RESUMEN
The microenvironment of malignant melanomas defines the properties of tumor blood vessels and regulates infiltration and vascular dissemination of immune and cancer cells, respectively. Previous research in other cancer entities suggested the complement system as an essential part of the tumor microenvironment. Here, we confirm activation of the complement system in samples of melanoma patients and murine melanomas. We identified the tumor endothelium as the starting point of the complement cascade. Generation of complement-derived C5a promoted the recruitment of neutrophils. Upon contact with the vascular endothelium, neutrophils were further activated by complement membrane attack complexes (MACs). MAC-activated neutrophils release neutrophil extracellular traps (NETs). Close to the blood vessel wall, NETs opened the endothelial barrier as indicated by an enhanced vascular leakage. This facilitated the entrance of melanoma cells into the circulation and their systemic spread. Depletion of neutrophils or lack of MAC formation in complement component 6 (C6)-deficient animals protected the vascular endothelium and prevented vascular intravasation of melanoma cells. Our data suggest that inhibition of MAC-mediated neutrophil activation is a potent strategy to abolish hematogenous dissemination in melanoma.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento , Endotelio Vascular , Trampas Extracelulares , Melanoma , Neutrófilos , Microambiente Tumoral , Animales , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas del Sistema Complemento , Endotelio Vascular/fisiopatología , Humanos , Melanoma/irrigación sanguínea , Melanoma/inmunología , Melanoma/patología , Ratones , Neutrófilos/inmunología , PermeabilidadRESUMEN
Clinically relevant brain metastases (BMs) frequently form in cancer patients, with limited options for effective treatment. Circulating cancer cells must first permanently arrest in brain microvessels to colonize the brain, but the critical factors in this process are not well understood. Here, in vivo multiphoton laser-scanning microscopy of the entire brain metastatic cascade allowed unprecedented insights into how blood clot formation and von Willebrand factor (VWF) deposition determine the arrest of circulating cancer cells and subsequent brain colonization in mice. Clot formation in brain microvessels occurred frequently (>95%) and specifically at intravascularly arrested cancer cells, allowing their long-term arrest. An extensive clot embedded â¼20% of brain-arrested cancer cells, and those were more likely to successfully extravasate and form a macrometastasis. Mechanistically, the generation of tissue factor-mediated thrombin by cancer cells accounted for local activation of plasmatic coagulation in the brain. Thrombin inhibition by treatment with low molecular weight heparin or dabigatran and an anti-VWF antibody prevented clot formation, cancer cell arrest, extravasation, and the formation of brain macrometastases. In contrast, tumor cells were not able to directly activate platelets, and antiplatelet treatments did reduce platelet dispositions at intravascular cancer cells but did not reduce overall formation of BMs. In conclusion, our data show that plasmatic coagulation is activated early by intravascular tumor cells in the brain with subsequent clot formation, which led us to discover a novel and specific mechanism that is crucial for brain colonization. Direct or indirect thrombin and VWF inhibitors emerge as promising drug candidates for trials on prevention of BMs.
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Coagulación Sanguínea , Neoplasias Encefálicas/sangre , Neoplasias de la Mama/patología , Melanoma/patología , Células Neoplásicas Circulantes/patología , Trombosis/sangre , Animales , Neoplasias Encefálicas/etiología , Neoplasias Encefálicas/patología , Neoplasias de la Mama/sangre , Neoplasias de la Mama/complicaciones , Puntos de Control del Ciclo Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Melanoma/sangre , Melanoma/complicaciones , Ratones , Trombosis/etiología , Trombosis/patología , Factor de von Willebrand/análisisRESUMEN
Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.
