Phenotypic and Mechanistic Characterization of Drug-Tolerant Persisters

Introduction

Drug-tolerant persisters are thought to represent a small subpopulation of cancer cells that can survive drug treatments despite the absence of resistance-mediating genetic alterations. Indeed, upon treatment withdrawal, these cells give rise to a new population with comparable drug sensitivity (as measured by IC50) to that of the treatment-naïve population. As persisters can give rise to genetically resistant cells, understanding the mechanisms that enable them to persist drug treatment is crucial for providing better treatment options for cancer patients.

Material and method

Using cancer cell lines as a model system, we have generated hundreds of single-cell derived colonies from each cell line and tested their persister percentage upon drug treatment. To identify single cell biomarkers of persistence, as well as the underlying molecular mechanisms of survival, we subjected colonies to karyotyping, RNA-Sequncing, proteomics, phospho-proteomics and apoptotic profiling by apoptosis antibody array. High throughput drug screens were used to find modulators of persister percentage in response to anti-cancer drugs.

Results and discussion

We found that persisters are not a well-defined subpopulation, but rather, a continuous trait in which every cell in the population has a specific “Chance to Persist” (CTP) a drug treatment. Clone-specific CTPs were found to be stable over months in culture, and the CTPs of their sub-clones averaged around the CTP of the mother clone with some drift to both directions. We show that there is no correlation between the IC50 of growth inhibition and CTP across clones. Indeed, while persister progenies have the same IC50 values as the treatment naïve population, their percentages of persisters are significantly higher. A detailed study of clones with a wide range of CTP values demonstrated that CTP does not correlate with growth rate, cell cycle status or EMT markers and that clones have a target-specific CTP value. We also found several biomarkers that correlate with CTP. A potential mechanism underlying the CTP of non-small cell lung cancer cells treated with EGFR inhibitors will also be discussed.

Conclusion

Overall, our study demonstrates that the chance to persist anti-cancer drugs does not depend on a pre-existing subpopulation of drug-tolerant persister cells that later give rise to sensitive cells, but rather, is a continuous and inheritable trait. Our findings have multiple clinical implications for cancer patient treatment.