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Caveolin-1 (Cav-1) is a fundamental constituent of caveolae, whose functionality and structure are strictly dependent on cholesterol. In this work the U18666A inhibitor was used to study the role of cholesterol transport in the endosomal degradative-secretory system in a metastatic human melanoma cell line (WM266-4). We found that U18666A induces a shift of Cav-1 from the plasma membrane to the endolysosomal compartment, which is involved, through Multi Vesicular Bodies (MVBs), in the formation and release of small extracellular vesicles (sEVs). Moreover, this inhibitor induces an increase in the production of sEVs with chemical-physical characteristics similar to control sEVs but with a different protein composition (lower expression of Cav-1 and increase of LC3II) and reduced transfer capacity on target cells. Furthermore, we determined that U18666A affects mitochondrial function and also cancer cell aggressive features, such as migration and invasion. Taken together, these results indicate that the blockage of cholesterol transport, determining the internalization of Cav-1, may modify sEVs secretory pathways through an increased fusion between autophagosomes and MVBs to form amphisome, which in turn fuses with the plasma membrane releasing a heterogeneous population of sEVs to maintain homeostasis and ensure correct cellular functionality.
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Vesículas Extracelulares , Melanoma , Humanos , Caveolina 1/metabolismo , Autofagossomos/metabolismo , Vesículas Extracelulares/metabolismo , Colesterol/metabolismoRESUMO
BACKGROUND: Glioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment. METHODS: In this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays. RESULTS: We confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration. CONCLUSION: In this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.
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The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), the main AP-endonuclease of the DNA base excision repair pathway, is a key molecule of interest to researchers due to its unsuspected roles in different nonrepair activities, such as: i) adaptive cell response to genotoxic stress, ii) regulation of gene expression, and iii) processing of microRNAs, which make it an excellent drug target for cancer treatment. We and others recently demonstrated that APE1 can be secreted in the extracellular environment and that serum APE1 may represent a novel prognostic biomarker in hepatocellular and non-small-cell lung cancers. However, the mechanism by which APE1 is released extracellularly was not described before. Here, using three different approaches for exosomes isolation: commercial kit, nickel-based isolation, and ultracentrifugation methods and various mammalian cell lines, we elucidated the mechanisms responsible for APE1 secretion. We demonstrated that APE1 p37 and p33 forms are actively secreted through extracellular vesicles (EVs), including exosomes from different mammalian cell lines. We then observed that APE1 p33 form is generated by proteasomal-mediated degradation and is enzymatically active in EVs. Finally, we revealed that the p33 form of APE1 accumulates in EVs upon genotoxic treatment by cisplatin and doxorubicin, compounds commonly found in chemotherapy pharmacological treatments. Taken together, these findings provide for the first time evidence that a functional Base Excision Repair protein is delivered through exosomes in response to genotoxic stresses, shedding new light into the complex noncanonical biological functions of APE1 and opening new intriguing perspectives on its role in cancer biology.
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DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Exossomos/enzimologia , Animais , Linhagem Celular , Reparo do DNA , HumanosRESUMO
Circulating endothelial progenitor cells (EPCs) have been suggested as a precious source for generating functionally competent endothelial cells (ECs), candidate for various clinical applications. However, the paucity of these progenitor cells and the technical difficulties for their in vitro growth represent a main limitation to their use. In the present study we hypothesized that the paracrine effects of human umbilical vein endothelial cells (HUVECs) may improve endothelial cell generation from cord blood (CB) EPCs. In line with this hypothesis we showed that HUVEC conditioned medium (CM) or co-culture with HUVECs markedly improved the proliferation and differentiation and delayed the senescence of CB EPCs. The endothelial-promoting effect of CM seems to be related to smaller vesicles including exosomes (sEV/exo) contained in this medium and transferred to CB CD34(+) EPCs: in fact, purified preparations of sEV/exo isolated from CM mimicked the effect of CM to sustain endothelial formation. These observations provided the interesting indication that mature ECs exert a stimulatory effect on endothelial cell differentiation from CD34(+) cells.
