Phosphatidylserine Synthase PTDSS1 Shapes the Tumor Lipidome to Maintain Tumor-Promoting Inflammation.

An altered lipidome in tumors may affect not only tumor cells themselves but also their microenvironment. In this study, a lipidomics screen reveals increased amounts of phosphatidylserine (PS), particularly ether-PS (ePS), in murine mammary tumors compared with normal tissue. PS was produced by phosphatidylserine synthase 1 (PTDSS1), and depletion of Ptdss1 from tumor cells in mice reduced ePS levels accompanied by stunted tumor growth and decreased tumor-associated macrophage (TAM) abundance. Ptdss1-deficient tumor cells exposed less PS during apoptosis, which was recognized by the PS receptor MERTK. Mammary tumors in macrophage-specific Mertk-/- mice showed similarly suppressed growth and reduced TAM infiltration. Transcriptomic profiles of TAMs from Ptdss1-knockdown tumors and Mertk-/- TAMs revealed that macrophage proliferation was reduced when the Ptdss1/Mertk pathway was targeted. Moreover, PTDSS1 expression correlated positively with TAM abundance but negatively with breast carcinoma patient survival. PTDSS1 thus may be a target to modify tumor-promoting inflammation. SIGNIFICANCE: This study shows that inhibiting the production of ether-phosphatidylserine by targeting phosphatidylserine synthase PTDSS1 limits tumor-associated macrophage expansion and breast tumor growth.

IL27Rα Deficiency Alters Endothelial Cell Function and Subverts Tumor Angiogenesis in Mammary Carcinoma.

IL-27 regulates inflammatory diseases by exerting a pleiotropic impact on immune cells. In cancer, IL-27 restricts tumor growth by acting on tumor cells directly, while its role in the tumor microenvironment is still controversially discussed. To explore IL-27 signaling in the tumor stroma, we used a mammary carcinoma syngraft approach in IL27Rα-deficient mice. Tumor growth in animals lacking IL27Rα was markedly reduced. We noticed a decrease in immune cell infiltrates, enhanced tumor cell death, and fibroblast accumulation. However, most striking changes pertain the tumor vasculature. Tumors in IL27Rα-deficient mice were unable to form functional vessels. Blocking IL-27-STAT1 signaling in endothelial cells in vitro provoked an overshooting migration/sprouting of endothelial cells. Apparently, the lack of the IL-27 receptor caused endothelial cell hyper-activation via STAT1 that limited vessel maturation. Our data reveal a so far unappreciated role of IL-27 in endothelial cells with importance in pathological vessel formation.

Downregulation of BTLA on NKT Cells Promotes Tumor Immune Control in a Mouse Model of Mammary Carcinoma.

Natural Killer T cells (NKT cells) are emerging as critical regulators of pro- and anti-tumor immunity, both at baseline and in therapeutic settings. While type I NKT cells can promote anti-tumor immunity, their activity in the tumor microenvironment may be limited by negative regulators such as inhibitory immune checkpoints. We observed dominant expression of B- and T-lymphocyte attenuator (BTLA) on type I NKT cells in polyoma middle T oncogene-driven (PyMT) murine autochthonous mammary tumors. Other immune checkpoint receptors, such as programmed cell death 1 (PD-1) were equally distributed among T cell populations. Interference with BTLA using neutralizing antibodies limited tumor growth and pulmonary metastasis in the PyMT model in a therapeutic setting, correlating with an increase in type I NKT cells and expression of cytotoxic marker genes. While therapeutic application of an anti-PD-1 antibody increased the number of CD8+ cytotoxic T cells and elevated IL-12 expression, tumor control was not established. Expression of ZBTB16, the lineage-determining transcription factor of type I NKT cells, was correlated with a favorable patient prognosis in the METABRIC dataset, and BTLA levels were instrumental to further distinguish prognosis in patents with high ZBTB16 expression. Taken together, these data support a role of BTLA on type I NKT cells in limiting anti-tumor immunity.

