Variability within rare cell states enables multiple paths toward drug resistance


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.

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Fig. 1: Rewind identifies rare cell states giving rise to vemurafenib-resistant colonies.
Fig. 2: A coordinated primed cell state characterized by high expression of multiple markers gives rise to vemurafenib resistance in WM989 A6-G3 cells.
Fig. 3: Resistance to vemurafenib is associated with single-cell variability in phosphorylated ERK levels 24 h after but not before treatment.
Fig. 4: Variation in gene expression among primed cells is associated with differences in resistant cell fate.
Fig. 5: Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant.
Fig. 6: DOT1Li inhibition enables a new subpopulation of cells to survive vemurafenib treatment without converting them into the known primed cell state.

Data availability

All RNA sequencing data generated for this study are available at the Gene Expression Omnibus (accession no. GSE161300). Additional sequencing and imaging data are available on Dropbox at and upon reasonable request to the corresponding author.

Code availability

Software used to segment cells and quantify RNA spots is available at Software used to stitch, segment and quantify scan images of resistant colonies is available at Additional custom image analysis scripts are available at The pipeline used for barcode sequencing analysis is available at


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We thank C. Bartman, A. Anguierra, J. Murray, N. Zhang, L. Cai, B. Stanger, A. Coté, K. Kiani, E. Sanford and Y. Goyal, along with other members of the Raj laboratory, for many useful suggestions. We thank the Flow Cytometry Core Laboratory at the Children’s Hospital of Philadelphia Research Institute for assistance in designing and performing FACS, including F. Tuluc for several helpful discussions. B.L.E. acknowledges support from NIH training grants F30 CA236129, T32 GM007170 and T32 HG000046; E.A.T. acknowledges support from R01 CA238237; I.P.D. acknowledges support from NIH 4DN U01 HL129998 and the NIH Center for Photogenomics (RM1 HG007743); C.L.J. acknowledges support from NIH T32 DK007780 and F30 HG010822; N.J. acknowledges support from NIH F30 HD103378; S.M.S. acknowledges support from DP5 OD028144; and A.R. acknowledges support from R01 CA238237, NIH Director’s Transformative Research Award R01 GM137425, R01 CA232256, NSF CAREER 1350601, P30 CA016520, SPORE P50 CA174523, NIH U01 CA227550, NIH 4DN U01 HL129998, the NIH Center for Photogenomics (RM1 HG007743) and the Tara Miller Foundation.

Author information




B.L.E. and A.R. conceived and designed the project with input from E.A.T. and S.M.S. B.L.E. performed experiments and analyses. I.P.D., C.L.J. and N.J. assisted with optimizing barcode RNA FISH protocols. C.J.C. assisted with cell and colony segmentation. A.R. supervised the project. B.L.E. and A.R. wrote the manuscript with feedback from S.M.S., C.L.J., N.J. and other members of the Raj laboratory.

Corresponding author

Correspondence to Arjun Raj.

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Competing interests

A.R. receives consulting income, and A.R. and S.M.S. receive royalties, related to Stellaris RNA FISH probes.

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Peer review information Nature Biotechnology thanks Leor Weinberger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Emert, B.L., Cote, C.J., Torre, E.A. et al. Variability within rare cell states enables multiple paths toward drug resistance. Nat Biotechnol (2021).

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