Topical imiquimod has therapeutic and immunomodulatory effects against intracranial tumors

Abstract

Topical imiquimod cream (trade name: Aldara) is a Toll-like receptor (TLR) 7 agonist that is approved for the treatment of cutaneous tumors. Aldara is also used as a vaccine adjuvant in clinical trials in patients with glioma and other tumors. The main mechanism of action ascribed to Aldara has been the local activation of TLR7(+) cells near the application site. Here we report the unexpected finding that Aldara has therapeutic and immunomodulatory activity as a single agent in mice bearing intracranial tumors. Repeated administration of Aldara onto the skin significantly increased the survival of mice bearing intracranial GL261 glioma and EMT6 breast carcinoma. Aldara treatment was associated with a reduction in the number CD4(+)Foxp3(+) regulatory T cells in the blood and brain tumor site. Mice treated with Aldara exhibited a generalized lymphopenia in the blood amidst an increase in brain tumor infiltrating CD4(+) and CD8(+) T cells and dendritic cells. Brain-infiltrating CD8(+) T cells were tumor reactive as demonstrated by degranulation and interferon-γ secretion in a GL261-dependent manner. In addition, soluble imiquimod directly inhibited the proliferation of GL261 cells in a TLR7-independent manner. This is the first report demonstrating that topical application of imiquimod can enhance T-cell responses to intracranial tumors as a single agent. The results must be interpreted with caution considering anatomical and biological differences between mice and humans. Nevertheless, Aldara that is being used as a vaccine adjuvant in clinical trials may have direct antitumor effects that are independent of exogenous antigen. Further studies in humans are warranted.

Figure 1. Aldara inhibits glioma growth

(A) B6 mice bearing GL261-Luc were treated by Aldara or saline weekly starting three days after tumor implantation. Kaplan Meier survival plot is shown (**p=0.0021; n=6/group). (B) Representative bioluminescent imaging of individual mice longitudinally over time illustrates tumor burden in each group. (C) Aggregate bioluminescent imaging data from mice in A–B. (**p<0.01; error bars represent standard deviation). These experiments were repeated three times with similar results.

Figure 2. Aldara selectively modulates the percentage and absolute number of leukocytes

On day 22 after GL261-Luc challenge, four mice from each group were sacrificed and leukocytes from the indicated tissues were analyzed by flow cytometry. (A) Representative gating scheme used to define leukocyte subsets in B-E. The percentage and absolute numbers were calculated in the blood (B), spleen (C), pooled cervical lymph nodes (D), and brain infiltrating leukocytes (E). All graphs represent the mean ± SEM (*p<0.05,**p<0.01,***p<0.001). The experiments were repeated at least twice with similar results.

Figure 3. Aldara increased the number of brain infiltrating tumor-reactive T cells

On day 22 after tumor implantation, brain-infiltrating lymphocytes (BIL) were dissociated from four mice in each group and analyzed by flow cytometry. (A) BIL were co-cultured with irradiated GL261-Luc target cells and the number of degranulating CD8 T cells was measured by flow cytometry as described in the methods. (B) BILs were co-cultured with B6 mouse splenocytes that had been pulsed with cell lysates from GL261-Luc or C1498 cells as a negative control. Four days later, soluble IFNγ was measured in the supernatant. Averages are graphed +/− SEM (*p<0.05,***p<0.001). The experiments were repeated at least twice with similar results.

Figure 4. Soluble imiquimod inhibits GL261 proliferation despite lack of TLR7 expression

(A) A bioluminescent cell viability assay was conducted whereby GL261-Luc cells were cultured in the indicated concentrations of IMQ for 72 hours. Data are normalized to cells treated with no IMQ as 100% viable. (B) RT-PCR was preformed for TLR7 using 10 ng of total RNA from GL261-Luc cells or B6 mouse splenocytes. Tubes run without the RT step were used as a “No RT control”.