Testing Adaptive Therapy Protocols Using Gemcitabine and Capecitabine in a Preclinical Model of Endocrine-Resistant Breast Cancer

Abstract

Adaptive therapy, an ecologically inspired approach to cancer treatment, aims to overcome resistance and reduce toxicity by leveraging competitive interactions between drug-sensitive and drug-resistant subclones, prioritizing patient survival and quality of life instead of killing the maximum number of cancer cells. In preparation for a clinical trial, we used endocrine-resistant MCF7 breast cancer to stimulate second-line therapy and tested adaptive therapy using capecitabine, gemcitabine, or their combination in a mouse xenograft model. Dose modulation adaptive therapy with capecitabine alone increased survival time relative to MTD but not statistically significantly (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, the survival time was significantly increased compared to high-dose combination therapy (HR = 0.07, 95% CI = 0.013-0.42, p = 0.003). Overall, the survival time increased with reduced dose for both single drugs (p < 0.01) and combined drugs (p < 0.001), resulting in tumors with fewer proliferation cells (p = 0.0026) and more apoptotic cells (p = 0.045) compared to high-dose therapy. Adaptive therapy favors slower-growing tumors and shows promise in two-drug alternating regimens instead of being combined.

Keywords: adaptive therapy; drug-resistant and drug-sensitive subclones; endocrine-resistant MCF7 breast cancer; high-dose therapy.

Figure 1

Schematic figure of comparing 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), suppressing the growth of less fit, resistant cells (red). (B) Dose modulation adaptive therapy raises the dose if the tumor grows and lowers the dose if the tumor shrinks. Previous mouse models have used a tumor burden change of 20% to trigger a change in dose [2]. Our simulation studies suggest a lower threshold is better [7], so we have used a 10% change in tumor burden to trigger a change in dose in this study. (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) [8,9].

Figure 2

(A) Drug dose–response curves for sensitive and resistant MCF7/luc cell lines to combination of fulvestrant and palbociclib. MCF7 resistant (MCF7 R) and sensitive (MCF7 S) cell lines showed IC50 values of 62.65 and 13.13 μM, respectively. The percentages of viability at 40, 50, and 60 μM were significantly different between MCF7 R and MCF7 S cell lines. Each data point represents eight replicates. (B) Drug dose–response curves for sensitive and resistant MCF7/luc cell lines to gemcitabine and capecitabine. Green lines show drug dose–response of endocrine-sensitive MCF7 cell lines to gemcitabine with IC50 value of 153 μM and capecitabine with IC50 value of 235 μM. Red lines show drug dose–response of endocrine-resistant (resistant to fulvestrant and palbociclib) MCF7 cell lines to gemcitabine with IC50 value of 134 μM and capecitabine with IC50 value of 128 μM. Note that, if anything, resistance to endocrine therapy led to increased sensitivity to antimetabolite therapy in these cells.

Figure 3

Survival analysis of single-drug therapies. (A) Survival analysis of capecitabine protocols using 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; 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 were statistically significantly different using Cox regressions.

Figure 4

Survival analysis of multidrug therapies. (A) Multidrug 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 a single drug. (B) Multidrug tandem adaptive therapy protocols versus MTD or no treatment (vehicle control). In the tandem and MTD protocols, both gemcitabine and capecitabine were given (and modulated) at the same time.

Figure 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 < 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 were a lot of variances associated with different protocols, there was still a strong trend that mice treated with more drugs had a shorter survival time (p = 0.0074, R² = 0.19).

Figure 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 across the 1–3 cell lines we were able to derive from the tumors for each condition. (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 based on eight replicates at each drug concentration in our drug dose–response experiments.

Figure 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.

Figure 8

Immunohistochemistry analysis. The percentage of (A) 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).

Figure 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.