Innate immune cells in breast cancer--from villains to heroes?

The innate immune system ensures effective protection against foreign pathogens and plays important roles in tissue remodeling. There are many types of innate immune cells, including monocytes, macrophages, dendritic cells, and granulocytes. Interestingly, these cells accumulate in most solid tumors, including those of the breast. There, they play a tumor-promoting role through secretion of growth and angiogenic factors, as well as immunosuppressive molecules. This is in strong contrast to the tumor-suppressing effects that innate immune cells exert in vitro upon proper activation. Therapeutic approaches have been developed with the aim of achieving similar suppressive activities in vivo. However, multiple factors in the tumor microenvironment, many of which are immunosuppressive, represent a major obstacle to effective treatment. Here, we discuss the potential of combating breast cancer through activation of the innate immune system, including possible strategies to enhance the success of immunotherapy.

Activating a collaborative innate-adaptive immune response to control metastasis.

Tumor-associated macrophages (TAMs) promote metastasis and inhibit T cells, but macrophages can be polarized to kill cancer cells. Macrophage polarization could thus be a strategy for controlling cancer. We show that macrophages from metastatic pleural effusions of breast cancer patients can be polarized to kill cancer cells with monophosphoryl lipid A (MPLA) and interferon (IFN) γ. MPLA + IFNγ injected intratumorally or intraperitoneally reduces primary tumor growth and metastasis in breast cancer mouse models, suppresses metastasis, and enhances chemotherapy response in an ovarian cancer model. Both macrophages and T cells are critical for the treatment's anti-metastatic effects. MPLA + IFNγ stimulates type I IFN signaling, reprograms CD206+ TAMs to inducible NO synthase (iNOS)+ macrophages, and activates cytotoxic T cells through macrophage-secreted interleukin-12 (IL-12) and tumor necrosis factor alpha (TNFα). MPLA and IFNγ are used individually in clinical practice and together represent a previously unexplored approach for engaging a systemic anti-tumor immune response.

Sphingolipid rheostat alterations related to transformation can be exploited for specific induction of lysosomal cell death in murine and human glioma.

The search for cancer cell-specific targets suffers from a lack of integrative approaches that take into account the relative contributions of several mechanisms or pathways involved in cell death. A systematic experimental and computational comparison of murine glioma cells with astrocytes, their nontransformed counterparts, identified differences in the sphingolipid (SL) rheostat linked to an increased lysosomal instability in glioma cells. In vitro and in silico analyses indicate that sphingosine metabolized in lysosomes was preferentially recycled into ceramide, the prodeath member of the rheostat, in astrocytes. In glioma cells, it preferentially was used for production of the prosurvival sphingosine-1-phosphate (S1P). A combination of tumor necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), and interferon gamma (IFN-gamma) strongly decreased S1P production that resulted in abnormal lysosome enlargement and cell death associated with mitochondrial dysfunction of glioma cells only. Lack of intracellular S1P in glioma cells was concomitant with protein and lipid accumulation in enlarged lysosomes, indicating a blockade in lysosome recycling, and hence a role for S1P in membrane trafficking. A pharmacological sphingosine kinase inhibitor efficiently replaced the TNF-alpha, LPS, and IFN-gamma combination and killed murine and human glioma cells without affecting astrocytes. Our study provides evidence for a novel mechanism of lysosomal death dependent upon the SL rheostat that can be specifically triggered in glioma cells. It further strengthens the potential of cancer therapies based on specific ceramide pathway alterations.

TNF-alpha- and TRAIL-resistant glioma cells undergo autophagy-dependent cell death induced by activated microglia.

The role of microglia, the brain resident macrophages, in glioma biology is still ill-defined. Despite their cytotoxic potential, these cells that significantly infiltrate the tumor mass seem to support tumor growth rather than tumor eradication. A proper activation of microglia anti-tumor activities within the tumor may provide a valuable additional arm of defense to immunotherapies against brain tumors. We herewith report a detailed characterization of (lipopolysaccharide and interferon-gamma)-induced anti-tumor activities of mouse primary microglia towards two TNF-alpha and TRAIL resistant glioma cell lines, in cell monolayer or spheroid cultures and in collagen-embedded tumor explants. Irrespective of the mouse strain, stimulated microglia secreted proteic factors that decreased proliferation and migration of these glioma cells and efficiently killed them. Death occurred specifically in glioma cells as demonstrated by the lack of toxicity of microglia supernatant towards primary cultures of astrocytes or neurons. Cell death was characterized by the early accumulation of acidic vesicles, phosphatidylserine exposure, appearance of double-membrane cytoplasmic vesicles, extensive zeiosis and a very late loss of DNA in cells that had lost membrane integrity. Inhibition of autophagosome formation efficiently protected glioma cells from death whereas caspase inhibition could only prevent DNA loss but not cytotoxicity. Death however, resulted from a blockade by microglia supernatant of the basal autophagic flux present in the glioma cells. These observations demonstrate that glioma cells resistant to apoptotic death ligands could be successfully and specifically killed through autophagy-dependent death induced by appropriately activated microglia.

