The TLR7 agonist imiquimod induces anti-cancer effects via autophagic cell death and enhances anti-tumoral and systemic immunity during radiotherapy for melanoma

Abstract

Toll-like receptor (TLR) ligands are strongly considered immune-adjuvants for cancer immunotherapy and have been shown to exert direct anti-cancer effects. This study was performed to evaluate the synergistic anti-cancer and anti-metastatic effects of the TLR7 agonist imiquimod (IMQ) during radiotherapy for melanoma. The pretreatment of B16F10 or B16F1 cells with IMQ combined with γ-ionizing radiation (IR) led to enhanced cell death via autophagy, as demonstrated by increased expression levels of autophagy-related genes, and an increased number of autophagosomes in both cell lines. The results also confirmed that the autophagy process was accelerated via the reactive oxygen species (ROS)-mediated MAPK and NF-κB signaling pathway in the cells pretreated with IMQ combined with IR. Mice subcutaneously injected with melanoma cells showed a reduced tumor growth rate after treatment with IMQ and IR. Treatment with 3-methyladenine (3-MA), ameliorated the anti-cancer effect of IMQ combined with IR. Additionally, the combination therapy enhanced anti-cancer immunity, as demonstrated by an increased number of CD8+ T cells and decreased numbers of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) in the tumor lesions. Moreover, the combination therapy decreased the number of metastatic nodules in the lungs of mice that were injected with B16F10 cells via the tail vein. In addition, the combination therapy enhanced systemic anti-cancer immunity by increasing the abundances of T cell populations expressing IFN-γ and TNF-α. Therefore, these findings suggest that IMQ could serve as a radiosensitizer and immune booster during radiotherapy for melanoma patients.

Trial Watch: Toll-like receptor agonists in cancer immunotherapy

“…Among these studies, we found of particular interest the work of: (1) Molgora and colleagues (Humanitas Clinical and Research Center, Rozzano, Italy), who demonstrated that interleukin-1 receptor 8 (IL1R8, also known as TIR8 or SIGIRR), which is known to impair the signal transduction cascades elicited by several TLRs as well as by various interleukin receptors, 116,117 negatively regulates the activity of NK cells as an immunosuppressive checkpoint; 118 (2) Lou and co-authors (Institute for Molecular Bioscience. The University of Queensland, Brisbane, Australia), who showed that SLP adaptor and CSK interacting membrane protein (SCIMP), a transmembrane adaptor protein, facilitates TLR4 signaling upon direct binding to its TIR domain;- 119 , who demonstrated that the combination of a TLR3 agonist (ARNAX), 129,130 a PD-L1-specific immune checkpoint blocker and a TAA-derived vaccine may be used to overcome resistance to PD-1-targeting therapies, at least in mice; 131 (9) Caronni and colleagues (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy), who found that lactic acid blocks the ability of DCs to produce type I IFN in response to TLR3 and STING agonists 132 in a mouse model of lung cancer; 133 confirming previous reports on the major influence of local metabolism on the immune functions of the tumor microenvironment; [134][135][136][137] (10) Dooduijn and collaborators (Leiden University Medical Center Leiden, The Netherlands), who showed that TLR7/TLR8 agonism, compared to TLR3 and TLR9 agonism, drives an NK cell-dependent immune response that can eradicate tumors that have escaped immunosurveillance following MHC Class I downregulation, 138 which is in line with data from other groups demonstrating the ability of TLR7 ligands to trigger NK celldependent tumor control; 75,[139][140][141][142] (11) Klein and co-authors (University Hospital Essen, Essen, Germany), who showed that mice lacking TLR3, TLR7 and TLR9 are able to reject syngeneic wild-type malignant cells upon the activation of a tumor-targeting immune response involving both CD4 + and CD8 + T lymphocytes, suggesting that endosomal TLRs may operate as part of immunological checkpoints, at least in some settings; 143 (12) Rashedi et al (University of Toronto, Toronto, Canada), who demonstrated that mesenchymal stromal cells recruit T REG cells- 144,145 upon TLR3 or TLR4 activation, as a consequence of Notch signaling modulation…”

A TLR7 agonist strengthens T and NK cell function during BRAF‐targeted therapy in a preclinical melanoma model

“…Upon screening for possible TLR ligands, we selected the TLR7 agonist (TLR7A) imiquimod as it has been used in the clinics for the treatment of nonmelanoma skin cancer but also melanoma . Furthermore, TLR7A has pleiotropic effects on T and NK cells and induces autophagic cell death in melanoma cells . Moreover, a recent study showed that a novel TLR7A reversed NK cell anergy and induced antitumor CD8 + T cell responses .…”

“…[28][29][30]36 Furthermore, TLR7A has pleiotropic effects on T and NK cells and induces autophagic cell death in melanoma cells. 37,38 Moreover, a recent study showed that a novel TLR7A reversed NK cell anergy and induced antitumor CD8 + T cell responses. 26,27 Therefore, we assessed if treatment with a TLR7A as an immune modulator would be efficient in prolonging sensitivity to BRAFi.…”

Immunomodulatory Effect of Imiquimod Through CCL22 Produced by Tumor-associated Macrophages in B16F10 Melanomas

“…The therapeutic effect of a TLR7 agonist (including IQM) or TLR7/8 dual agonist on melanoma growth in vivo has been previously reported (2,4,7,33). These reports revealed the immunostimulatory effect of these agonists through effector cells, such as CD8 + T cells, plasmacytoid DCs or M1 macrophages, and decrease the Tregs and MDSC in the tumor lesion, leading to the inhibition of B16F10 melanoma growth in vivo.…”