[18F]-FLT-PET to evaluate how the sequencing of chemotherapies impacts the efficacy of combination treatment in mouse models of triple-negative breast cancer

Abstract

Introduction: Triple-negative breast cancer (TNBC) lacks targeted therapies due to an absence of biomarkers, making chemotherapy the primary treatment option for early-stage cancer. This study evaluates whether the order and sequence of combination chemotherapy-doxorubicin (DRB) and paclitaxel (PTX)-affects treatment efficacy in TNBC.

Methods: In vitro and in vivo models (MDA-MB-231 human and 4T1 syngeneic mouse TNBC) were used to assess treatment efficacy across three groups: saline control, DRB→PTX, and PTX→DRB. [¹⁸F]fluorothymidine (FLT) Positron emission tomography (PET) imaging was performed at baseline, day 3, and day 6 to monitor changes in tumor proliferation, and flow cytometry on day 6 examined immune profile differences in endpoint cohorts. Statistical significance was evaluated using the ANOVA and Kolmogorov-Smirnov test.

Results: In vitro experiments showed PTX→DRB treatment significantly reduced S/G2/M cell cycles and cancer cell viability. The MDA-MB-231 tumor model showed that PTX→DRB treatment significantly decreased cell proliferation and tumor heterogeneity comparing day 6 to baseline. In 4T1 models, DRB→PTX suppressed tumor growth and enhanced B cell and macrophage recruitment in immunocompetent but not immunocompromised mice. In both models, [¹⁸F]-FLT-PET plays a crucial role in directing the sequencing of chemotherapy in TNBC.

Conclusions: Our study highlights the immune system's critical role in enhancing chemotherapy's efficacy. It provides compelling evidence that imaging can guide the sequencing of therapies by tracking changes in cellular proliferation and the heterogeneity of tumor response. This approach underscores the potential to refine treatment strategies for improved therapeutic outcomes.

Keywords: cell cycle; doxorubicin; immune cells; molecular imaging; paclitaxel; triple-negative breast cancer.

Fig. 1

Paclitaxel prior to doxorubicin showed a decreased ratio of cell cycle S/G2/M and caused more cell death in TNBC. (a) Schematic diagram of in vitro experiment of MDA-MB-231-FUCCI to assess sequencing of PTX (100nM) or DRB (20nM) treatment. (b) Representative images show RFP (which represents cell cycle G1) and GFP (which represents cell cycle S, G2, M) signals dramatically reduced in the PTX→DRB group. (c) PTX prior to DRB treatment shifted the cell cycle and largely reduced the S/G2/M phase. (d) PTX prior to DRB significantly reduced MDA-MB-231 cell viability (n=4). PTX, paclitaxel. DRB, doxorubicin. ns, non-significant; ****, p<0.0001.

Fig. 2

PTX prior to DRB treatment reveals decreased [¹⁸F]-FLT uptake in the MDA-MB-231 model. (a) Schematic diagram of experimental timeline. (b) From day 0 to day 6, tumor volume measured by calipers showed no significant changes. (c) Representative [¹⁸F]-FLT PET/CT images. The red arrow pointed at tumors. (d) Representative histogram plots of [¹⁸F]-FLT SUV in TNBC tumors with three different treatments (saline, DRB→PTX, PTX→DRB) on day 0 (black), day 3 (blue), and day 6 (red) showed increased cell proliferative heterogeneity in saline and DRB→PTX treated tumor but not in PTX→DRB treated tumor. (e-f) Tumor [¹⁸F]-FLT SUV_mean (e) and SUV_max (f) was normalized to muscle FLT SUV_mean and SUV_max respectively. PTX prior to DRB group showed significantly reduced SUV_max compared to saline group (p=0.03, n=12). ns, non-significant; *, p<0.05.

Fig. 3

Doxorubicin prior to paclitaxel treatment showed decreased [¹⁸F]-FLT uptake in the 4T1 model. (a) Schematic diagram of experimental timeline. (b) Tumor volume measurements showed the significance of tumor volume decrease in the DRB→PTX group. (c) Representative [¹⁸F]-FLT PET/CT images. The red arrow pointed at tumors. (d-e) Tumor [¹⁸F]-FLT SUV_mean (d) and SUV_max (e) changes from day 0 to day 6. The DRB→PTX group showed significantly reduced SUV_mean and SUV_max compared to the saline group. (f-h) Representative histogram plots of [¹⁸F]-FLT SUV in 4T1 tumors with three different treatments (saline, DRB→PTX, PTX→DRB) on day 0 (black), day 3 (blue), and day 6 (red) showed decreased cell proliferative heterogeneity in DRB→PTX treated tumor.

Fig. 4

Doxorubicin prior to paclitaxel treatment showed decreased cell proliferation in immunocompetent but not in immunocompromised mice. (a) Tumor volume measurements showed a significant decrease in tumor size in BALB/c mice (left) but not in athymic nude mice (right) when comparing the DRB→PTX treatment group to the saline group. (b) Flow cytometry with propidium iodide staining in BALB/c mice on day 6 of treatment showed a significant increase in the G1 phase and a significant decrease in the G2/M phases in the DRB→PTX group compared to the saline and PTX→DRB groups. (c) Flow cytometry with propidium iodide staining on day 6 of treatment revealed no significant difference in cell cycles among the treatment groups in athymic nude mice. *, p<0.05; **, p<0.01.

Fig. 5

Doxorubicin prior to paclitaxel in 4T1 tumors shows no change in intratumoral T cells but increased presence of B cells and recruited macrophages in immunocompetent mice but not in immunocompromised mice. (a) Quantification of intratumoral CD3+, CD4+, or CD8+ T cells (% of CD45+ or CD3+) of 4T1 tumors in BALB/c mice treated with either saline, PTX→DRB, or DRB→PTX. No significant differences (ns) were observed across the groups in CD3+, CD4+, or CD8+ T cell populations. (b) Quantification of intratumoral CD45+ cells revealed a significant increase in the DRB→PTX group. (c) Quantification of intratumoral B220+ B Cells (% of CD45+CD3-) or Ly6c+ recruited macrophages (% of CD45+CD11b+F4/80+) in 4T1 tumors in both BALB/c or athymic nude mice treated with either saline, PTX→DRB, or DRB→PTX. In BALB/c mice, B220+ and Ly6c+ populations were significantly increased when treated with DRB→PTX, indicating differential immune recruitment of immune subsets. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.00001.