End-stage dying glioma cells are engulfed by mouse microglia with a strain-dependent efficacy.

Microglia phagocytic activity for apoptotic glioma cells is hardly analysed inspite of its relevance to tissue damage prevention. We provide evidence for a phosphatidylserine-independent clearance of mouse glioma cells at an advanced stage of death, suggesting microglia recognition of late apoptotic markers. Dying cells were immediately cleared or stayed for hours in that stage before engulfment occurred. This phagocytic activity was restricted to a microglia subset representing 30 to 70% of the population according to the used strain. Expression of receptors involved in late apoptotic markers recognition therefore seems confined to a subpopulation of microglia and to be strain-dependent.

Circular interpretation of regression coefficients.

The interpretation of the effect of predictors in projected normal regression models is not straight-forward. The main aim of this paper is to make this interpretation easier such that these models can be employed more readily by social scientific researchers. We introduce three new measures: the slope at the inflection point (bc ), average slope (AS) and slope at mean (SAM) that help us assess the marginal effect of a predictor in a Bayesian projected normal regression model. The SAM or AS are preferably used in situations where the data for a specific predictor do not lie close to the inflection point of a circular regression curve. In this case bc is an unstable and extrapolated effect. In addition, we outline how the projected normal regression model allows us to distinguish between an effect on the mean and spread of a circular outcome variable. We call these types of effects location and accuracy effects, respectively. The performance of the three new measures and of the methods to distinguish between location and accuracy effects is investigated in a simulation study. We conclude that the new measures and methods to distinguish between accuracy and location effects work well in situations with a clear location effect. In situations where the location effect is not clearly distinguishable from an accuracy effect not all measures work equally well and we recommend the use of the SAM.

Microglia isolated from patients with glioma gain antitumor activities on poly (I:C) stimulation.

The role of microglia, the brain-resident macrophages, in glioma biology is still a matter of debate. Clinical observations and in vitro studies in the mouse model indicate that microglia and macrophages that infiltrate the brain tumor tissue in high numbers play a tumor-supportive role. Here, we provide evidence that human microglia isolated from brain tumors indeed support tumor cell growth, migration, and invasion. However, after stimulation with the Toll-like receptor 3 agonist poly (I:C), microglia secrete factors that exerted toxic and suppressive effects on different glioblastoma cell lines, as assessed in cytotoxicity, migration, and tumor cell spheroid invasion assays. Remarkably, these effects were tumor-specific because the microglial factors impaired neither growth nor viability of astrocytes and neurons. Culture supernatants of tumor cells inhibited the poly (I:C) induction of this microglial M1-like, oncotoxic profile. Microglia stimulation before coculture with tumor cells circumvented the tumor-mediated suppression, as demonstrated by the ability to kill and phagocytose glioma cells. Our results show, for the first time to our knowledge, that human microglia exert tumor-supporting functions that are overridden by tumor-suppressing activities gained after poly (I:C) stimulation.

Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.

Little is known about the dynamics of cancer cell death in response to therapy in the tumor microenvironment. Intravital microscopy of chemotherapy-treated mouse mammary carcinomas allowed us to follow drug distribution, cell death, and tumor-stroma interactions. We observed associations between vascular leakage and response to doxorubicin, including improved response in matrix metalloproteinase-9 null mice that had increased vascular leakage. Furthermore, we observed CCR2-dependent infiltration of myeloid cells after treatment and that Ccr2 null host mice responded better to treatment with doxorubicin or cisplatin. These data show that the microenvironment contributes critically to drug response via regulation of vascular permeability and innate immune cell infiltration. Thus, live imaging can be used to gain insights into drug responses in situ.

Danger signaling protein HMGB1 induces a distinct form of cell death accompanied by formation of giant mitochondria.

Cells dying by necrosis release the high-mobility group box 1 (HMGB1) protein, which has immunostimulatory effects. However, little is known about the direct actions of extracellular HMGB1 protein on cancer cells. Here, we show that recombinant human HMGB1 (rhHMGB1) exerts strong cytotoxic effects on malignant tumor cells. The rhHMGB1-induced cytotoxicity depends on the presence of mitochondria and leads to fast depletion of mitochondrial DNA, severe damage of the mitochondrial proteome by toxic malondialdehyde adducts, and formation of giant mitochondria. The formation of giant mitochondria is independent of direct nuclear signaling events, because giant mitochondria are also observed in cytoplasts lacking nuclei. Further, the reactive oxygen species scavenger N-acetylcysteine as well as c-Jun NH(2)-terminal kinase blockade inhibited the cytotoxic effect of rhHMGB1. Importantly, glioblastoma cells, but not normal astrocytes, were highly susceptible to rhHMGB1-induced cell death. Systemic treatment with rhHMGB1 results in significant growth inhibition of xenografted tumors in vivo. In summary, rhHMGB1 induces a distinct form of cell death in cancer cells, which differs from the known forms of apoptosis, autophagy, and senescence, possibly representing an important novel mechanism of specialized necrosis. Further, our findings suggest that rhHMGB1 may offer therapeutic applications in treatment of patients with malignant brain tumors.