Asunto(s)
Selectina E , Neoplasias Pulmonares , Animales , Bortezomib/farmacología , Antígeno CA-19-9/farmacología , Adhesión Celular , Selectina E/genética , Selectina E/metabolismo , Humanos , Ligandos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Metástasis de la Neoplasia , Oligosacáridos , Antígeno Sialil Lewis XRESUMEN
Ingenol mebutate (IM) is highly effective in the treatment of human papillomavirus (HPV)-induced anogenital warts (AGW) leading to fast ablation within hours. However, the exact mode of action is still largely unknown. We performed dermoscopy, in vivo confocal microscopy (CLM), histology, immunohistochemistry, and immunofluorescence to gain insights in mechanisms of IM treatment in AGW. In addition, we used in vitro assays (ELISA, HPV-transfection models) to further investigate in vivo findings. IM treatment leads to a strong recruitment of neutrophils with thrombosis of small skin vessels within 8 h, in a sense of immunothrombosis. In vivo and in vitro analyses showed that IM supports a prothrombotic environment by endothelial cell activation and von Willebrand factor (VWF) secretion, in addition to induction of neutrophil extracellular traps (NETosis). IM superinduces CXCL8/IL-8 expression in HPV-E6/E7 transfected HaCaT cells when compared to non-infected keratinocytes. Rapid ablation of warts after IM treatment can be well explained by the observed immunothrombosis. This new mechanism has so far only been observed in HPV-induced lesions and is completely different from the mechanisms we see in the treatment of transformed keratinocytes in actinic keratosis. Our initial findings indicate an HPV-specific effect, which could be also of interest for the treatment of other HPV-induced lesions. Larger studies are now needed to further investigate the potential of IM in different HPV tumors.
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Condiloma Acuminado , Diterpenos , Queratosis Actínica , Infecciones por Papillomavirus , Anomalías Cutáneas , Verrugas , Humanos , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/tratamiento farmacológico , Condiloma Acuminado/tratamiento farmacológico , Diterpenos/farmacología , Diterpenos/uso terapéutico , Queratosis Actínica/tratamiento farmacológico , Papillomaviridae , NecrosisRESUMEN
RATIONALE: Adhesion molecules are key elements in stroke-induced brain injury by regulating the migration of effector immune cells from the circulation to the lesion site. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an adhesion molecule highly expressed on endothelial cells and leukocytes, which controls the final steps of trans-endothelial migration. A functional role for PECAM-1 in post-ischemic brain injury has not yet been demonstrated. OBJECTIVE: Using genetic Pecam-1 depletion and PECAM-1 blockade using a neutralizing anti-PECAM-1 antibody, we evaluated the role of PECAM-1 mediated trans-endothelial immune cell migration for ischemic injury, delayed brain atrophy, and brain immune cell infiltrates. Trans-endothelial immune cell migration was furthermore evaluated in cultured human cerebral microvascular endothelial cells. METHODS AND RESULTS: Transient middle cerebral artery occlusion (tMCAO) was induced in 10-12-week-old male Pecam-1-/- and Pecam-1+/+ wildtype mice. PECAM-1 levels increased in the ischemic brain tissue due to the infiltration of PECAM-1+ leukocytes. Using magnetic resonance imaging, we observed smaller infarct volume, less edema formation, and less brain atrophy in Pecam-1-/- compared with Pecam-1+/+ wildtype mice. The transmigration of leukocytes, specifical neutrophils, was selectively reduced by Pecam-1-/-, as shown by immune fluorescence and flow cytometry in vivo and transmigration assays in vitro. Importantly, inhibition with an anti-PECAM-1 antibody in wildtype mice decreased neutrophil brain influx and infarct. CONCLUSION: PECAM-1 controls the trans-endothelial migration of neutrophils in a mouse model of ischemic stroke. Antibody blockade of PECAM-1 after stroke onset ameliorates stroke severity in mice, making PECAM-1 an interesting target to dampen post-stroke neuroinflammation, reduce ischemic brain injury, and enhance post-ischemic brain remodeling.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Movimiento Celular , Células Endoteliales , Endotelio Vascular , Masculino , Ratones , Ratones Noqueados , Neutrófilos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Migración Transendotelial y TransepitelialRESUMEN
Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation.
