The RNA-binding protein tristetraprolin (TTP) destabilizes target messenger RNAs (mRNAs) containing AU-rich elements within their 3' untranslated region. Thereby, it controls the expression of multiple inflammatory and tumor-associated transcripts. Moreover, a loss of TTP in tumors predicts disease-associated survival. Although tumor intrinsic functions of TTP have previously been studied, the impact of TTP on the interaction of tumors with their microenvironment remains elusive. As immune cell infiltration into tumors is a critical determinant for tumor progression, this study aimed at determining the influence of tumor cell TTP on the interaction between tumor and immune cells, specifically monocytes (MO)/macrophages (MΦ). Knockdown (k/d) of TTP in T47D breast cancer cells enhanced tumor growth both in vitro and in vivo and increased infiltration of MO into 3D tumor spheroids in vitro and of MΦ into tumor xenografts in vivo. Enhanced migration of MO toward supernatants of TTP-deficient tumor spheroids was determined as the underlying principle. Interestingly, we noticed interleukin-16 (IL-16) mRNA stabilization when TTP was depleted. In line, IL-16 protein levels were elevated in TTP-deficient spheroids and their supernatants as well as in TTP k/d tumor xenografts and critically contributed to the enhanced chemotactic behavior. In summary, we show that the loss of TTP in tumors not only affects tumor cell proliferation and survival but also enhances infiltration of MO/MΦ into the tumors, which is typically associated with poor prognosis. Moreover, we identified IL-16 as a critical TTP-regulated chemotactic factor that contributes to MO/MΦ migration.

• Transcriptome analysis of CD206+ and CD206-macrophages reveals differential phenotype. • CD06+ macrophages coexpressing either MORC4 or SERPINH1 show a positive correlation with patient survival. • CD206-macrophages expressing MHCII are correlated with negative patient survival.

Prostaglandin E2 (PGE2) favors multiple aspects of tumor development and immune evasion. Therefore, microsomal prostaglandin E synthase (mPGES-1/-2), is a potential target for cancer therapy. We explored whether inhibiting mPGES-1 in human and mouse models of breast cancer affects tumor-associated immunity. A new model of breast tumor spheroid killing by human PBMCs was developed. In this model, tumor killing required CD80 expression by tumor-associated phagocytes to trigger cytotoxic T cell activation. Pharmacological mPGES-1 inhibition increased CD80 expression, whereas addition of PGE2, a prostaglandin E2 receptor 2 (EP2) agonist, or activation of signaling downstream of EP2 reduced CD80 expression. Genetic ablation of mPGES-1 resulted in markedly reduced tumor growth in PyMT mice. Macrophages of mPGES-1−/− PyMT mice indeed expressed elevated levels of CD80 compared to their wildtype counterparts. CD80 expression in tumor-spheroid infiltrating mPGES-1−/− macrophages translated into antigen-specific cytotoxic T cell activation. In conclusion, mPGES-1 inhibition elevates CD80 expression by tumor-associated phagocytes to restrict tumor growth. We propose that mPGES-1 inhibition in combination with immune cell activation might be part of a therapeutic strategy to overcome the immunosuppressive tumor microenvironment.

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IntroductionMammalian hematopoiesis in the BM is regulated among others by the oxygen (O 2 ) availability. O 2 concentrations in the BM range from anoxia to 6% opposed to 4%-14% in well-oxygenated tissues, including the blood. 1,2 Recent data indicate that O 2 gradients within the BM participate in keeping hematopoietic stem cells (HSCs) in a low-replicating pluripotent state. HSCs are located in an extremely hypoxic niche as demonstrated by dye-perfusion and engraftment studies. 3,4 Hypoxia-inducible factors 1-3 (HIF-1-HIF-3) are stabilized by a low pO 2 to induce adaptive gene expression. They are heterodimers consisting of distinct O 2 -sensitive ␣-subunits and a stable common ␤-subunit, also known as aryl hydrocarbon receptor nuclear translocator (ARNT). 5 HIF-1 and HIF-2 were recently connected to HSC biology. In their hypoxic niche, HIF-1 maintains HSC quiescence, 6 whereas HIF-2 maintains their self-renewing capacity. 7 HIF-1 also affects embryonic hematopoiesis as demonstrated by defective myeloid and erythroid progenitor formation in HIF-1␣ Ϫ/Ϫ as well as ARNT Ϫ/Ϫ embryos. 8 In adult hematopoiesis, HIF-1 is essential for B-cell progenitor proliferation and mature B-cell subclass differentiation. 9 However, its involvement in mononuclear phagocyte development is unknown. Previous findings indicated defective development of human plasmacytoid dendritic cells (pDCs) under hypoxia in vitro. 10 Therefore, we asked whether HIF-1 regulates DC lineage differentiation in mice. Methods AnimalsHIF-1␣ fl/fl or HIF-2␣ fl/fl mice 11,12 were bred with LysM-Cre transgenic mice 13 in the C57BL/6 background. Age-matched C57BL/6 wild-type (WT) mice were controls. ID2 Ϫ/Ϫ mice and their respective WT control were in the NMRI background. 14 The guidelines of the Hessian animal care and use committee were followed. DC generation from BMFor DC generation in vitro, 2 ϫ 10 6 total BM cells/mL in RPMI 1640 with 10% FCS and 200 ng/mL recombinant murine fms-related tyrosine kinase 3-ligand (Flt3-L, PeproTech) were cultured in 6-well Ultra-Low attachment plates (Corning) for up to 9 days 15 at various O 2 levels as indicated, using a InVivo 2 400 hypoxia workstation (Ruskinn Technologies). Alternatively, cells were cultured with 100M dimethyloxallyl glycine (DMOG, from Biomol). Sorted monocyte/DC progenitors/common DC progenitors (MDPs/ CDPs; 10 4 cells/well) were cultured with 200 ng/mL Flt3-L in 24-well Ultra-Low attachment plates. Flow cytometry and cell sortingBM cells, spleen, or whole blood cells were stained with fluorochromeconjugated antibodies and analyzed on a LSRII/Fortessa flow cytometer (BD Biosciences). MDP/CDP were sorted from lineage Ϫ cell-enriched BM (lineage cell depletion kit and AutoMACS cell separator from Miltenyi Biotec) using a FACSAria III cell sorter (BD Biosciences). For details (antibodies, surface markers, intracellular transcription factor staining procedures), see supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). pDCs were i...