RESUMO
Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.
Assuntos
Melanoma , Humanos , Camundongos , Animais , Melanoma/patologia , Células Endoteliais/metabolismo , Linfócitos T CD8-Positivos , NF-kappa B/metabolismo , Autofagia , Imunoterapia , Microambiente TumoralRESUMO
Polychromatic flowcytometry is increasingly used for simultaneously analyzing multiple intracellular and cell-surface proteins on a given cell population. Here we describe a flowcytometry-based method to analyze various proteins on the surface of endothelial cells (which comprise of less than 0.5% of the tumor microenvironment) and concurrently sort the live endothelial cells for the downstream applications such as gene expression by conventional quantitative PCR or by single-cell RNA sequencing.
Assuntos
Células Endoteliais , Neoplasias , Contagem de Células , Células Endoteliais/patologia , Citometria de Fluxo/métodos , Humanos , Proteínas de Membrana , Neoplasias/patologia , Microambiente TumoralRESUMO
Autophagy, the major lysosomal pathway for the degradation and recycling of cytoplasmic materials, is increasingly recognized as a major player in endothelial cell (EC) biology and vascular pathology. Particularly in solid tumors, tumor microenvironmental stress such as hypoxia, nutrient deprivation, inflammatory mediators, and metabolic aberrations stimulates autophagy in tumor-associated blood vessels. Increased autophagy in ECs may serve as a mechanism to alleviate stress and restrict exacerbated inflammatory responses. However, increased autophagy in tumor-associated ECs can re-model metabolic pathways and affect the trafficking and surface availability of key mediators and regulators of the interplay between EC and immune cells. In line with this, heightened EC autophagy is involved in pathological angiogenesis, inflammatory, and immune responses. Here, we review major cellular and molecular mechanisms regulated by autophagy in ECs under physiological conditions and discuss recent evidence implicating EC autophagy in tumor angiogenesis and immunosurveillance.
Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Células Endoteliais/imunologia , Hipóxia/genética , Neoplasias/genética , Neovascularização Patológica/genética , Animais , Autofagia/imunologia , Proteínas Relacionadas à Autofagia/imunologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/patologia , Regulação Neoplásica da Expressão Gênica , Homeostase/genética , Homeostase/fisiologia , Humanos , Hipóxia/imunologia , Hipóxia/patologia , Vigilância Imunológica/genética , Lisossomos/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Neoplasias/patologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Transdução de Sinais , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Abdominal aortic aneurysms (AAAs) are a potentially deathly disease, needing surgical or endovascular treatment. To evaluate potentially new diagnostic tools and treatments, a large animal model, which resembles not only the morphological characteristics but also the pathophysiological background, would be useful. METHODS: Rodent animal aneurysm models were extrapolated to sheep. Four groups were created: intraluminal infusion with an elastase-collagenase solution (n = 4), infusion with elastase-collagenase solution combined with proximal stenosis (n = 7), aortic xenograft (n = 3), and elastase-collagenase-treated xenograft (n = 4). At fixed time intervals (6, 12, and 24 weeks), computer tomography and autopsy with histological evaluation were performed. RESULTS: The described models had a high perioperative mortality (45%), due to acute aortic thrombosis or fatale hemorrhage. A maximum aortic diameter increase of 30% was obtained in the protease-stenosis group. In the protease-treated groups, some histological features of human AAAs, such as inflammation, thinning of the media, and loss of elastin could be reproduced. In the xenotransplant groups, a pronounced inflammatory reaction was visible at the start. In all models, inflammation decreased and fibrosis occurred at long follow-up, 24 weeks postoperatively. CONCLUSIONS: None of the extrapolated small animal aneurysm models could produce an AAA in sheep with similar morphological features as the human disease. Some histological findings of human surgical specimens could be reproduced in the elastase-collagenase-treated groups. Long-term histological evaluation indicated stabilization and healing of the aortic wall months after the initial stimulus.
Assuntos
Aorta Abdominal/cirurgia , Aneurisma da Aorta Abdominal/induzido quimicamente , Colagenases , Elastase Pancreática , Animais , Aorta Abdominal/diagnóstico por imagem , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/patologia , Aortografia/métodos , Angiografia por Tomografia Computadorizada , Modelos Animais de Doenças , Progressão da Doença , Estudos de Viabilidade , Feminino , Xenoenxertos , Ratos , Carneiro Doméstico , Fatores de TempoRESUMO
BACKGROUND: Abdominal aortic aneurysm (AAA) is a prevalent disease affecting around 5% of the population aged more than 65 years. The exact etiology and physiopathology of AAA still raises questions, and elective surgery is currently the only treatment option for this often progressive disease. In this study, we hypothesized and tested a pathophysiological model that depicts AAA as an inflammation-triggered autoimmune disease with remnant vessel wall peptide fragments as the antigen. METHODS: A pilot study with male AAA patients (n = 14) and male controls (n = 8) was conducted. In both study groups, peripheral blood monocytes and plasma were separated from whole blood by centrifugation. An ELISpot test was performed on cultured white blood cells for the presence of elastin-specific T-lymphocytes. An Enzyme-linked immuno sorbent assay (ELISA) was performed on plasma for the presence of elastin-specific IgG molecules. RESULTS: ELISpot interferon-gamma secretion in AAA (7.7 ± 9.5%) and control (4.6 ± 3.5%) and ELISA anti-elastin IgG titer in AAA (77.5 ± 17.8%) and control (78.2 ± 31.5%) were not significantly different (P = 0.94 and P = 0.55, respectively). Both results are expressed as a percentage relative to the respective positive and negative control. CONCLUSIONS: The results of our pilot study did not indicate a clear and invariable autoimmune process directed against remnant elastin peptide fragments. Further research into the model mechanics and a possible antigen is still necessary. In the mean time, the model as presented here already offers a pathophysiological framework to further research into the possible remnant epitope-driven AAA etiology.