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Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Antígenos CD34 , Células Cultivadas , Células Endoteliais/citologia , Exossomos , Sangue Fetal/citologia , Humanos , Comunicação ParácrinaRESUMO
Blood and airway eosinophilia represent markers for the endotype-driven treatment of allergic asthma. Little is known on mechanisms that link eosinophils and airway epithelial cells before and after these cells are infiltrated by eosinophils during allergic response. Given that innate immune mechanisms, mainly mediated by epithelial-derived cytokines (IL-33, IL-25, TSLP), induce eosinophil-maturing/attractive substances, we thought to evaluate the crosstalk between eosinophils and airway epithelial cells in the context of IL-33-mediated allergic inflammation. DUOX1 was previously described in clinically relevant aspects of allergic inflammation in a HDM -induced allergic asthma mice model, and in patients with chronic sinusitis or allergic asthma. Thus, we evaluated the involvement of HDM and eosinophils in the regulation of DUOX1 in airway epithelial cells. To recapitulate the lung environment present at the allergen challenge time in acute asthma, we set up an in vitro model based on murine bone marrow-derived eosinophils differentiated with IL-5 and then activated with IL-33 (EOs33) and TC1 or C57 airway epithelial cells. We found that treatment of epithelial cells with HDM induced an eosinophil-attractive environment and increased DUOX1 expression. Importantly, we found that the co-culture of airway epithelial cells with EOs33 or with conditioned medium from EOs33 enhanced the expression of DUOX1, which was further increased by combined stimulation (HDM plus EOs33). Our results suggest that lung recruited EOs once activated by IL-33 could be involved in a crosstalk loop with airway epithelial cells by DUOX1-mediated IL-33 secretion.
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The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW 4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.
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Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Transporte Proteico , Humanos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células HeLa , Endossomos/metabolismo , Células A549 , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Corpos Multivesiculares/metabolismo , Reparo por Excisão , Ácidos HidroxâmicosRESUMO
Immune cell-derived extracellular vesicles (EV) affect tumor progression and hold promise for therapeutic applications. Eosinophils are major effectors in Th2-related pathologies recently implied in cancer. Here, we evaluated the anti-tumor activities of eosinophil-derived EV following activation with the alarmin IL-33. We demonstrate that IL-33-activated mouse and human eosinophils produce higher quantities of EV with respect to eosinophils stimulated with IL-5. Following incorporation of EV from IL-33-activated eosinophils (Eo33-EV), but not EV from IL-5-treated eosinophils (Eo5-EV), mouse and human tumor cells increased the expression of cyclin-dependent kinase inhibitor (CDKI)-related genes resulting in cell cycle arrest in G0/G1, reduced proliferation and inhibited tumor spheroid formation. Moreover, tumor cells incorporating Eo33-EV acquired an epithelial-like phenotype characterized by E-Cadherin up-regulation, N-Cadherin downregulation, reduced cell elongation and migratory extent in vitro, and impaired capacity to metastasize to lungs when injected in syngeneic mice. RNA sequencing revealed distinct mRNA signatures in Eo33-EV and Eo5-EV with increased presence of tumor suppressor genes and enrichment in pathways related to epithelial phenotypes and negative regulation of cellular processes in Eo33-EV compared to Eo5-EV. Our studies underscore novel IL-33-stimulated anticancer activities of eosinophils through EV-mediated reprogramming of tumor cells opening perspectives on the use of eosinophil-derived EV in cancer therapy.
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Eosinófilos , Vesículas Extracelulares , Interleucina-33 , Animais , Interleucina-33/metabolismo , Camundongos , Eosinófilos/metabolismo , Humanos , Vesículas Extracelulares/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Reprogramação CelularRESUMO
Caveolae have been indicated as a center of cytoskeleton regulation for Src kinase/Rho GTPase signaling. In addition, Src recruitment on intact cortical actin cytoskeleton appears to be required for bFGF/FGFR signal activation. Recently, we established a relationship between caveolin-1 (Cav-1) expression and cell migration in human malignant melanoma, constitutively activated by a bFGF autoregulatory loop. This work intends to investigate whether caveolae's asset, through bFGF/FGFR/c-Src/Rho signaling, could be related to melanoma cell anchorage. Accordingly, we revealed the existence of a FGFR/Src kinase pathway in Cav-1 enriched detergent-resistant membranes (DRMs) of Me665/1 metastatic melanoma cells, as confirmed by FGFR silencing. Moreover, we determined the expression and phosphorylation levels of Cav-1/Src/Erk signal pathway as a function of FGFR activation and cell density. A sucrose density gradient ultracentrifugation was employed to monitor Cav-1 membrane association and buoyancy in Me665/1 cells treated for actin fragmentation or for altered phosphorylation signals. As a result, melanoma cells show remarkable resistance to Cav-1 disassembly, together with persisting cell signal activity, being Src and Cav-1 crucial modulators of Rho GTPases. In conclusion, our study primarily highlights, in a metastatic melanoma cell line expressing caveolin, the circumstances whereby caveola structural and functional endurance enables the FGFR/Src/Rho GTPases pathway to keep on cell progression.
