Variability within rare cell states enables multiple paths toward drug resistance

Abstract

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 FISH to directly capture rare cells that give rise to cellular behaviors of interest. Applied to BRAF V600E melanoma, we trace drug-resistant cell fates back to single-cell gene expression differences in their drug-naive precursors (initial frequency of ~1:1000–1:10,000 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 between several distinct subpopulations predict future differences in phenotypic behavior, such as proliferative capacity of distinct resistant clones following drug treatment. Our results reveal hidden, rare-cell variability that underlies a range of latent phenotypic outcomes upon drug exposure.

Cell type determination for cardiac differentiation occurs soon after seeding of human induced pluripotent stem cells

“…Even if we were to take the B1 and B2 results without caveats, we could only interpret them to mean that a small subset of hiPS cells may be primed to take on certain ranges of final expression states. In contrast, when performing this analysis on barcoded vemurafenib-treated melanoma cells, a system with known heritable predetermination of the final cell state (Shaffer et al 2020; Schuh et al 2020; Emert et al 2021), barcoded clones in one split were far more similar (i.e. had lower Jensen-Shannon distance values) to the related clones in the other split than to clones labeled by a distinct barcode (Supplemental figure 4A).…”

“…Schematic of barcoding approach for labeling hiPS cells prior to cardiac differentiation. For the experiment shown, we transduced 4,000-8,000 hiPS cells at an MOI of ~0.03 with the Rewind (Emert et al 2021) barcode library. After 3 days (3-4 population doublings), we seeded cells for cardiac differentiation.…”

“…To package lentivirus we adapted protocols that have been previously described (Emert et al 2021; Torre et al 2021). Prior to plasmid transfection, we grew HEK293FT cells to 80-90% confluency in 10×15-cm plates in DMEM containing 10% FBS without antibiotics.…”

“…Barcode libraries were constructed as previously described (Emert et al 2021). Full protocol available at https://www.protocols.io/view/barcode-plasmid-library-cloning-4hggt3w.…”

A lineage tree-based hidden Markov model to quantify cellular heterogeneity and plasticity

“…Collecting data in which cells are tracked before and after drug treatment would enable some linkage between the pre-and post-exposure phenotypes of cells. 55 Finally, while we identify these states as subpopulations of cells that are phenotypically distinct, we currently cannot comment on the molecular factors leading to these phenotypes. However, we expect that single cell molecular analyses such as transcriptional pro!ling, tied with molecular barcodes, should allow us to align molecular and phenotypic states in the same populations of cells.…”