Killing Is Not Enough: How Apoptosis Hijacks Tumor-Associated Macrophages to Promote Cancer Progression.

Macrophages are a group of heterogeneous cells of the innate immune system that are crucial to the initiation, progression, and resolution of inflammation. Moreover, they control tissue homeostasis in healthy tissue and command a broad sensory arsenal to detect disturbances in tissue integrity. Macrophages possess a remarkable functional plasticity to respond to irregularities and to initiate programs that allow overcoming them in order to return back to normal. Thus, macrophages kill malignant or transformed cells, rearrange extracellular matrix, take up and recycle cellular as well as molecular debris, initiate cellular growth cascades, and favor directed migration of cells. As an example, apoptotic death of bystander cells is sensed by macrophages, initiating functional responses that support all hallmarks of cancer. In this chapter, we describe how tumor cell apoptosis hijacks tumor-associated macrophages to promote tumor growth. We propose that tumor therapy should not only kill malignant cells but also target the interaction of the host with apoptotic cancer cells, as this might be efficient to limit the protumor action of apoptotic cells and boost the antitumor potential of macrophages. Leaving the apoptotic cell/macrophage interaction untouched might also limit the benefit of conventional tumor cell apoptosis-focused therapy since surviving tumor cells might receive overwhelming support by the wound healing response that apoptotic tumor cells will trigger in local macrophages, thereby enhancing tumor recurrence.

HIF-1α is a negative regulator of plasmacytoid DC development in vitro and in vivo.

Hypoxia-inducible factors (HIFs) regulate hematopoiesis in the embryo and maintain hematopoietic stem cell function in the adult. How hypoxia and HIFs contribute to hematopoietic lineage differentiation in the adult is ill defined. Here we provide evidence that HIF-1 limits differentiation of precursors into plasmacytoid dendritic cells (pDCs). Low oxygen up-regulated inhibitor of DNA binding 2 (ID2) and suppressed Flt3-L-induced differentiation of bone marrow cells to pDCs in wild-type but not HIF-1α(fl/fl) LysM-Cre bone marrow cells. Moreover, pDC differentiated normally in hypoxic ID2(-/-) bone marrow cultures. Finally, we observed elevated pDC frequencies in bone marrow, blood, and spleen of HIF-1α(fl/fl) LysM-Cre and ID2(-/-), but not HIF-2α(fl/fl) LysM-Cre mice. Our data indicate that the low oxygen content in the bone marrow might limit pDC development. This might be an environmental mechanism to restrict the numbers of these potentially autoreactive cells.

Apoptotic tumor cells induce IL-27 release from human DCs to activate Treg cells that express CD69 and attenuate cytotoxicity.

Intrinsic immunosuppression is a major obstacle for successful cancer therapy. The mechanisms for the induction and regulation of immunosuppression in humans are ill defined. A microenvironmental component that might prevent antitumor immunity is the presence of dying tumor cells, which are abundant following conventional cancer ablation methods such as chemo- or radiotherapy. Shedding of apoptotic debris and/or secretion of factors to the tumor bed or draining lymph nodes thus might have a profound impact on professional phagocytes, such as DCs, and subsequent priming of lymphocytes. Here, we exposed human DCs to supernatants of live, apoptotic, or necrotic human breast cancer cells and cocultured them with autologous T cells. Priming with apoptotic debris prevented DCs from establishing cytotoxicity toward live human tumor cells by inducing a Treg-cell population, defined by coexpression of CD39 and CD69. Immunosuppression via Treg cells was transferable and required the release of sphingosine-1-phosphate (S1P) from apoptotic cells, acting via S1P receptor 4 on DCs to induce IL-27 secretion. We propose that CD69 expression on CD39(+) Treg cells enables them to interact with CD73-expressing CD8(+) T cells to generate adenosine, thereby suppressing cytotoxicity. These findings aid the understanding of how dying tumor cells limit antitumor immunity.

