RESUMO
BACKGROUND AND AIMS: Dickkopf-1 (DKK1) is associated with poor prognosis in intrahepatic cholangiocarcinoma (iCCA), but the mechanisms behind this are unclear. Here, we show that DKK1 plays an immune regulatory role in vivo and inhibition reduces tumour growth. METHODS: Various in vivo GEMM mouse models and patient samples were utilized to assess the effects of tumour specific DKK1 overexpression in iCCA. DKK1-driven changes to the tumour immune microenvironment were characterized by immunostaining and gene expression analysis. DKK1 overexpressing and damage-induced models of iCCA were used to demonstrate the therapeutic efficacy of DKK1 inhibition in these contexts using the anti-DKK1 therapeutic, DKN-01. RESULTS: DKK1 overexpression in mouse models of iCCA drives an increase in chemokine and cytokine signalling, the recruitment of regulatory macrophages, and promotes the formation of a tolerogenic niche with higher numbers of regulatory T cells. We show a similar association of DKK1 with FOXP3 and regulatory T cells in patient tissue and gene expression data, demonstrating these effects are relevant to human iCCA. Finally, we demonstrate that inhibition of DKK1 with the monoclonal antibody mDKN-01 is effective at reducing tumour burden in two distinct mouse models of the disease. CONCLUSION: DKK1 promotes tumour immune evasion in iCCA through the recruitment of immune suppressive macrophages. Targeting DKK1 with a neutralizing antibody is effective at reducing tumour growth in vivo. As such, DKK1 targeted and immune modulatory therapies may be an effective strategy in iCCA patients with high DKK1 tumour expression or tolerogenic immune phenotypes.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Peptídeos e Proteínas de Sinalização Intercelular , Animais , Humanos , Camundongos , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fenótipo , Microambiente TumoralRESUMO
The pioneering cell biologist Michael Abercrombie first described the process of contact inhibition of locomotion more than 50 years ago when migrating fibroblasts were observed to rapidly change direction and migrate away upon collision. Since then, we have gleaned little understanding of how contact inhibition is regulated and only lately observed its occurrence in vivo. We recently revealed that Drosophila macrophages (haemocytes) require contact inhibition for their uniform embryonic dispersal. Here, to investigate the role that contact inhibition plays in the patterning of haemocyte movements, we have mathematically analysed and simulated their contact repulsion dynamics. Our data reveal that the final pattern of haemocyte distribution, and the details and timing of its formation, can be explained by contact inhibition dynamics within the geometry of the Drosophila embryo. This has implications for morphogenesis in general as it suggests that patterns can emerge, irrespective of external cues, when cells interact through simple rules of contact repulsion.
Assuntos
Padronização Corporal/fisiologia , Movimento Celular/fisiologia , Inibição de Contato/fisiologia , Drosophila/embriologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Comunicação Celular/fisiologia , Movimento Celular/genética , Rastreamento de Células , Simulação por Computador , Inibição de Contato/genética , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiologia , Embrião não Mamífero , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemócitos/citologia , Hemócitos/metabolismo , Hemócitos/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Modelos Teóricos , Proteína Vermelha FluorescenteRESUMO
Metastasis-associated macrophages (MAM) promote persistent growth of breast cancer cells at the metastatic site and are, thus, an attractive therapeutic target to treat breast cancer metastasis, a leading cause of cancer-related death in women. However, the precise mechanisms behind MAM-mediated metastatic tumor outgrowth have not been fully elucidated. Using mouse models of metastatic breast cancer, we showed that MAMs uniquely expressed hepatocyte growth factor (HGF) in metastatic tumors. We also demonstrated that a selected population of cancer cells with high metastatic potential (cancer cells that can establish metastatic tumors in mice with higher number and incidence than parental cells) had higher expression of HGF receptor, MNNG HOS transforming gene (MET), and were more responsive to HGF released from macrophages compared with the parental cells. Blockade of MET signaling in cancer cells suppressed metastatic tumor expansion, in part, through activation of natural killer cells. Results from this study suggest an approach to prevent life-threatening metastatic tumor formation using blockade of MAM-induced MET signal activation in metastatic cancer cells.
Assuntos
Fator de Crescimento de Hepatócito/genética , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/patologia , Proteínas Proto-Oncogênicas c-met/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Células Matadoras Naturais , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Dendritic cells (DC) are specialized sentinel cells that bridge the innate and adaptive immune response and play a crucial role in shaping the adaptive immune response. Vitamin D, a known epidemiological risk factor for the development of several autoimmune diseases, influences the development of dendritic cells. Consequently, vitamin D metabolites are frequently used in protocols to develop therapeutic dendritic cell therapies for autoimmune diseases. However, the mechanisms by which vitamin D modulates DC function remain poorly understood. We investigated the effects of vitamin D on murine CD11c+ bone marrow derived DC (BMDC) function by analyzing global gene expression in CD11c+ BMDC generated in the presence (VitD-CD11c+BMDC) or absence (Veh-CD11c+BMDC) of the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Seven genes were significantly increased in expression in both immature and LPS-matured VitD-CD11c+BMDC, one of which was CD31, a member of the immunoglobulin superfamily. Gene knockdown of CD31 enhanced the ability of VitD-CD11c+BMDC to prime naïve CD4+ T cells in vitro; conversely, increased expression of CD31 on vehicle treated CD11c+BMDC restrained their T cell priming abilities. Time-lapse imaging of BMDC and CD4+ T cells during in vitro priming revealed that CD31 reduced the BMDC-T cell interaction time. Finally, we confirmed a similar effect of 1,25(OH)2D3 on human CD34+ cell-derived CD11c+DC, whereby DC generated in the presence of 1,25(OH)2D3 had increased CD31 expression. In summary, we show that both mouse and human DC generated in the presence of 1,25(OH)2D3 upregulate CD31 expression, resulting in a reduced ability to prime CD4+ T cells by impairing a stable cell-cell contact.