Asunto(s)
Melanoma/metabolismo , Agregación Plaquetaria , Factor de von Willebrand/metabolismo , Proteínas ADAM/sangre , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Animales , Coagulación Sanguínea , Plaquetas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Activación Enzimática , Fibrinolíticos/farmacología , Heparina de Bajo-Peso-Molecular/farmacología , Humanos , Melanoma/sangre , Melanoma/patología , Ratones , Ratones Transgénicos , Microvasos/metabolismo , Neovascularización Patológica/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-ret/metabolismo , Tinzaparina , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In this study, it is shown that the cytotoxic response of cells as well as the uptake kinetics of nanoparticles (NPs) is cell type dependent. We use silica NPs with a diameter of 310 nm labeled with perylene dye and 304 nm unlabeled particles to evaluate cell type-dependent uptake and cytotoxicity on human vascular endothelial cells (HUVEC) and cancer cells derived from the cervix carcinoma (HeLa). Besides their size, the particles are characterized concerning homogeneity of the labeling and their zeta potential. The cellular uptake of the labeled NPs is quantified by imaging the cells via confocal microscopy in a time-dependent manner, with subsequent image analysis via a custom-made and freely available digital method, Particle_in_Cell-3D. We find that within the first 4 h of interaction, the uptake of silica NPs into the cytoplasm is up to 10 times more efficient in HUVEC than in HeLa cells. Interestingly, after 10 or 24 h of interaction, the number of intracellular particles for HeLa cells by far surpasses the one for HUVEC. Inhibitor studies show that these endothelial cells internalize 310 nm SiO2 NPs via the clathrin-dependent pathway. Remarkably, the differences in the amount of taken up NPs are not directly reflected by the metabolic activity and membrane integrity of the individual cell types. Interaction with NPs leads to a concentration-dependent decrease in mitochondrial activity and an increase in membrane leakage for HUVEC, whereas HeLa cells show only a reduced mitochondrial activity and no membrane leakage. In addition, silica NPs lead to HUVEC cell death while HeLa cells survive. These findings indicate that HUVEC are more sensitive than HeLa cells upon silica NP exposure.
Asunto(s)
Nanopartículas/metabolismo , Nanopartículas/toxicidad , Dióxido de Silicio/química , Muerte Celular/efectos de los fármacos , Células HeLa , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Cinética , Nanopartículas/químicaRESUMEN
Tumor cell extravasation is a critical step in the metastatic cascade and requires interaction between the tumor cell and the endothelium. Although cancer progression depends on a complex network of mechanisms, including inflammation and coagulation, the involvement of tumor-induced endothelium activation and the subsequent release of procoagulatory factors in this process are not well understood. Using tissue sections from patients with malignant melanoma, immunofluorescence studies for the presence of von Willebrand factor (VWF) clearly demonstrated endothelium activation and the formation of ultra-large VWF fibers in these patients. In vitro analyses revealed that supernatants from highly invasive melanoma cells induced an acute endothelium activation measured by VWF, P-selectin, and angiopoietin-2 release. Proteome profiling identified vascular endothelial growth factor A (VEGF-A) as the main mediator of endothelium activation. Inhibition and knock-down of VEGF-A in melanoma cells led to a rigorous decrease in VWF exocytosis. Selective small-interfering RNA to matrix metalloproteinase-2 (MMP-2) inhibited endothelium activation, and this effect correlated with reduced VEGF-A content in the supernatants of melanoma cells. Further experiments showed that active MMP-2 regulates VEGF-A in melanoma cells on a transcriptional level via an integrin αvß5/phosphoinositide-3-kinase-dependent pathway. In conclusion, these results indicate an important role of VEGF-A in acute endothelium activation and provide clear evidence that MMP-2 plays a pivotal role in the autocrine regulation of VEGF-A expression in melanoma cells.
