Cancer dormancy is closely associated with cellular quiescence, a state whereby cells exit the cell cycle and are reversibly arrested in G0 phase. Curative cancer treatment thus requires therapies that either sustain the dormant state of quiescent cancer cells, or preferentially, eliminate them. However, the mechanisms responsible for the quiescent survival of these cells remain obscure. We took advantage that quiescent cells characteristically negative for the proliferation marker Ki67 and express high levels of the cyclin-dependent kinase (CDK) inhibitor p27 to develop a CRISPR/Cas9-based system to fuse a green fluorescent protein (EGFP) gene with endogenous CDKN1B, the gene encoding p27, and a red fluorescent protein (mCherry) gene with endogenous MKI67, the gene encoding Ki67 in the genome of human melanoma cells. Flow cytometry were used to isolate the viable quiescent (p27high/Ki67low) cells. The isolated quiescent and cycling cancer cells were subjected to RNA-seq By use of CRISPR-Cas9 genome editing, we introduced sequences encoding EGFP at the locus of CDKN1B, the gene encoding p27, and sequences encoding mCherry at the locus of MKI67, the gene encoding Ki67, into Mel-RM melanoma cells. The cycling (C, EGFP-p27lowmCherry-Ki67high cells ) and quiescent (Q, EGFP-p27highmCherry-Ki67low cells ) populations were isolated from this dually edited cells and subjected to RNA sequencing.