Tracking of Melanoma Cell Plasticity by Transcriptional Reporters

Clonal evolution and cellular plasticity are the genetic and non-genetic driving forces of tumor heterogeneity, which in turn determine tumor cell responses towards therapeutic drugs. Several lines of evidence suggest that therapeutic interventions foster the selection of drug-resistant neural crest stem-like cells (NCSCs) that establish minimal residual disease (MRD) in melanoma. Here, we establish a dual-reporter system, enabling the tracking of NGFR expression and mRNA stability and providing insights into the maintenance of NCSC states. We observed that a transcriptional reporter that contained a 1-kilobase fragment of the human NGFR promoter was activated only in a minor subset (0.72 ± 0.49%, range 0.3-1.5), and ~2-4% of A375 melanoma cells revealed stable NGFR mRNA. The combination of both reporters provides insights into phenotype switching and reveals that both cellular subsets gave rise to cellular heterogeneity. Moreover, whole transcriptome profiling and gene-set enrichment analysis (GSEA) of the minor cellular subset revealed hypoxia-associated programs that might serve as potential drivers of an in vitro switching of NGFR-associated phenotypes and relapse of post-BRAF inhibitor-treated tumors. Concordantly, we observed that the minor cellular subset increased in response to dabrafenib over time. In summary, our reporter-based approach provides insights into plasticity and identified a cellular subset that might be responsible for the establishment of MRD in melanoma.