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Caveolina 1/metabolismo , Melanoma/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Actinas/metabolismo , Caveolina 1/genética , Contagem de Células , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Citoesqueleto/genética , Citoesqueleto/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Melanoma/genética , Melanoma/patologia , Fosforilação , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transdução de Sinais , Proteínas rho de Ligação ao GTP/genética , Quinases da Família src/genéticaRESUMO
The mechanisms of melanoma progression have been extensively studied in the last decade, and despite the diagnostic and therapeutic advancements pursued, malignant melanoma still accounts for 60% of skin cancer deaths. Therefore, research efforts are required to better define the intercellular molecular steps underlying the melanoma development. In an attempt to represent the complexity of the tumour microenvironment (TME), here we analysed the studies on melanoma in acidic and hypoxic microenvironments and the interactions with stromal and immune cells. Within TME, acidity and hypoxia force melanoma cells to adapt and to evolve into a malignant phenotype, through the cooperation of the tumour-surrounding stromal cells and the escape from the immune surveillance. The role of tumour exosomes in the intercellular crosstalk has been generally addressed, but less studied in acidic and hypoxic conditions. Thus, this review aims to summarize the role of acidic and hypoxic microenvironment in melanoma biology, as well as the role played by melanoma-derived exosomes (Mexo) under these conditions. We also present a perspective on the characteristics of acidic and hypoxic exosomes to disclose molecules, to be further considered as promising biomarkers for an early detection of the disease. An update on the use of exosomes in melanoma diagnosis, prognosis and response to treatment will be also provided and discussed.
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Ácidos/metabolismo , Progressão da Doença , Exossomos/metabolismo , Melanoma/patologia , Microambiente Tumoral , Hipóxia Celular/genética , Redes Reguladoras de Genes , Humanos , Melanoma/genética , Melanoma/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.
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Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Progressão da Doença , Humanos , Concentração de Íons de Hidrogênio , Lipídeos/química , Melanoma/patologia , Microscopia Confocal/métodos , Modelos Biológicos , Metástase Neoplásica , Prótons , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Espectrometria de Fluorescência/métodosRESUMO
Colorectal cancer (CRC) is the second leading cause of cancer mortality in both men and women worldwide. Survival of patients is significantly associated with disease stage at diagnosis. Recent studies highlighted a role of exosomes in CRC development and progression, thus raising the interest on these nanosized vesicular structures as possible biomarkers. Exosomes contain a large variety of molecules, including proteins, lipids and nucleic acids, that are exchanged between cells either within tumor microenvironment or at distant sites from the primary tumor, where they prepare a suitable soil for tumor metastases. The present review summarizes the principal effects of exosomes on CRC development, progression, and provides an update of the most recent findings on the use of exosomal molecules as diagnostic, prognostic and predictive biomarkers in CRC.
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Neoplasias Colorretais/fisiopatologia , Neoplasias Colorretais/terapia , Progressão da Doença , Exossomos/fisiologia , Animais , Biomarcadores Tumorais , Humanos , Camundongos , Prognóstico , Microambiente TumoralRESUMO
Tumor microenvironment (TME) is a complex milieu in which tumor grows, develops and progresses through a complex bi-directional cross-talk with immune-, stromal cells, and the extracellular matrix (ECM). In this context, tumor-derived exosomes (TE) drive the fate of tumor cells through a stimulatory or inhibitory role on immune system. In fact, TE can induce the apoptosis of cells of the immune surveillance, and enhance the proliferation and survival of stromal cells that sustain tumor development. However, depending on the molecular cargo, TE are also able to stimulate anti-tumor immune response. TME is mainly characterized by the acidic pH that contributes to tumor development, through multiple mechanisms. Among these, the impairment of tumor immune surveillance does occur within acidic TME, and is directly mediated by acidic pH or by molecular cargo carried by TE. Little is known about the role of TE in immunomodulation in acidic conditions. The present review summarizes the studies describing the role of microenvironmental acidity and TE in immune system modulation.
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Caveolin-1 (Cav-1), a member of the caveolin family, regulates caveolae-associated signaling proteins, which are involved in many biological processes, including cancer development. Cav-1 was found to exert a complex and ambiguous role as oncogene or tumor suppressor depending on the cellular microenvironment. Here we investigated Cav-1 expression and function in a panel of melanomas, finding its expression in all the cell lines. The exception was the primary vertical melanoma cell line, WM983A, characterized by the lack of Cav-1, and then utilized as a recipient for Cav-1 gene transduction to address a series of functional studies. The alleged yet controversial role of phospho (Ph)-Cav-1 on cell regulation was also tested by transducing the nonphosphorylatable Cav-1Y14A mutant. Wild-type Cav-1, but not mutated Cav-1Y14A, increased tumorigenicity as indicated by enhanced proliferation, migration, invasion and capacity of forming foci in semisolid medium. Accordingly, Cav-1 silencing inhibited melanoma cell growth reducing some of the typical traits of malignancy. Finally, we detected a secreted fraction of Cav-1 associated with cell released microvesicular particles able to stimulate in vitro anchorage independence, migration and invasion in a paracrine/autocrine fashion and, more important, competent to convey metastatic asset from the donor melanoma to the less aggressive recipient cell line. A direct correlation between Cav-1 levels, the amount of microvesicles released in the culture medium and MMP-9 expression was also observed.