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Endotelio Vascular/enzimología , Endotelio Vascular/patología , Metaloproteinasa 2 de la Matriz/metabolismo , Melanoma/enzimología , Melanoma/patología , Receptores de Vitronectina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Metaloproteinasa 14 de la Matriz/metabolismo , Melanoma/irrigación sanguínea , Melanoma/genética , Ratones , Modelos Biológicos , Invasividad Neoplásica , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: An increased incidence of thrombotic complications associated with an increased mortality rate has been observed under immune checkpoint inhibition (ICI). Recent investigations on the coagulation pathways have highlighted the direct role of key coagulatory proteins and platelets in cancer initiation, angiogenesis and progression. The aim of this study was to evaluate the prognostic value of von Willebrand factor (vWF) and its regulatory enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), D-dimers and platelets in a cohort of patients with metastatic melanoma receiving ICI. METHODS: In a prospective cohort of 83 patients with metastatic melanoma, we measured the systemic levels of vWF-antigen (vWF:Ag), ADAMTS13 activity, D-dimers and platelets, before the beginning of the treatment (baseline), and 6, 12 and 24 weeks after. In parallel, we collected standard biological parameters used in clinical routine to monitor melanoma response (lactate deshydrogenase (LDH), S100). The impact of neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) on overall survival (OS) in patients receiving ICI was assessed. Univariable and multivariable Cox proportional models were then used to investigate any potential association of these parameters to clinical progression (progression-free survival (PFS) and OS). Baseline values and variations over therapy course were compared between primary responders and resistant patients. RESULTS: Patients with melanoma present with dysregulated levels of vWF:Ag, ADAMTS13 activity, D-dimers, LDH, S100 and CRP at the beginning of treatment. With a median clinical follow-up of 26 months, vWF:Ag interrogated as a continuous variable was significantly associated with PFS in univariate and multivariate analysis (HR=1.04; p=0.007). Lower values of vWF:Ag at baseline were observed in the primary responders group (median: 29.4 µg/mL vs 32.9 µg/mL; p=0.048) when compared with primary resistant patients. As for OS, we found an association with D-dimers and ADAMTS13 activity in univariate analysis and vWF:Ag in univariate and multivariate analysis including v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and Eastern Cooperative Oncology Group (ECOG) performance status. Follow-up over the course of treatment depicts different evolution profiles for vWF:Ag between the primary response and resistance groups. CONCLUSIONS: In this prospective cohort, coagulatory parameters such as ADAMTS13 activity and D-dimers are associated with OS but baseline vWF:Ag levels appeared as the only parameter associated with response and OS to ICI. This highlights a potential role of vWF as a biomarker to monitor ICI response of patients with malignant melanoma.
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Melanoma , Factor de von Willebrand , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Melanoma/tratamiento farmacológico , Pronóstico , Estudios Prospectivos , Factor de von Willebrand/metabolismoRESUMEN
Crossing the blood-brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and endpoint in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (EC) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by cross-talk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it. SIGNIFICANCE: Tracking single extravasating cancer cells using multimodal correlative microscopy uncovers a brain seeding mechanism involving endothelial remodeling driven by cancer cell-derived MMP9, which might enable the development of approaches to prevent brain metastasis. See related commentary by McCarty, p. 1167.
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Neoplasias Encefálicas , Endotelio Vascular , Humanos , Endotelio Vascular/patología , Células Endoteliales/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/patología , Línea Celular TumoralRESUMEN
Advances in understanding the molecular mechanisms of tumor progression have achieved impressive progress in the treatment of cancer and so-called immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. Indeed, antibody-based drugs blocking immune escape of tumor cells by modulation of T cell responses are increasingly utilized for a wide range of tumor entities. Nonetheless, response rates remain limited, and the development of secondary resistance is a common problem. In addition, by increasing the immune response a variety of severe side effects are provoked. Next to autoimmune responses, activation of the complement system and skin toxicity, an increased incidence for thrombotic complications has been observed associated with an increased mortality rate. Based on this, it can be postulated that the interplay of coagulation with inflammation in the tumor microenvironment is relevant for each step in the tumor life cycle. This review focuses on the coagulation as central player fostering mechanisms associated with tumor progression. Thus, a better understanding of the molecular pathways involved in the complex interaction of circulating tumor cells, the plasmatic coagulation and immune cells may help to improve therapeutic concepts reducing mortality and morbidity associated with cancer.