Assuntos
Células Dendríticas/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Vitamina D/análogos & derivados , Vitaminas/farmacologia , Animais , Antígeno CD11c/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Feminino , Humanos , Camundongos Transgênicos , Regulação para Cima/efeitos dos fármacos , Vitamina D/farmacologiaRESUMO
The roles of tumor-associated macrophages (TAMs) and circulating monocytes in human cancer are poorly understood. Here, we show that monocyte subpopulation distribution and transcriptomes are significantly altered by the presence of endometrial and breast cancer. Furthermore, TAMs from endometrial and breast cancers are transcriptionally distinct from monocytes and their respective tissue-resident macrophages. We identified a breast TAM signature that is highly enriched in aggressive breast cancer subtypes and associated with shorter disease-specific survival. We also identified an auto-regulatory loop between TAMs and cancer cells driven by tumor necrosis factor alpha involving SIGLEC1 and CCL8, which is self-reinforcing through the production of CSF1. Together these data provide direct evidence that monocyte and macrophage transcriptional landscapes are perturbed by cancer, reflecting patient outcomes.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Reprogramação Celular , Macrófagos/metabolismo , Monócitos/metabolismo , Comunicação Parácrina , Transcrição Gênica , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Macrófagos/patologia , Terapia de Alvo Molecular , Monócitos/patologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , Células THP-1 , Microambiente TumoralRESUMO
Although the link between inflammation and cancer initiation is well established, its role in metastatic diseases, the primary cause of cancer deaths, has been poorly explored. Our previous studies identified a population of metastasis-associated macrophages (MAMs) recruited to the lung that promote tumor cell seeding and growth. Here we show that FMS-like tyrosine kinase 1 (Flt1, also known as VEGFR1) labels a subset of macrophages in human breast cancers that are significantly enriched in metastatic sites. In mouse models of breast cancer pulmonary metastasis, MAMs uniquely express FLT1. Using several genetic models, we show that macrophage FLT1 signaling is critical for metastasis. FLT1 inhibition does not affect MAM recruitment to metastatic lesions but regulates a set of inflammatory response genes, including colony-stimulating factor 1 (CSF1), a central regulator of macrophage biology. Using a gain-of-function approach, we show that CSF1-mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity of FLT1-inhibited MAMs. Thus, CSF1 is epistatic to FLT1, establishing a link between FLT1 and inflammatory responses within breast tumor metastases. Importantly, FLT1 inhibition reduces tumor metastatic efficiency even after initial seeding, suggesting that these pathways represent therapeutic targets in metastatic disease.
Assuntos
Neoplasias da Mama/metabolismo , Macrófagos/metabolismo , Neoplasias Mamárias Animais/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Comunicação Autócrina/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/patologia , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Fluorescence localization after photobleaching is a new method for localized photolabeling and subsequent tracking of specific molecules within living cells. The molecular species to be located carries two different fluorophores that can be imaged independently but simultaneously by fluorescence microscopy. For the method to work, these two fluorophores should be accurately colocalized throughout the cell so that their images are closely matched. One of the fluorophores (the target fluorophore) is then rapidly photobleached at a chosen location. The unbleached (reference) fluorophore remains colocalized with the target fluorophore; thus, the subsequent fate of the photobleached molecules can be revealed by processing simultaneously acquired digital images of the two fluorophores. Here we demonstrate the simplicity and effectiveness of the FLAP method in revealing both fast and slow molecular dynamics in living cells using a Zeiss LSM 510 laser scanning confocal microscope.
Assuntos
Recuperação de Fluorescência Após Fotodegradação/métodos , Animais , Células Cultivadas , Recuperação de Fluorescência Após Fotodegradação/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Modelos Biológicos , SoftwareRESUMO
Transformed rat fibroblasts expressing two variants of green fluorescent protein, each fused to beta-actin, were used to study actin dynamics during cell protrusion. The recently developed FLAP (fluorescence localization after photobleaching) method permits the tracking of one fluorophore after localized photobleaching by using the other as a colocalized reference. Here, by visualizing the ratio of bleached to total molecules, we found that actin was delivered to protruding zones of the leading edge of the cell at speeds that exceeded 5 micrometers per second. Monte Carlo modeling confirmed that this flow cannot be explained by diffusion and may involve active transport.