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Published September 13, 2019 | Submitted
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Trajectories from Snapshots: Integrated proteomic and metabolic single-cell assays reveal multiple independent adaptive responses to drug tolerance in a BRAF-mutant melanoma cell line


The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge, with relevance towards understanding biological changes ranging from cellular differentiation to epigenetic (adaptive) responses of diseased cells to drugging. We report on a combined experimental and theoretic method for determining the trajectories that specific highly plastic BRAFV600E mutant patient-derived melanoma cancer cells take between drug-naive and drug-tolerant states. Recent studies have implicated non-genetic, fast-acting resistance mechanisms are activated in these cells following BRAF inhibition. While single-cell highly multiplex omics tools can yield snapshots of the cell state space landscape sampled at any given time point, individual cell trajectories must be inferred from a kinetic series of snapshots, and that inference can be confounded by stochastic cell state switching. Using a microfludic-based single-cell integrated proteomic and metabolic assay, we assayed for a panel of signaling, phenotypic, and metabolic regulators at four time points during the first five days of drug treatment. Dimensional reduction of the resultant data set, coupled with information theoretic analysis, uncovered a complex cell state landscape and identified two distinct paths connecting drug-naive and drug-tolerant states. Cells are shown to exclusively traverse one of the two pathways depending on the level of the lineage restricted transcription factor MITF in the drug-naive cells. The two trajectories are associated with distinct signaling and metabolic susceptibilities, and are independently druggable. Our results update the paradigm of adaptive resistance development in an isogenic cell population and offer insight into the design of more effective combination therapies.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. bioRxiv preprint first posted online Sep. 12, 2019. We thank members of the Heath and Baltimore laboratories for helpful comments on the manuscript. We acknowledge the following agencies and foundations for support: NIH Grants U54 CA199090 (to J.R.H., D.B., W.W., and A.R.), U01 CA217655 (to J.R.H. and W.W.), P01 CA168585 (to A.R.), R35 CA197633 (to A.R.), and U54CA209971 (to S.K.P.); the Dr. Robert Vigen Memorial Fund, the Ressler Family Fund, and Ken and Donna Schultz (A.R.); the Jean Perkins Foundation (J.R.H.); ISB Innovator Award (Y.S.). L.R. was supported by the V Foundation-Gil Nickel Family Endowed Fellowship and a scholarship from SEOM. M.E.K. was supported by the National Cancer Institute of the National Institutes of Health under Award Number F99 CA212231 and Stanford University's Diversifying Academia, Recruiting Excellence Fellowship. We acknowledge Rochelle Diamond and the Caltech Flow Cytometry Cell Sorting Facility for FACS analysis and advice. Author Contributions: J.R.H. and Y.S. conceived the study. Y.S. and G.D. designed the experiments. Y.S., G.D, H.C., R.Z, M.X., J.W., S.W., J.L., K.T., J.L., L.K., A.X., and L.R. performed the experiments. M.E.K., Y.S., W.W. and R.L. analyzed and interpreted the data. J.R. H., W.W., S.K.P., G.P.N, S.H., A.R., and M.E. provided conceptual advice on the data analysis and interpretation. Y.S., G.D., M.E.K. and J.R.H. wrote the manuscript. J.R.H. and D.B. supervised this study. Competing Interests: J.R.H. and A.R. are affiliated with Isoplexis, which is seeking to commercialize the single-cell barcode chip technology. All other authors declare no competing financial interests.

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