Technical advance: Generation of human pDC equivalents from primary monocytes using Flt3-L and their functional validation under hypoxia.

The division of labor between DC subsets is evolutionarily well-defined. mDC are efficient in antigen presentation, whereas pDC act as rheostats of the immune system. They activate NK cells, cause bystander activation of mDC, and interact with T cells to induce tolerance. This ambiguity positions pDC at the center of inflammatory diseases, such as cancer, arthritis, and autoimmune diseases. The ability to generate human mDC ex vivo made it possible to engineer them to suit therapy needs. Unfortunately, a similar, easily accessible system to generate human pDC is not available. We describe a method to generate human pDC equivalents ex vivo, termed mo-pDC from peripheral blood monocytes using Flt3-L. mo-pDC showed a characteristic pDC profile, such as high CD123 and BDCA4, but low CD86 and TLR4 surface expression and a low capacity to induce autologous lymphocyte proliferation and to phagocytose apoptotic debris in comparison with mDC. Interestingly, mo-pDC up-regulated the pDC lineage-determining transcription factor E2-2 as well as expression of BDCA2, which is under the transcriptional control of E2-2 but not its inhibitor ID2, during differentiation. mo-pDC produced high levels of IFN-alpha when pretreated overnight with TNF-alpha. Under hypoxia, E2-2 was down-regulated, and ID2 was induced in mo-pDC, whereas surface expression of MHCI, CD86, and BDCA2 was decreased. Furthermore, mo-pDC produced high levels of inflammatory cytokines when differentiated under hypoxia compared with normoxia. Hence, mo-pDC can be used to study differentiation and functions of human pDC under microenvironmental stimuli.

Sphingosine kinase 2 deficient tumor xenografts show impaired growth and fail to polarize macrophages towards an anti-inflammatory phenotype.

A challenging task of the immune system is to fight cancer cells. However, a variety of human cancers educate immune cells to become tumor supportive. This is exemplified for tumor-associated macrophages (TAMs), which are polarized towards an anti-inflammatory and cancer promoting phenotype. Mechanistic explanations, how cancer cells influence the macrophage phenotype are urgently needed to address potential anti-cancer strategies along this line. One potential immune modulating compound, sphingosine-1-phosphate (S1P), was recently highlighted in both tumor growth and immune modulation. Using a xenograft model in nude mice, we demonstrate a supportive role of sphingosine kinase 2 (SphK2), one of the S1P-producing enzymes for tumor progression. The growth of SphK2-deficient MCF-7 breast tumor xenografts was markedly delayed when compared with controls. Infiltration of macrophages in SphK2-deficient and control tumors was comparable. However, TAMs from SphK2-deficient tumors displayed a pronounced anti-tumor phenotype, showing an increased expression of pro-inflammatory markers/mediators such as NO, TNF-alpha, IL-12 and MHCII and a low expression of anti-inflammatory IL-10 and CD206. These data suggest a role for S1P, generated by SphK2, in early tumor development by affecting macrophage polarization.

Tumor-associated macrophages as targets for tumor immunotherapy.

Restoration of one of the major physiological functions of the body's immune response, the rejection of malignant cells, is a promising yet challenging task for cancer therapy. Prinicipally, immunotherapeutic approaches make use of cells of the adaptive immune system, since antigen-based tumor rejection might be the most specific approach. However, other immune cell populations, such as tumor-associated macrophages (TAMs), contribute significantly to protumor mechanisms elicited by a distorted immune response. In this review, we summarize the current knowledge about the pathology of TAMs and discuss potential therapeutic approaches to overcome TAM-mediated tumor promotion. Hereby, we focus on TAM phenotypes that were observed in the clinically relevant stages of cancer progression. The function of macrophages and other inflammatory cells in the onset of cancer has been discussed elsewhere.