Rethinking the adenosine-A2AR checkpoint: implications for enhancing anti-tumor immunotherapy.

Adenosine signaling through A2AR serves as a negative regulator of the immune system. Unique to this suppressive pathway is its ability to impact numerous stromal and immune cells. Additionally, tumors exhibit elevated concentrations of adenosine further advancing the pathway's potential as a powerful target for activating anti-tumor immunity. The promise of this therapeutic strategy has been repeatedly demonstrated in mice, but has so far only yielded limited success in the clinic. Nonetheless, it is notable that many of these observed clinical responses have been in individuals resistant to prior immunotherapy. These observations suggest this pathway is indeed involved in tumor immune evasion. Thus, identifying the disparities between the translational and clinical implementation of this therapy becomes necessary. To this end, this review will revisit how and where adenosine-A2AR signaling regulates the immune system and anti-tumor immunity so as to reveal opportunities for improving the translational success of this immunotherapy.

Tumor necrosis factor-alpha-converting enzyme activities and tumor-associated macrophages in breast cancer.

The role of the tumor microenvironment especially of tumor-associated macrophages (TAMs) in the progression and metastatic spread of breast cancer is well established. TAMs have primarily a M2 (wound-healing) phenotype with minimal cytotoxic activities. The mechanisms by which tumor cells influence TAMs to display a pro-tumor phenotype are still debated although the key roles of immunomodulatory cytokines released by tumor cells, including colony-stimulating factor 1, tumor necrosis factor (TNF) and soluble TNF receptors 1/2, soluble vascular cell adhesion molecule 1, soluble interleukin 6 receptor and amphiregulin, have been demonstrated. Importantly, these factors are released through ectodomain shedding by the activities of the tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17). The role of TACE activation leading to autocrine effects on tumor progression has been extensively studied. In contrast, limited information is available on the role of tumor cell TACE activities on TAMs in breast cancer. TACE inhibitors, currently in clinical trials, will certainly affect TAMs and subsequently treatment outcomes based on the substrates it releases. Furthermore, whether targeting a subset of the molecules shed by TACE, specifically those leading to TAMs with altered functions and phenotype, holds greater therapeutic promises than past clinical trials of TACE antagonists' remains to be determined. Here, the potential roles of TACE ectodomain shedding in the breast tumor microenvironment are reviewed with a focus on the release of tumor-derived immunomodulatory factors shed by TACE that directs TAM phenotypes and functions.

Breast tumor cell TACE-shed MCSF promotes pro-angiogenic macrophages through NF-κB signaling.

Most deaths associated with breast cancer, the most common malignancy in women, are caused by metastasis. Tumor associated macrophages significantly contribute to breast cancer progression and development of metastasis through the promotion of angiogenesis which involves a central regulator of macrophage functions: nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Macrophages are activated by macrophage colony stimulating factor (MCSF) and chemokine (C-C motif) ligand 2 (CCL2) to secrete angiogenic factors including vascular endothelial growth factor (VEGF). The release of MCSF from tumor cells is mediated by ectodomain shedding through tumor necrosis factor alpha converting enzyme activation (TACE). Here we determined whether tumor cells TACE-shed MCSF promotes angiogenesis through activation of the NF-κB pathway in macrophages and the subsequent release of VEGF. These interactions were modeled in vitro using a panel of mammary cells mimicking the breast cancer progression from normal murine mammary gland cells to metastatic 4T1 cells along with J774 macrophages, all derived from BALB/c mice. TACE and MCSF expressions were higher in metastatic cells compared to epithelial cells (p < 0.05). Tumor conditioned medias activated the expression of VEGF by macrophages through stimulation of the NF-κB pathway and resulting macrophage secretions that promoted high levels of endothelial cell tubes. Furthermore, the combinations of CCL2, also highly expressed by tumor cells, and MCSF promoted pro-angiogenic macrophages. These results highlight the key role of tumor cell TACE-shed MCSF and secreted CCL2 in stimulating pro-angiogenic macrophages.

Soluble tumor necrosis factor receptors shed by breast tumor cells inhibit macrophage chemotaxis.

Breast tumor cells alter their microenvironment in part through the expression of protumor molecules that influence macrophages during tumor progression and metastasis. Macrophage recruitment is stimulated by chemotactic factors, including tumor necrosis factor alpha (TNF-α), which also stimulates the cytotoxic/tumor cell killing macrophage phenotype. Through TNF-α converting enzyme (TACE/ADAM17) activities, breast tumor cells shed membrane-bound proteins, including their TNF receptors (sTNFR1/2), which serve as decoys sequestering TNF-α and preventing TNF-α-driven apoptosis of tumor cells, thereby decreasing TNF-α bioavailability. Here we investigated the levels of sTNFRs shed by breast tumor cells and determined the effects of shed sTNFRs on macrophage migration toward TNF-α. TNF-α and sTNFRs concentrations were measured in murine normal epithelial, stromal, and mammary tumor cells. The migration of murine macrophages towards TNF-α in the presence of tumor derived soluble factors (TDSFs) shed by TACE was determined. TNF-α concentrations secreted by tumor and normal epithelial cells were below the detection limit contrasting with stromal cells, especially macrophages, which expressed higher levels of TNF-α (P<0.001). Regardless of the cell tested, treatment with the TACE inhibitor TAPI-0 led to a significant decrease in sTNFR2 shed (P<0.05). The dose-dependent macrophage migration toward TNF-α prevented by incubation with TDSFs was not observed with TDSFs collected following TAPI-0 treatment (P<0.05). Furthermore, the TNF-α-driven increased pAkt expression in macrophage was inhibited by TACE shed TDSFs (P<0.05). These results highlight the role of tumor-shed sTNFRs in TNF-α -driven macrophage chemotaxis.