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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.
Tumor development usually follows predictable paths where tumor cells acquire common characteristics and features known as the hallmarks of cancer. Recently, additional characteristics have been added to these hallmarks since solid tumors are composed of a very heterogeneous population of transformed, formerly normal tissue cells and stromal cells, e.g. immune cells and fibroblasts. Compelling evidence suggests that stromal cells and tumor cells maintain a symbiotic relationship to build up the tumor microenvironment and to fuel tumor growth. In cancer therapies, common features of tumors such as unrestricted cell growth, suppression of immunological responses, and the ability to form new blood vessels (angiogenesis) have emerged as the main targets of interest. The lipid mediator prostaglandin E2 (PGE2) is known to promote all these features and thus, is connected to cancer progression in general. Its synthesis is triggered in response to stress factors or during inflammation. Inducible PGE2 production relies on the enzymes cyclooxygenase 2 (COX-2) and microsomal prostanglandin E synthase 1 (mPGES-1), which are simultaneously expressed in response to a variety of different stimuli and are functionally coupled. Inhibition of COX-2 with non-steroidal antiinflammatory drugs (NSAIDs) for cancer treatment is, however, limited by cardiovascular risks, since selective COX-2 inhibition disrupts the prostacyclin/thromboxane balance. Therefore targeting mPGES-1 downstream of COX-2 for PGE2 inhibition was evaluated in this work in different steps of carcinogenesis. Knockdown of mPGES-1 in DU145 prostate cancer cells revealed that the mPGES-1 status did not affect growth of monolayer tumor cells, but significantly impaired 3D growth of multi-cellular tumor spheroids (MCTS). Spheroid formation induced COX-2 in DU145 and other prostate cancer spheroids. High levels of PGE2 were detected in supernatants of DU145 MCTS as opposed to monolayer DU145 cells. Pharmacological inhibition of COX-2 and mPGES-1 confirmed the pivotal role of PGE2 for DU145 MCTS growth. Besides promoting spheroid growth, MCTS-derived PGE2 also inhibited cytotoxic T lymphocyte (CTL) activation. When investigating the mechanisms of COX-2 induction during spheroid formation, the typical tumor microenvironmental factors such as glucose deprivation, hypoxia or tumor cell apoptosis failed to enhance COX-2. Interestingly, when interfering with apoptosis in DU145 spheroids, the pan-caspase inhibitor Z-VAD-FMK triggered a Summary 12 shift towards necrosis, thus enhancing COX-2 expression. Coculturing viable DU145 monolayer cells with isolated heat-shocked-treated necrotic DU145 cells, but not with necrotic cell supernatants, induced COX-2 and PGE2, confirming the impact of necrosis for MCTS growth and CTL inhibition. As mentioned, in vivo tumors are very heterogenous mixtures of tumor cells and stromal cells e.g. immune cells. Hence, the interaction of the immune system with tumors was investigated in further experiments. When coculturing MCF-7 breast cancer spheroids with human peripheral blood mononuclear cells (PBMCs), only low levels of PGE2 were detected, since MCF-7 cells did not upregulate COX-2 during spheroid formation and did not induce PGE2 production by PBMCs. Under inflammatory conditions, by adding the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) to cocultures, PGE2 production was triggered, spheroid sizes were reduced, and numbers of high levels of granzyme B expressing (GrBhi) CTLs were increased, while CD80 expression by tumor-associated phagocytes was also elevated. Inhibition of CD80 but not CD86 diminished numbers of GrBhi CTLs and attenuated spheroid lysis. To determine the role of ctivation-induced PGE2 production, use of the COX-2 inhibitor celecoxib and the experimental mPGES-1 inhibitor C3 further increased CD80 expression. Addition of PGE2, the prostaglandin E2 (EP2) receptor agonist butaprost, and the phosphodiesterase 4 (PDE4) inhibitor rolipram reduced LPS/C3-triggered CD80 expression, confirming the impact of COX- 2/mPGES-1-derived PGE2 on shaping phagocyte phenotypes in an EP2/cAMP-dependent manner. In a spontaneous breast cancer model (MMTV-PyMT), mPGES-1-deficiency significantly delayed tumor growth in mice, confirming an overall protumorigenic role of mPGES-1 in breast cancer development in vivo. However in tumors of mPGES-1-/- mice, tumor-infiltrating phagocytes expressed low levels of CD80 similar to their wildtype counterparts. These data suggest that the immunosuppressive microenvironment does not allow for immunostimulatory effects by mPGES-1 inhibition without an activating stimulus. Evidences in this study recommend the application of mPGES-1 inhibitors for treating cancer diseases, since mPGES-1 promotes tumor growth in multiple steps of carcinogenesis, ranging from well-characterized effects of tumor cell growth to immune suppression of CTL activity and phagocyte polarization. Regarding the latter, blunting PGE2 during immune activation may limit the tumor-favoring features of inflammation and improve the efficiency of TLR4 based immune therapies.
Background: Breast cancer is the leading cause of cancer-related deaths in women, demanding new treatment options. With the advent of immune checkpoint blockade, immunotherapy emerged as a treatment option. In addition to lymphocytes, tumor-associated macrophages exert a significant, albeit controversial, impact on tumor development. Pro-inflammatory macrophages are thought to hinder, whereas anti-inflammatory macrophages promote tumor growth. However, molecular markers to identify prognostic macrophage populations remain elusive. Methods: We isolated two macrophage subsets, from 48 primary human breast tumors, distinguished by the expression of CD206. Their transcriptomes were analyzed via RNA-Seq, and potential prognostic macrophage markers were validated by PhenOptics in tissue microarrays of patients with invasive breast cancer. Results: Normal human breast tissue contained mainly CD206+ macrophages, while increased relative amounts of CD206− macrophages were observed in tumors. The presence of CD206+ macrophages correlated with a pronounced lymphocyte infiltrate and subsets of CD206+ macrophages, expressing SERPINH1 and collagen 1, or MORC4, were unexpectedly associated with improved survival of breast cancer patients. In contrast, MHCIIhi CD206− macrophages were linked with a poor survival prognosis. Conclusion: Our data highlight the heterogeneity of tumor-infiltrating macrophages and suggest the use of multiple phenotypic markers to predict the impact of macrophage subpopulations on cancer prognosis. We identified novel macrophage markers that correlate with the survival of patients with invasive mammary carcinoma.