Dynamic Phenotypic Switching and Group Behavior Help Non-Small Cell Lung Cancer Cells Evade Chemotherapy

Abstract

Drug resistance, a major challenge in cancer therapy, is typically attributed to mutations and genetic heterogeneity. Emerging evidence suggests that dynamic cellular interactions and group behavior also contribute to drug resistance. However, the underlying mechanisms remain poorly understood. Here, we present a new mathematical approach with game theoretical underpinnings that we developed to model real-time growth data of non-small cell lung cancer (NSCLC) cells and discern patterns in response to treatment with cisplatin. We show that the cisplatin-sensitive and cisplatin-tolerant NSCLC cells, when co-cultured in the absence or presence of the drug, display dynamic group behavior strategies. Tolerant cells exhibit a 'persister-like' behavior and are attenuated by sensitive cells; they also appear to 'educate' sensitive cells to evade chemotherapy. Further, tolerant cells can switch phenotypes to become sensitive, especially at low cisplatin concentrations. Finally, switching treatment from continuous to an intermittent regimen can attenuate the emergence of tolerant cells, suggesting that intermittent chemotherapy may improve outcomes in lung cancer.

Keywords: chemoresistance; cisplatin; evolutionary game theory; group behavior; lung cancer; persister trait; phenotypic switching.

Figure 1

Schematic summarizing the experiment and the source of data used in developing the theoretical cell growth models. (A) The schematic representation of the different incubation duration, ratios, and treatments used for generating the data to develop the mathematical model. (B) schematic describing the principles on which the mathematical model PSMSR was developed; (C) panel representing the functional form of PSMSR (please see the main text for further details).

Figure 2

Behavior of cisplatin-sensitive (S) and tolerant (T) NSCLC cells in 2D co-culture. (A) Schematic representation of the experimental design of co-culturing S and T cells in a ratio of 1:1 and collection of data points. Proliferation of sensitive (red) and tolerant (green) cells under different culture conditions in the absence or presence of cisplatin. Two-way ANOVA test (multiple comparison) showing statistical significance **** p < 0.0001. (B) Sensitive and tolerant cells were plated in increasing T:S ratios and cultured for three weeks. Proliferation rate of sensitive cells (red) and tolerant cells (green) in heterotypic culture over the course of 144 h. (C) Fold change in cell count of sensitive cells (red) and tolerant cells (green) in heterotypic culture was measured after 144 h for ratios 1:1, 2:1, 4:1 and 8:1. Two-way ANOVA was used for calculating statistical significance **** p < 0.0001, ns—not significant. (D) Fold change in cell count of sensitive cells (purple) and tolerant cells (blue) in heterotypic culture was measured after 144 h in presence of cisplatin for ratios 1:1, 2:1, 4:1 and 8:1. Two-way ANOVA was used for calculating statistical significance *** p < 0.0001, ns—not significant. (E) Change in tolerant/sensitive cells ratio with (orange) and without (black) 5 μM cisplatin over the course of 144 h was measured. Statistical significance * p ≤ 0.05, **** p < 0.0001, ns—not significant. (F) Schematic representation of the conditioned medium experiment. (G) The left line graph representing the effect of tolerant cell conditioned medium on sensitive cells, and the right line graph representing the inhibitory effect of tolerant cell conditioned medium on sensitive cells growth. (H) Schematic representation of conditioned medium experiment to correlate the stoichiometry between cell number and inhibitory effect secreted by sensitive cells. (I) The bar graph representing the inhibitory effect of condition medium on different cell number of tolerant or sensitive cells. Statistical significance information can be found in Supplementary Tables S2 and S3.