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Coagulación Sanguínea/inmunología , Heparina de Bajo-Peso-Molecular/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inflamación/sangre , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Anticoagulantes/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Humanos , Inflamación/inmunología , Inflamación/patología , Neoplasias/inmunología , Trombosis/sangre , Trombosis/tratamiento farmacológico , Trombosis/patología , Escape del Tumor/efectos de los fármacosRESUMEN
Breast cancer (BC) is the second-most common cause of brain metastases (BM) and BCBM patients have a reduced quality of life and a poor prognosis. Hyaluronan (HA), and in particular the hyaluronidase Hyal-1, has been already linked to the development of BCBM, and therefore presents an interesting opportunity to develop new effective therapeutic options. HA metabolism was further discovered by the CRISPR/Cas9-mediated knockout of HYAL1 and the shRNA-mediated down-regulation of HA-receptor CD44 in the brain-seeking triple-negative breast cancer (TNBC) cell line MDA-MB-231-BR. Therefore, the impact of Hyal-1 on adhesion, disruption, and invasion through the brain endothelium, both in vitro and in vivo, was studied. Our analysis points out a key role of Hyal-1 and low-molecular-weight HA (LMW-HA) in the formation of a pericellular HA-coat in BC cells, which in turn promotes tumor cell adhesion, disruption, and migration through the brain endothelium in vitro as well as the extent of BM in vivo. CD44 knockdown in MDA-MB-231-BR significantly reduced the pericellular HA-coat on these cells, and, consequently, tumor cell adhesion and invasion through the brain endothelium. Thus, the interaction between Hyal-1-generated LMW-HA fragments and the HA-receptor CD44 might represent a potential target for future therapeutic options in BC patients with a high risk of cerebral metastases formation.
Asunto(s)
Neoplasias Encefálicas , Ácido Hialurónico , Hialuronoglucosaminidasa , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/genética , Hialuronoglucosaminidasa/metabolismo , Calidad de Vida , ARN Interferente Pequeño , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Immune checkpoint inhibition (ICI) has yielded remarkable results in prolonging survival of metastatic melanoma patients but only a subset of individuals treated respond to therapy. Success of ICI treatment appears to depend on the number of tumor-infiltrating effector T-cells, which are known to be influenced by activated eosinophils. To verify the co-occurrence of activated eosinophils and T-cells in melanoma, immunofluorescence was performed in 285 primary or metastatic tumor tissue specimens from 118 patients. Moreover, eosinophil counts and activity markers such as eosinophil cationic protein (ECP) and eosinophil peroxidase (EPX) were measured in the serum before therapy start and before the 4th infusion of ICI in 45 metastatic unresected melanoma patients. We observed a positive correlation between increased tumor-infiltrating eosinophils and T-cells associated with delayed melanoma progression. High baseline levels of eosinophil count, serum ECP and EPX were linked to prolonged progression-free survival in metastatic melanoma. Our data provide first indications that activated eosinophils are related to the T-cell-inflamed tumor microenvironment and could be considered as potential future prognostic biomarkers in melanoma.
RESUMEN
Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients' survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression.
