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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.
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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.
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Antígeno B7-1/antagonistas & inhibidores , Neoplasias de la Mama/metabolismo , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Macrófagos/metabolismo , Animales , Antígeno B7-1/biosíntesis , Antígeno B7-1/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Dinoprostona/metabolismo , Femenino , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Células MCF-7 , Macrófagos/inmunología , Ratones , Ratones Noqueados , Prostaglandina-E Sintasas , Transducción de Señal , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Microambiente TumoralRESUMEN
NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species, has recently been reported to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a proinflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8(+) T cell activation by using an antigen-driven coculture model consisting of Nrf2(-/-) and Nrf2(+/+) bone marrow-derived macrophages (BMDMΦ) and transgenic OT-1 CD8(+) T cells. OT-1 CD8(+) T cells encode a T cell receptor that specifically recognizes MHC class I-presented ovalbumin OVA(257-264) peptide, thereby causing a downstream T cell activation. Interestingly, coculture of OVA(257-264)-pulsed Nrf2(-/-) BMDMΦ with transgenic OT-1 CD8(+) T cells attenuated CD8(+) T cell activation, proliferation, and cytotoxic function. Since the provision of low-molecular-weight thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8(+) T cell activation, we quantified the amounts of intracellular and extracellular GSH and Cys in both cocultures. Indeed, GSH levels were strongly decreased in Nrf2(-/-) cocultures compared to wild-type counterparts. Supplementation of thiols in Nrf2(-/-) cocultures via addition of glutathione ester, N-acetylcysteine, ß-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH-synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine antiporter xCT in Nrf2(+/+) BMDMΦ mimicked the effect of Nrf2(-/-) BMDMΦ on CD8(+) T cell function. Our findings demonstrate that reduced levels of GCLM as well as xCT in Nrf2(-/-) BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8(+) T cell function.
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Médula Ósea/inmunología , Linfocitos T CD8-positivos/inmunología , Cistina/metabolismo , Glutatión/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Macrófagos/inmunología , Factor 2 Relacionado con NF-E2/fisiología , Animales , Antioxidantes/metabolismo , Apoptosis , Western Blotting , Médula Ósea/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Células Cultivadas , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/metabolismo , Técnicas para Inmunoenzimas , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Estrés Oxidativo , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2 ) supports the growth of a spectrum of cancers. The potential benefit of COX-2-inhibiting non-steroidal anti-inflammatory drugs (NSAIDs) for cancer treatment is however limited by their well-known cardiovascular side-effects. Therefore, targeting microsomal PGE synthase 1 (mPGES-1), the downstream enzyme in the COX-2-dependent pathway of PGE2 production might be attractive, although conflicting data regarding a potential tumor-supporting function of mPGES-1 were reported. We determined the impact of mPGES-1 in human DU145 prostate cancer cell growth. Surprisingly, knockdown of mPGES-1 did not alter growth of DU145 monolayer cells, but efficiently inhibited the growth of DU145 multicellular tumor spheroids (MCTS). Opposed to MCTS, monolayer cells did not secrete PGE2 due to a lack of COX-2 expression, which was induced during spheroid formation. Pharmacological inhibition of COX-2 and mPGES-1 supported the crucial role of PGE2 for growth of MCTS. The functionality of spheroid-derived PGE2 was demonstrated by its ability to inhibit cytotoxic T cell activation. When investigating mechanisms of spheroid-induced COX-2 induction, we observed that among microenvironmental factors neither glucose deprivation, hypoxia nor tumor cell apoptosis enhanced COX-2 expression. Interestingly, interfering with apoptosis in spheroids triggered a shift towards necrosis, thus augmenting COX-2 expression. We went on to demonstrate that necrotic cells induced COX-2 mRNA expression and PGE2 secretion from live tumor cells. In conclusion, necrosis-dependent COX-2 upregulation in MCTS promoted PGE2 -dependent tumor growth and inhibited activated cytotoxic T cells. Hence, blocking mPGES-1 as a therapeutic option may be considered for COX-2/mPGES-1-positive solid cancers.
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Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Esferoides Celulares/patología , Línea Celular Tumoral , Proliferación Celular , Humanos , Oxidorreductasas Intramoleculares/genética , Activación de Linfocitos , Masculino , Necrosis , Prostaglandina-E Sintasas , Neoplasias de la Próstata/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esferoides Celulares/metabolismo , Linfocitos T/inmunologíaRESUMEN
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.
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Neoplasias de la Mama/metabolismo , Interleucina-16/biosíntesis , Macrófagos/fisiología , Monocitos/fisiología , Tristetraprolina/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Quimiotaxis de Leucocito , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Interleucina-16/genética , Ratones , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Pronóstico , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Esferoides Celulares/citología , Trasplante Heterólogo , Microambiente Tumoral , Regulación hacia ArribaRESUMEN
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.
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Médula Ósea/patología , Hipoxia de la Célula , Células Dendríticas/patología , Células Madre Hematopoyéticas/patología , Sistema Hematopoyético/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Oxígeno/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proliferación Celular , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Humanos , Técnicas In Vitro , Proteína 2 Inhibidora de la Diferenciación/fisiología , Integrasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Extensive research in the last two decades implemented that the inflammatory cell infiltrate, especially in solid tumors, is a major determinant for patient prognosis. Mononuclear phagocytes, i.e. monocytes/macrophages, dendritic cells and myeloid-derived suppressor cells, constitute the majority of tumor-associated immune cells. Instead of inducing anti-tumor immunity, mononuclear phagocytes are functionally subverted by tumor microenvironmental factors to support each stage of oncogenesis. Although mechanisms how tumors program their inflammatory infiltrate to support tumor development are ill-defined, few master regulators are beginning to emerge. One of them is the inflammatory eicosanoid prostaglandin E(2) (PGE(2)), produced by tumor cells or the infiltrating immune cells. In this review we summarize the impact of PGE(2) on mononuclear phagocytes in inflammation and cancer and discuss potential implications for cancer therapy.