Figure 3

Tolerant cells reversibly switch their phenotype to become sensitive with intermittent therapy. (A–C) Bar graph showing the ratio of tolerant versus sensitive cell population over a period of 10 days. The cell ratio for the “Continuous” group wherein the cells were continuously treated with cisplatin is shown in blue and the ratio for the “Intermittent” group wherein the cells were treated with cisplatin for two days and released in fresh medium (intermittent) is shown in black. (D–F) Media from “Intermittent—2 cycles” group was removed after four days of cisplatin treatment and replaced with fresh medium and the cells were allowed to grow until confluent. These cells were monitored in real-time to determine the ratio of tolerant versus sensitive over the course of 25 days. Similarly, the cells that only received cisplatin once (“Intermittent—1 cycle”) throughout the experiment were also followed for 25 days. (G) Sensitive (S, red fluorescence) and tolerant (T, green fluorescence) cells were mixed at S:T ratio of 4:1 and microinjected into the perivitelline space of zebrafish larvae 48-h post fertilization (hpf). Twenty-four hours after microinjection, larvae were randomly divided into three groups: Group 1 received no drug treatment (Untreated), Group 2 received cisplatin 20 µM for three days and released with no drug for two days (Intermittent), and Group 3 received cisplatin 20 µM continuously for five days (Continuous). Ratio of tolerant versus sensitive cells was determined by measuring fluorescence intensity. One way ANOVA was used for calculating statistical significance ** p < 0.05, *** p < 0.001, ns—not significant (H–J) Effect of suberoylanilide hydroxamic acid (SAHA) on cisplatin-sensitive (H23), -tolerant (H2009), and -resistant (H1993) cells, demonstrating that SAHA can reverse the phenotype of H2009 from tolerant state to sensitive state. Statistical significance information can be found in Supplementary Table S4.

Figure 4

Cooperativity and stress response as described by the PSMSR model. (A) Schematic describing the PSMSR model; initially, the sensitive and the tolerant cells proliferate independently; as stress builds up, sensitive cells switch their phenotype to tolerant cells and vice versa; tolerant cells remove stress and maintain a small population, while enabling the sensitive cells to proliferate. (B,C) Fitting of the phenotype-switch model to the cellular growth curves of sensitive and tolerant cell populations, where the cells were mixed at different proportions and counting was started immediately; the colors represent the growth curves from different initial seeding proportions, as indicated in the legend (sensitive to tolerant cell seeding ratios); (D–F) predicted evolution of phenotypic switching and stress in monotypic cultures; (D,E) populations of sensitive and (switched) tolerant phenotypes with time, when seeded with sensitive cells only; (F) stress as function of time; (G–I) predicted evolution of switched phenotypes and stress in heterotypic culture experiments, where cell growth was monitored immediately after mixing; (G) fraction of sensitive cells that have switched to the tolerant phenotype, as function of time; (H) fraction of tolerant cells that have switched to the sensitive phenotype, as function of time; (I) stress with time; colors are according to the initial seeding ratio of sensitive to tolerant cells as shown in the legend; the total cell population in each case was close to 5000; (J,K) evolving game strategy landscape of cellular population due to stress and phenotypic switching; the heatmaps of time varying payoff values representative of inter-species competition/cooperation are shown as function of the sensitive-to-tolerant seeding ratio; payoff values are derived by fitting the PSMSR model to the competitive Lotka–Volterra equations; orange areas in the maps represent competitive behavior, green areas represent cooperative behavior; (J) α₁₂ representing the effect of tolerant cells towards the sensitive cells; (K) α₂₁ representing the effect of sensitive cells towards the tolerant cells.

Figure 5

Mathematical model for cisplatin resistance. (A) Schematic demonstration of AUC and cellular death rate as function of AUC; (B,C) fitting of the experimental growth data where the cells were co-cultured for three weeks; (B): sensitive cells; (C): tolerant cells; circles and lines represent the experimental and fitted trends respectively; (D) SCALE parameter as measure of cisplatin sensitivity for the sensitive and the tolerant cells; the error bars represent 95% confidence limits (E,F) simulation of intermittent and continuous cisplatin treatment according to the protocols described in Figure 3; the initial sensitive to tolerant cell ratio was set to 4:1 with a total cell population of 50,000. (G) An illustrative model depicting the presence (and absence) of group behavior among sensitive and tolerant cells under varying conditions of stress and effects of continuous versus intermittent therapy.