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Heparitina Sulfato , Melanoma , Factor de von Willebrand , Animales , Adhesión Celular , Heparitina Sulfato/metabolismo , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Unión Proteica , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: The prognosis of patients with brain metastases (BM) is poor despite advances in our understanding of the underlying pathophysiology. The high incidence of thrombotic complications defines tumor progression and the high mortality rate. We, therefore, postulated that von Willebrand factor (VWF) promotes BM via its ability to induce platelet aggregation and thrombosis. METHODS: We measured the abundance of VWF in the blood and intravascular platelet aggregates of patients with BM, and determined the specific contribution of endothelial and platelet-derived VWF using in vitro models and microfluidics. The relevance for the brain metastatic cascade in vivo was demonstrated in ret transgenic mice, which spontaneously develop BM, and by the intracardiac injection of melanoma cells. RESULTS: Higher levels of plasma VWF in patients with BM were associated with enhanced intraluminal VWF fiber formation and platelet aggregation in the metastatic tissue and peritumoral regions. Platelet activation triggered the formation of VWF multimers, promoting platelet aggregation and activation, in turn enhancing tumor invasiveness. The absence of VWF in platelets, or the blocking of platelet activation, abolished platelet aggregation, and reduced tumor cell transmigration. Anticoagulation and platelet inhibition consistently reduced the number of BM in preclinical animal models. CONCLUSIONS: Our data indicate that platelet-derived VWF is involved in cerebral clot formation and in metastatic growth of melanoma in the brain. Targeting platelet activation with low-molecular-weight heparins represents a promising therapeutic approach to prevent melanoma BM.
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Cancer-related venous thromboembolisms (VTE) are associated with metastasis and reduced survival in patients with urothelial cancer of the bladder. Although previous reports suggest the contribution of tissue factor and podoplanin, the mechanistic linkage between VTE and bladder cancer cell-derived molecules is unknown. Therefore, we compared distinct procoagulant pathways in four different cell lines. In vitro findings were further confirmed by microfluidic experiments mimicking the pathophysiology of tumor blood vessels and in tissue samples of patients with bladder cancer by transcriptome analysis and immunohistology. In vitro and microfluidic experiments identified bladder cancer-derived VEGF-A as highly procoagulant because it promoted the release of von Willebrand factor (VWF) from endothelial cells and thus platelet aggregation. In tissue sections from patients with bladder cancer, we found that VWF-mediated blood vessel occlusions were associated with a poor outcome. Transcriptome data further indicate that elevated expression levels of enzymes modulating VEGF-A availability were significantly connected to a decreased survival in patients with bladder cancer. In comparison with previously postulated molecular players, we identified tumor cell-derived VEGF-A and endothelial VWF as procoagulant mediators in bladder cancer. Therapeutic strategies that prevent the VEGF-A-mediated release of VWF may reduce tumor-associated hypercoagulation and metastasis in patients with bladder cancer. IMPLICATIONS: We identified the VEGF-A-mediated release of VWF from endothelial cells to be associated with bladder cancer progression.
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Carcinoma de Células Transicionales/metabolismo , Células Endoteliales/citología , Neoplasias de la Vejiga Urinaria/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor de von Willebrand/metabolismo , Carcinoma de Células Transicionales/genética , Línea Celular Tumoral , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas Analíticas Microfluídicas , Metástasis de la Neoplasia , Proteómica , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
The glycocalyx regulates the interaction of mammalian cells with extracellular molecules, such as cytokines. However, it is unknown to which extend the glycocalyx of distinct cancer cells control the binding and uptake of nanoparticles. In the present study, exome sequencing data of cancer patients and analysis of distinct melanoma and bladder cancer cell lines suggested differences in cancer cell-exposed glycocalyx components such as heparan sulphate. Our data indicate that glycocalyx differences affected the binding of cationic chitosan nanocapsules (Chi-NCs). The pronounced glycocalyx of bladder cancer cells enhanced the internalisation of nanoencapsulated capsaicin. Consequently, capsaicin induced apoptosis in the cancer cells, but not in the less glycosylated benign urothelial cells. Moreover, we measured counterion condensation on highly negatively charged heparan sulphate chains. Counterion condensation triggered a cooperative binding of Chi-NCs, characterised by a weak binding rate at low Chi-NC doses and a strongly increased binding rate at high Chi-NC concentrations. Our results indicate that the glycocalyx of tumour cells controls the binding and biological activity of nanoparticles. This has to be considered for the design of tumour cell directed nanocarriers to improve the delivery of cytotoxic drugs. Differential nanoparticle binding may also be useful to discriminate tumour cells from healthy cells.
