Inflammatory macrophage derived TNFα downregulates estrogen receptor α via FOXO3a inactivation in human breast cancer cells.

Patients with estrogen receptor α positive (ERα+) breast cancer can respond to endocrine therapy, but treatment resistance is common and associated with downregulation of ERα expression in the dormant residual cells. Here we show, using long-term NSG xenograft models of human breast cancer and primary human monocytes, in vitro primary cell cultures and tumors from breast cancer patients, that macrophage derived tumor necrosis factor alpha (TNFα) downregulates ERα in breast cancer cells via inactivation of the transcription factor Forkhead box O transcription factor 3a (FOXO3a). Moreover, presence of tumor associated macrophages in the primary tumor of breast cancer patients, was associated with ERα negativity, and with worse prognosis in patients with ERα+ tumors. We propose that pro-inflammatory macrophages, despite being tumoricidal, may have direct effects on tumor progression and endocrine resistance in breast cancer patients. Our findings suggest that TNFα antagonists should be evaluated for treatment of ERα+ breast cancer.

Clinical relevance of systemic monocytic-MDSCs in patients with metastatic breast cancer.

The overall aim of this prospective study was to delineate the role of monocytic myeloid-derived suppressor cells (Mo-MDSCs) in patients with metastatic breast cancer (MBC). MDSCs are a heterogeneous group of immunosuppressive cells often enriched in different malignancies which hold prognostic and predictive value for clinical outcomes. Here, we assessed the clinical significance of Mo-MDSCs in 54 patients with de novo or distant recurrent MBC. We show that high levels of Mo-MDSCs significantly correlated with de novo MBC (metastatic disease at initial diagnosis), estrogen receptor (ER) negativity, and liver- and bone metastasis. A trend towards an association between high levels of Mo-MDSCs and survival (P = 0.053) was also found in patients with distant recurrent ER-positive MBC. We therefore propose that an increased population of Mo-MDSCs may be related to the metastatic or immunoregulatory switch associated with transition to a more systemic disease. Our data imply that high levels of systemic Mo-MDSCs represent patients with more aggressive disease and worse outcome.

Wnt5a is a TLR2/4-ligand that induces tolerance in human myeloid cells.

Innate immune responses are rapid, dynamic and highly regulated to avoid overt reactions. This regulation is executed by innate immune tolerance mechanisms that remain obscure. Wnt5a is a signalling protein mainly involved in developmental processes and cancer. The effect of Wnt5a on inflammatory myeloid cells is controversial. Here, we combine primary cell cultures, in vitro binding studies, mass spectrometry and Drosophila protein modelling to show that Wnt5a is a direct ligand of toll-like receptor (TLR) 2 and 4. The binding promotes a MyD88-non-canonical nuclear factor of kappa B (NFκB) and AP-1 signalling cascade, with contradictory profiles in mouse (pro-inflammatory) and human (anti-inflammatory) myeloid immune cells. These data reveal that the true nature of Wnt5a in inflammatory cells, is to regulate TLR signals, and in human myeloid cells it acts as an endogenous, tolerance-associated molecular pattern (TAMP), inducing IL-10 and innate immune tolerance.

Docetaxel promotes the generation of anti-tumorigenic human macrophages.

The taxanes Docetaxel and Paclitaxel are two of the standard chemotherapies for patients with metastatic breast cancer. The functional effect of Docetaxel and Paclitaxel on human innate immune cells of the myeloid lineage is not well established, nor is the effects these agents have on differentiation of monocytes into macrophages and dendritic cells. Therefore, the aim with this project was to determine the effects of Docetaxel and Paclitaxel on primary human monocyte differentiation, activation and function. For this purpose, primary human monocytes were isolated from healthy donors and cultured with or without Docetaxel and Paclitaxel. We found that Docetaxel promoted the differentiation of primary human monocytes into pro-inflammatory macrophages with an M1 phenotype and an ability to present antigens to T cells. Monocytes treated with Docetaxel also displayed an elevated secretion of IL-8 and IL-1ß, but did not promote generation of monocytic myeloid-derived suppressor cells. In conclusion, Docetaxel appears to have an immune stimulatory effect that would be beneficial for an anti-tumorigenic type of immune response, whereas Paclitaxel seems to have less effect on myeloid cells.

S100A9 expressed in ER(-)PgR(-) breast cancers induces inflammatory cytokines and is associated with an impaired overall survival.

BACKGROUND: Breast cancer is the most common cancer form among women today. Depending on hormone receptor status, breast cancers are divided into different subtypes with vastly varying prognosis. S100A9 is a calcium-binding protein that is associated with inflammation and expressed not only in myeloid cells but also in some tumours. The role for S100A9 in the malignant cells is not well characterised; however, previous studies have shown that the protein could have important immune-modulating properties. METHODS: Using a human breast cancer cohort consisting of 144 tumour samples and in vitro analysis of human breast cancer cell lines, we investigated the expression and function of S100A9 in human breast cancer. RESULTS: We show that S100A9 expression in breast cancer correlated with the ER(-)PgR(-) breast tumour subtype (P<0.001) and with Ki67 (P=0.024) and was expressed both in the malignant cells and in the tumour-infiltrating anti-inflammatory CD163(+) myeloid cells (P<0.001). Stromal expression of S100A9 also correlated to nodal stage, tumour size and Her2 positivity. Within the ER(-)PgR(-) subgroup, all Her2(+) and EGFR(+) tumours expressed S100A9 in the cytoplasm. Both cytoplasmic staining in the malignant cells as well as stromal S100A9 expression in myeloid cells correlated with a decreased overall survival in breast cancer patients. Furthermore, rS100A9 homodimers induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1ß) in a TLR4- and EGFR-dependent manner in human breast cancer cells in vitro. CONCLUSION: We suggest that S100A9 could be viewed as a novel therapeutic target for patients with ER(-)PgR(-) breast cancers.

Expression of functional toll like receptor 4 in estrogen receptor/progesterone receptor-negative breast cancer.

INTRODUCTION: Toll-like receptors (TLRs) are a family of pattern recognition receptors that are expressed on cells of the innate immune system. The ligands can be pathogen derived (pathogen associated molecular patterns; PAMPs) or endogenous (damage associated molecular patterns; DAMPs) that when bound induces activation of nuclear factor kappa B (NF-κB) and transcription of pro-inflammatory genes. TLRs have also been discovered in various malignant cell types, but with unknown function. METHODS: In this study we performed a detailed analysis of TLR and co-receptor expression pattern and function in breast cancer. Expression patterns were examined using real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) on three estrogen receptor-positive (ER(+)) and four estrogen receptor/progesterone receptor-negative (ER(-)/PR(-); ER/PR-negative) breast cancer cell lines, and a breast cancer cohort consisting of 144 primary breast cancer samples. The function was investigated using in vitro assays comprising PAMP/DAMP-stimulation, downstream signaling and TLR-silencing experiments. RESULTS: We found that TLR4 was expressed in a biologically active form and responded to both PAMPs and DAMPs primarily in ER/PR-negative breast cancers. Stimulation of TLR2/4 in vitro induced expression of pro-inflammatory genes and a gene expression analysis of primary breast cancers showed a strong correlation between TLR4 expression and expression of pro-inflammatory mediators. In line with this, TLR4 protein expression correlated with a decreased survival. CONCLUSIONS: These findings suggest that TLR4 is expressed in a functional form in ER/PR-negative breast cancers. Studies regarding TLR4-antagonist therapies should be focusing on ER/PR-negative breast cancer particularly.