Synergy of topical toll-like receptor 7 agonist with radiation and low-dose cyclophosphamide in a mouse model of cutaneous breast cancer

Abstract

Purpose: This study tested the hypothesis that topical Toll-like receptor (TLR) 7 agonist imiquimod promotes antitumor immunity and synergizes with other treatments in a model of skin-involving breast cancer.

Experimental design: TSA mouse breast carcinoma cells were injected s.c. into syngeneic mice. Imiquimod 5% or placebo cream was applied topically on the shaved skin overlying tumors three times/wk. In some experiments, local ionizing radiation therapy (RT) was delivered to the tumor in three fractions of 8 Gy, given on consecutive days. Cyclophosphamide was given intraperitoneally (i.p.) in one dose of 2 mg/mouse. Mice were followed for tumor growth and survival.

Results: Treatment with imiquimod significantly inhibited tumor growth, an effect that was associated with increased tumor infiltration by CD11c(+), CD4(+), and CD8(+) cells, and abolished by depletion of CD8(+) cells. Administration of imiquimod in combination with RT enhanced significantly tumor response compared with either treatment alone (P < 0.005), and 11% to 66% of irradiated tumors completely regressed. Importantly, the addition of topical imiquimod also resulted in growth inhibition of a secondary tumor outside of the radiation field. Low-dose cyclophosphamide given before start of treatment with imiquimod and RT further improved tumor inhibition and reduced tumor recurrence. Mice that remained tumor-free rejected a tumorigenic inoculum of TSA cells, showing long-term immunologic memory.

Conclusions: Topical imiquimod inhibits tumor growth and synergizes with RT. Addition of cyclophosphamide further increases the therapeutic effect and induces protective immunologic memory, suggesting that this combination is a promising strategy for cutaneous breast cancer metastases.

Figure 1. Topical imiquimod inhibits growth of TSA tumors in vivo

(A) Treatment schema. Placebo (PLA) or imiquimod (IMQ) 5% cream was applied topically as indicated. (B) Tumor growth in mice treated with PLA (closed circles), IMQ (closed squares), IMQ and CD4⁺ cell depletion (closed triangles), or IMQ and CD8⁺ cell depletion (closed diamonds). Upper panel, data show the mean ±SEM of 10 mice/group. Lower panel, CD4⁺ and CD8⁺ cell depletion was started at day 7. Data show the mean ±SEM of 6 mice/group. (C, D) Immune cells infiltration of tumors from mice treated with PLA or IMQ at day 26. (C) Representative fields (200X) showing CD11c⁺, CD8⁺ and CD4⁺ cells (orange). Nuclei were stained with DAPI (green). (D) Number of CD11c⁺, CD8⁺ and CD4⁺ cells infiltrating TSA tumors quantified in three mice/group. (E) IL-10 concentration measured at day 26 in tumor lysates, serum, and supernatants of TDLN and spleen cells stimulated ex vivo with PMA and ionomycin in 6 mice/group. Data are representative of two experiments. ***, p<0.0005: **, p<0.005, *, p<0.05.

Figure 2. RT and imiquimod synergize in inducing TSA tumor regression

(A) Treatment schema. PLA or IMQ 5% cream was applied topically and RT was given in 3 fractions of 8 Gy as indicated. (B) Tumor growth delay in mice treated with PLA (closed circles), IMQ (open circles), RT+PLA (closed squares), and RT+IMQ (open squares). Data are the mean ± SEM. The number of mice with complete tumor regression over the total number of mice per group is indicated. (C) Representative photographs of tumors during treatment and four days after end of treatment, showing progressive ulcerated tumors that are smaller in mice treated with monotherapy but only show complete regression in the RT+IMQ combination group. Data are representative of two experiments. ***, p<0.0005.

Figure 3. Topical imiquimod sensitizes a secondary tumor to the abscopal effect induced by treatment with IMQ+RT of a primary tumor

(A) Treatment schema. Mice were injected s.c. with TSA cells into the right (defined as “primary” tumor) and left (defined as “secondary” tumor) flank on days 0 and 2, respectively. PLA or IMQ 5% cream was applied topically as indicated. RT was administered locally exclusively to the primary tumor as indicated. (B) Schematic representation of the tumor model. Primary (C) and secondary (D) tumor volumes at end of experiment (day 25) in mice receiving IMQ to the primary only or to both primary and secondary tumors, as indicated. Number above the columns indicates mice with complete tumor regression over the total number of mice treated. Data show the mean ±SEM of 10-12 mice/group, and are from two independent experiments combined. ***, p<0.0005: **, p<0.005.

Figure 4. Topical imiquimod upregulates MHC class I molecule and ICAM-1 on tumor cells and induces tumor-specific T cell responses in TDLN in combination with RT

(A) Mice were injected with TSA cells on day 0. PLA or IMQ 5% cream was applied topically on days 10, 12, 14 and 17, and tumors were harvested on day 18. Obtained single cell suspensions were stained with Pacific Blue-anti-CD45, PE-anti-H2-K^d, FITC-anti-H2-L^d and FITC-anti-ICAM-1. Histograms show the expression of H2-K^d, H2-L^d, and ICAM-1, on the gated CD45-negative TSA cells treated with PLA (grey lines) or IMQ (black lines). Cells stained with isotype control (dashed grey lines). Bar graphs show mean fluorescence intensity (MFI) of 3 samples ± SD. (B) Production of IFN-γ by TDLN cells stimulated with the tumor-specific CTL epitope AH1 (full circles) or control peptide (empty circles). TDLN cells were harvested at day 26 from LN draining the primary tumor of mice treated RT+imiquimod, as in figure 3. Each symbol represents one mouse. Horizontal lines indicate the mean of AH1 (solid line) or control (dotted line) peptide-stimulated cultures. *, p<0.05.

Figure 5. Low dose CY reduces Treg cells and IL-10 production, and enhances tumor-specific IFN-γ production by TDLN

(A, B) Mice were injected s.c. with TSA cells on day 0. CY was administered i.p. on day 9 in a single dose of 100 mg/kg, and blood and spleen were analyzed on day 12 for the presence of Treg cells by staining for CD4, CD25 and FoxP3. Data are the mean ± SD of 3 mice/group, and are representative of two experiments. (C) Tumor growth delay in mice treated with PLA (open squares), IMQ (closed triangles), CY+PLA (open circles), and CY+IMQ (closed squares). PLA and IMQ were administered as in figure 1. CY was given once at day 9. Data are the mean ± SEM of 7 mice/group. (D) Production of IFN-γ by TDLN cells harvested at day 18 and stimulated with the tumor-specific CTL epitope AH1 (full circles) or control peptide (empty circles). Each symbol represents one mouse. Horizontal lines indicate the mean of AH1 (solid line) or control (dotted line) peptide-stimulated cultures. (E, F) IL-10 concentration measured at day 18 in supernatants of ex vivo stimulated spleen cells (E) and serum (F). Each symbol represents one mouse. Data are representative of two experiments. ***, p<0.0005: **, p<0.005, *, p<0.05.

Figure 6. Low dose CY improves tumor inhibition by IMQ+RT

(A) Treatment schema. (B) Tumor growth delay and survival (C) in mice treated with PLA (N=17), IMQ+RT (N=9), CY+PLA+RT (N=13), and CY+IMQ+RT (N=14), as indicated. Only a subset of mice in the control group was followed for survival. The arrow indicates the day of re-challenge of mice that had remained tumor-free. Data show the mean ±SEM of 9-17 mice/group, and are from two independent experiments combined. ***, p<0.0005: **, p<0.005.