Variability within rare cell states enables multiple paths toward drug resistance

Nat Biotechnol. 2021 Jul;39(7):865-876. doi: 10.1038/s41587-021-00837-3. Epub 2021 Feb 22.


Molecular differences between individual cells can lead to dramatic differences in cell fate, such as death versus survival of cancer cells upon drug treatment. These originating differences remain largely hidden due to difficulties in determining precisely what variable molecular features lead to which cellular fates. Thus, we developed Rewind, a methodology that combines genetic barcoding with RNA fluorescence in situ hybridization to directly capture rare cells that give rise to cellular behaviors of interest. Applying Rewind to BRAFV600E melanoma, we trace drug-resistant cell fates back to single-cell gene expression differences in their drug-naive precursors (initial frequency of ~1:1,000-1:10,000 cells) and relative persistence of MAP kinase signaling soon after drug treatment. Within this rare subpopulation, we uncover a rich substructure in which molecular differences among several distinct subpopulations predict future differences in phenotypic behavior, such as proliferative capacity of distinct resistant clones after drug treatment. Our results reveal hidden, rare-cell variability that underlies a range of latent phenotypic outcomes upon drug exposure.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Cell Line
  • Cell Survival / drug effects*
  • Drug Resistance, Neoplasm*
  • Extracellular Signal-Regulated MAP Kinases / genetics
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • Integrin alpha3 / genetics
  • Integrin alpha3 / metabolism
  • Melanoma
  • Phosphorylation
  • Single-Cell Analysis
  • Vemurafenib / pharmacology*


  • Antineoplastic Agents
  • ITGA3 protein, human
  • Integrin alpha3
  • Vemurafenib
  • Extracellular Signal-Regulated MAP Kinases