Drug-tolerant persister generation

Generation of Drug-Tolerant Persister Cells by Prolonged Drug Treatment

Background

Drug-tolerant persister (DTP) cells represent a small fraction of cancer cells capable of surviving prolonged exposure to drug concentrations that strongly inhibit or eliminate most of the parental population. Drug tolerance can be transient and reversible, although surviving populations may also contain cells with more stable resistance mechanisms. This protocol describes a general approach for generating drug-tolerant persister cells from adherent cancer cell lines, adapted from previously published methods (Sharma et al., 2010; Haven et al., 2016; Hangauer et al., 2017; França et al., 2024).

Materials and Reagents

• Drug-naive cancer cell line of interest
• Appropriate complete cell culture medium
• Drug of interest and corresponding vehicle
• Tissue culture dishes (100 or 150 mm)
• Multiwell plates for dose-response assays
• Phosphate-buffered saline (PBS)
• Trypsin-EDTA or appropriate cell dissociation reagent
• Cell viability assay reagents (e.g., CellTiter-Glo or equivalent)
• Cell counter or hemocytometer
• Standard cell culture equipment

Procedure

A. Determination of drug sensitivity and selection of treatment concentration

1. Determine the baseline drug sensitivity of the parental cell line using a dose-response viability assay. Seed cells into multiwell plates at an appropriate density, allow them to adhere overnight, and treat them with serial dilutions of the drug, including vehicle-treated controls.
2. Following an appropriate treatment period (e.g., 72h, 6 days, or 9 days), measure cell viability using an established assay (e.g., CellTiter-Glo). Normalize the measurements to vehicle-treated controls and generate a dose-response curve to estimate the half-maximal inhibitory concentration (IC50) and, when possible, the concentration corresponding to 90% inhibition (IC90).
3. Based on the dose-response results, select a drug concentration that produces strong inhibition of the parental population. Concentrations at or above the IC90 can serve as a useful starting point for generating persisters, but the optimal concentration should be determined empirically for each cell line and drug combination.
4. If necessary, perform preliminary prolonged-treatment experiments to identify a concentration that consistently eliminates or strongly inhibits most drug-sensitive cells while allowing a small viable population to survive.

Note: The IC50 and IC90 provide reference points rather than strict thresholds for persister generation. Drug concentrations and response kinetics vary substantially between cell lines and compounds, and inhibition measured in short-term assays may not fully predict survival during prolonged treatment. Therefore, the selected concentration and time of drug exposure should be validated under the experimental conditions used to generate persisters.

B. Generation of drug-tolerant persister cells

1. Seed drug-naive cells into tissue culture dishes at a density sufficient to recover surviving cells following prolonged drug exposure. For reference, approximately 1 × 10⁶ cells per 150 mm dish have been used successfully, although the seeding density should be adjusted according to the growth characteristics of the cell line and the expected surviving fraction.
2. Allow cells to adhere overnight under standard culture conditions.
3. Replace the medium with a fresh complete medium containing the selected drug concentration. Maintain parallel vehicle-treated control cultures.
4. Treat cells continuously with the selected drug for a period sufficient to eliminate or strongly inhibit the majority of drug-sensitive cells and establish a residual surviving population. Treatment duration should be optimized for each cell line and drug combination. Published protocols commonly use approximately 9 days of treatment, although shorter or longer periods may be appropriate depending on the experimental model and treatment response.
5. Replace the culture medium with fresh drug-containing medium at regular intervals (e.g., every 2–3 days), adjusting the frequency according to drug stability, medium requirements, and cell culture conditions.
6. Monitor cell morphology, survival, and proliferation periodically using microscopy or an appropriate viability assay. During treatment, most drug-sensitive cells are expected to undergo cell death or growth arrest, while a smaller fraction may remain viable.
7. At the selected experimental endpoint, carefully remove the medium and non-adherent cellular debris. Viable cells remaining attached to the culture surface following sustained drug exposure are defined as drug-tolerant persisters (DTPs).
8. Collect the surviving cells using an appropriate dissociation reagent (e.g., Trypsin) or process them directly for downstream experiments, including viability assays, transcriptomic profiling, or other molecular and functional analyses.
9. When required, quantify the viable persister population by cell counting or an appropriate viability assay. The number of surviving cells can be expressed relative to the initial number of seeded cells, with the understanding that this measurement does not account for cell division or death occurring during treatment.

Note: Treatment duration should be established empirically based on the dynamics of the cellular response. Ideally, the endpoint should capture a small, viable residual population after substantial depletion of drug-sensitive cells, before extensive expansion of resistant populations. Prolonged drug exposure may promote the emergence of proliferating drug-tolerant expanded persisters (DTEPs) or more stably resistant cells. 

C. Optional: Assessment of drug-tolerance reversibility

1. Following the initial drug treatment, remove the drug-containing medium and replace it with fresh complete medium without drug.
2. Allow surviving cells to recover under drug-free conditions for a period appropriate for the cell line. The recovery period should allow sufficient regrowth for subsequent analysis and may be defined by elapsed time or population doublings.
3. Perform a new drug dose-response viability assay on the recovered population and compare its sensitivity with that of the original drug-naive parental cells.
4. If desired, perform additional treatment-recovery cycles using the same drug concentration and experimental conditions to assess the reproducibility of persister generation and changes in drug sensitivity following repeated exposure.
5. Compare the dose-response profiles of parental and recovered populations. Restoration of drug sensitivity following withdrawal is consistent with reversible drug tolerance.

Note: Reversibility of drug sensitivity supports a transient drug-tolerant phenotype but does not conclusively establish its underlying mechanism. Repeated treatment-recovery experiments can assess the reproducibility and stability of the response but cannot, on their own, exclude selection of pre-existing resistant subpopulations. 

Important Considerations

• Perform experiments using independent biological replicates, preferably at least three.
• Drug concentration, treatment duration, and initial seeding density should be optimized for each cell line and compound.
• The treatment endpoint should be selected according to the dynamics of drug-induced cell depletion and survival, rather than a universally prescribed duration.
• Distinguish the initial residual persister population from proliferating drug-tolerant expanded persisters or stably resistant populations that may emerge during prolonged treatment.
• When comparing experimental conditions, maintain consistent treatment schedules, document the total duration of drug exposure, and include appropriate vehicle-treated controls.

References

1. Sharma SV et al. (2010). A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 141, 69–80. https://doi.org/10.1016/j.cell.2010.02.027
2. Haven B et al. (2016). Registered report: A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. eLife 5, e09462. https://doi.org/10.7554/eLife.09462
3. Hangauer MJ et al. (2017). Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature 551, 247–250. https://doi.org/10.1038/nature24297
4. França GS et al. (2024). Cellular adaptation to cancer therapy along a resistance continuum. Nature 631, 876–883. https://doi.org/10.1038/s41586-024-07690-9