Testing Adaptive Therapy Protocols using Gemcitabine and Capecitabine on a Mouse Model of Endocrine-Resistant Breast Cancer

Abstract

Highly effective cancer therapies often face limitations due to acquired resistance and toxicity. Adaptive therapy, an ecologically inspired approach, seeks to control therapeutic resistance and minimize toxicity by leveraging competitive interactions between drug-sensitive and drug-resistant subclones, prioritizing patient survival and quality of life over maximum cell kill. In preparation for a clinical trial in breast cancer, we used large populations of MCF7 cells to rapidly generate endocrine-resistance breast cancer cell line. We then mimicked second line therapy in ER+ breast cancers by treating the endocrine-resistant MCF7 cells in a mouse xenograft model to test adaptive therapy with capecitabine, gemcitabine, or the combination of those two drugs. Dose-modulation adaptive therapy with capecitabine alone increased survival time relative to MTD, but not statistically significant (HR: 0.22, 95% CI 0.043- 1.1 P = 0.065). However, when we alternated the drugs in both dose modulation (HR = 0.11, 95% CI: 0.024 - 0.55, P = 0.007) and intermittent adaptive therapies significantly increased survival time compared to high dose combination therapy (HR = 0.07, 95% CI: 0.013 - 0.42; P = 0.003). Overall, survival time increased with reduced dose for both single drugs (P < 0.01) and combined drugs (P < 0.001). Adaptive therapy protocols resulted in tumors with lower proportions of proliferating cells (P = 0.0026) and more apoptotic cells (P = 0.045). The results show that Adaptive therapy outperforms high-dose therapy in controlling endocrine-resistant breast cancer, favoring slower-growing tumors, and showing promise in two-drug alternating regimens.

Fig. 1.. Schematic Figure of Comparison Adaptive Therapy Protocols with Standard Therapy.

(A) Standard therapy selects for cells (red) that are resistant to treatment and tumor relapse. Adaptive therapy maintains a stable tumor volume by preserving drug-sensitive cells (blue) that suppress the growth of less fit, resistant cells (red). (B) Dose modulation adaptive therapy that raises the dose if the tumor grows and lowers the dose if the tumor shrinks. Previous mouse models used a tumor burden change of 20% to trigger a change in dose (2). Our simulation studies suggest a lower threshold is better (17), so here we have used a 10% change in tumor burden to trigger a change in dose. C. Intermittent adaptive therapy stops dosing altogether if the tumor burden falls below a threshold (e.g., 50% of its initial value), and restarts treatment if the tumor recovers (e.g., to 100% of its initial value) (6,9).

Fig. 2.. Drug dose-response curves for sensitive and resistant MCF7/luc cell lines.

MCF7 resistant (MCF7 R) and sensitive (MCF7 S) cell lines showed an IC50 value of 62.65 uM and 13.13 uM respectively. The percentage of viability at 40 uM, 50 uM, and 60 uM were significantly different between MCF7 R and MCF7 S cell lines. Each data point represents 8 replicates.

Fig. 3.. Survival analysis of Single-drug therapies.

(A) Survival analysis of capecitabine protocols. Cox Regression (CAP Dose modulation relative to no treatment HR: 0.24, 95% CI 0.042– 1.4, P = 0.1; CAP Intermittent relative to no treatment HR: 0.96, 95% CI 0.247– 3.7, P = 0.95; CAP MTD relative to no treatment HR: 1.2, 95% CI 0.323– 4.5, P = 0.78). Cox Regression (CAP Dose modulation relative to MTD HR: 0.22, 95% CI 0.043– 1.1 P = 0.065; CAP Intermittent relative to MTD HR: 0.78, 95% CI 0.236– 2.6, P = 0.68). (B) Survival analysis of gemcitabine protocols. None of the protocols are statistically significantly different using Cox regressions.

Fig. 4.. Survival analysis of multi-drug therapies.

(A) Multi-drug ping-pong adaptive therapy protocols versus MTD or no treatment (vehicle control). In ping-pong protocols, only one drug is used at a time. Here MTD is the application of MTD of both gemcitabine and capecitabine using the same dose scheduling as single drug MTD. (B) Multi-drug tandem adaptive therapy protocols versus MTD or no treatment (vehicle control). In the tandem and MTD protocols, both gemcitabine and capecitabine are given (and modulated) at the same time.

Fig. 5.. Correlation Between the Percentage of Maximum Tolerated Drug Dose Used with Survival Time.

(A) Survival time as a function of the amount of combined gemcitabine and capecitabine that was given per day. Mice that were given more chemotherapy tended to have a shorter survival time (P-value <0.0001, R² = 0.56). (B) Survival time as a function of the amount of single drug (either gemcitabine or capecitabine) that was given per day. Although there is a lot of variances associated with different protocols, there was still a strong trend that mice treated with more drug had a shorter survival time (P-value = 0.0074, R² = 0.19).

Fig. 6.. IC50 values for cell lines derived from tumors under different treatment conditions.

In each panel, the protocols have been ordered from lowest to highest mean IC50 values (bars indicate means). (A) Capecitabine IC50 values for mice treated with capecitabine alone. (B) Gemcitabine IC50 values for mice treated with gemcitabine alone. (C) Combined capecitabine and gemcitabine IC50 values for mice treated with both drugs together. Error bars show the 95% confidence intervals on the IC50 values.

Fig. 7.. Correlation Between IC50 Values with both Tumor Burden and Drug Dose.

(A) The relationship between the tumor burden at death and the IC50 values of the cell lines derived from those tumors. (B) The relationship between the average amount of drug used per day to treat a mouse and the resulting IC50 of the cells derived from that mouse’s tumor.

Fig. 8.. Immunohistochemistry Analysis.

(A) The percentage of proliferating (Ki-67 positive) cells and (B) apoptotic (caspase-3 positive) cells in the tumors at the end of the different treatment protocols. Protocols have been ordered by increasing mean values (shown by the bars).

Fig. 9.. Computational Simulations Match the Rank Orders for the Different Adaptive Therapy Protocols.

Comparison of (A) simulation results to (B) mouse experimental results for adaptive therapy and MTD protocols using capecitabine alone. (C) Comparison of simulation results to D. mouse experimental results for adaptive therapy and MTD protocols using both capecitabine and gemcitabine.