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Antipruriginosos/administración & dosificación , Antipruriginosos/farmacocinética , Capsaicina/administración & dosificación , Capsaicina/farmacocinética , Quitosano/química , Glicocálix/metabolismo , Nanocápsulas/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Especificidad de Órganos , Unión Proteica , Electricidad Estática , Nanomedicina TeranósticaRESUMEN
The dynamic change from a globular conformation to an elongated fiber determines the ability of von Willebrand factor (VWF) to trap platelets. Fiber formation is favored by the anchorage of VWF to the endothelial cell surface, and VWF-platelet aggregates on the endothelium contribute to inflammation, infection, and tumor progression. Although P-selectin and ανß3-integrins may bind VWF, their precise role is unclear, and additional binding partners have been proposed. In the present study, we evaluated whether the endothelial glycocalyx anchors VWF fibers to the endothelium. Using microfluidic experiments, we showed that stabilization of the endothelial glycocalyx by chitosan oligosaccharides or overexpression of syndecan-1 (SDC-1) significantly supports the binding of VWF fibers to endothelial cells. Heparinase-mediated degradation or impaired synthesis of heparan sulfate (HS), a major component of the endothelial glycocalyx, reduces VWF fiber-dependent platelet recruitment. Molecular interaction studies using flow cytometry and live-cell fluorescence microscopy provided further evidence that VWF binds to HS linked to SDC-1. In a murine melanoma model, we found that protection of the endothelial glycocalyx through the silencing of heparanase increases the number of VWF fibers attached to the wall of tumor blood vessels. In conclusion, we identified HS chains as a relevant binding factor for VWF fibers at the endothelial cell surface in vitro and in vivo.
Asunto(s)
Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Glicocálix/metabolismo , Factor de von Willebrand/metabolismo , Animales , Plaquetas/metabolismo , Femenino , Expresión Génica , Humanos , Ratones , Adhesividad Plaquetaria , Unión Proteica , Transporte de Proteínas , Sindecano-1/metabolismoRESUMEN
Tumor progression is associated with aberrant hemostasis, and patients with malignant diseases have an elevated risk of developing thrombosis. A crosstalk among the vascular endothelium, components of the coagulation cascade, and cancer cells transforms the intravascular milieu to a prothrombotic, proinflammatory, and cell-adhesive state. We review the existing evidence on activation of the coagulation system and its implication in genitourinary malignancies and discuss the potential therapeutic benefit of antithrombotic agents. A literature review was performed searching the Medline database and the Cochrane Library for original articles and reviews. A second search identified studies reporting on oncological benefit of anticoagulants in genitourinary cancer. An elevated expression of procoagulatory tissue factor on tumor cells and tumor-derived microparticles seems to stimulate cancer development and progression. Several components of the hemostatic system, including D-dimers, von Willebrand Factor, thrombin, fibrin-/ogen, soluble P-selectin, and prothrombin fragments 1 + 2 were either overexpressed or overactive in genitourinary cancers. Hypercoagulation was in general associated with a poorer prognosis. Experimental models and small trials in humans showed reduced cancer progression after treatment with anticoagulants. Main limitations of these studies were heterogeneous experimental methodology, small patient numbers, and a lack of prospective validation. In conclusion, experimental and clinical evidence suggests procoagulatory activity of genitourinary neoplasms, particularly in prostate, bladder and kidney cancer. This may promote the risk of vascular thrombosis but also metastatic progression. Clinical studies linked elevated biomarkers of hemostasis with poor prognosis in patients with genitourinary cancers. Thus, anticoagulation may have a therapeutic role beyond prevention of thromboembolism.