Pre-existing cell states predict resistance to multiple treatments

Pre-existing differences between individual cancer cells can predict which cells will become resistant upon the application of treatment. This understanding has been furthered by novel methods in DNA barcoding that allow tracking of clones and their cell states during treatment. However, previous studies using these techniques have been limited in their scope, focusing on how single cell-states lead to resistance to a single treatment. In this study, we performed multi-treatment, high-throughput clonal tracking and single-cell RNA-sequencing to trace rare clones through the development of resistance to many different treatments in parallel with the goal of identifying cell states associated with multi-treatment resistance. We found that clones that will go on to develop resistance to one treatment had an increased chance of separately developing resistance to other treatments with diverse mechanisms of action. Additionally, we identified high CD44 expression in treatment-naive cells as a predictor of future resistance to multiple different treatments. Furthermore, for cells within the same treatment condition, we found that differences in gene expression states prior to treatment can lead cells to follow divergent paths towards their ultimate resistance fate. This work provides a framework for extracting targetable gene expression states from complex resistance dynamics across multiple treatments to eliminate multi-treatment resistance. Overall design: Trascribed clonal barcodes were introduced into melanoma cells and allowed to double so that there were multiple cells with each barcode. A subset of the population was then collected for scRNA-seq (called the untreated.naive sample). The remaining cells were then split six ways for treatment with four weeks of treatment with dabrafenib, trametinib, CoCl2, or acidic media or 2 weeks of treatment with cisplatin or doxorubicin followed by two weeks of treatment holiday. After four weeks, we performed scRNA-seq on the treated, now-resistant cells.