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Biomarcadores Tumorais/metabolismo , Caveolina 1/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Western Blotting , Caveolina 1/genética , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Melanoma/patologia , Invasividade Neoplásica , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/patologiaRESUMO
Previous studies suggested an important role for vascular endothelial growth factor (VEGF) and its receptors in postnatal haemopoiesis. However, it is unclear how VEGF receptor (VEGFR) signalling could interact with that issued from the activation of haematopoietic growth factor receptors. To elucidate this point we explored VEGF-R2 and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) membrane localization and cell signalling in TF1-KDR cells (TF1 leukaemic cells that overexpress VEGF-R2/KDR). Activation of either GM-CSFR or VEGF-R2 was shown to determine the migration of both receptor elements (VEGF-R2 and the common beta-chain of the GM-CSFR) to lipid rafts. The study of receptor phosphorylation showed that GM-CSF induced the phosphorylation of its own receptor and the transphosphorylation of VEGF-R2; on the other hand, VEGF triggered the phosphorylation of its receptor and transphosphorylated the beta-chain of the GM-CSFR. Co-stimulation of TF1-KDR cells with both GM-CSF and VEGF-A resulted in massive migration of both the common GM-CSFR beta-chain and VEGF-R2 to lipid rafts and sustained p38 mitogen-activated protein kinase activation. Disruption of lipid rafts inhibited the capacity of both GM-CSF and VEGF-A to activate p38. Experiments with specific p38 inhibitors showed that p38 activation was required to sustain the VEGF- and GM-CSF-dependent proliferation of TF1-KDR and the survival of primary acute myeloid leukaemia blasts.
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Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Interleucina-3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Microdomínios da Membrana/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Apoptose , Transporte Biológico , Western Blotting/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Ativação Enzimática , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imidazóis/farmacologia , Imunofenotipagem , Imunoprecipitação , Leucemia Mieloide Aguda/patologia , Fosforilação , Piridinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Eosinophils are major effectors of Th2-related pathologies, frequently found infiltrating several human cancers. We recently showed that eosinophils play an essential role in anti-tumor responses mediated by immunotherapy with the 'alarmin' intereukin-33 (IL-33) in melanoma mouse models. Here, we analyzed the mechanisms by which IL-33 mediates tumor infiltration and antitumor activities of eosinophils. We show that IL-33 recruits eosinophils indirectly, via stimulation of tumor cell-derived chemokines, while it activates eosinophils directly, up-regulating CD69, the adhesion molecules ICAM-1 and CD11b/CD18, and the degranulation marker CD63. In co-culture experiments with four different tumor cell lines, IL-33-activated eosinophils established large numbers of stable cell conjugates with target tumor cells, with the polarization of eosinophil effector proteins (ECP, EPX, and granzyme-B) and CD11b/CD18 to immune synapses, resulting in efficient contact-dependent degranulation and tumor cell killing. In tumor-bearing mice, IL-33 induced substantial accumulation of degranulating eosinophils within tumor necrotic areas, indicating cytotoxic activity in vivo. Blocking of CD11b/CD18 signaling significantly reduced IL-33-activated eosinophils' binding and subsequent killing of tumor cells, indicating a crucial role for this integrin in triggering degranulation. Our findings provide novel mechanistic insights for eosinophil-mediated anti-tumoral function driven by IL-33. Treatments enabling tumor infiltration and proper activation of eosinophils may improve therapeutic response in cancer patients.
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BACKGROUND: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. METHODS: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. RESULTS: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. CONCLUSIONS: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.
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Exossomos/metabolismo , Melanoma/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Chaperona BiP do Retículo Endoplasmático , Humanos , Melanoma/patologia , Microscopia Confocal , Metástase Neoplásica , Microambiente TumoralRESUMO
Resistance to IFN-I-induced antineoplastic effects has been reported in many tumors and arises, in part, from epigenetic silencing of IFN-stimulated genes by DNA methylation. We hypothesized that restoration of IFN-stimulated genes by co-administration of the demethylating drug 5-aza-2'-deoxycitidine (decitabine [DAC]) may enhance the susceptibility to IFN-I-mediated antitumoral effects in melanoma. We show that combined administration of IFN-I and DAC significantly inhibits the growth of murine and human melanoma cells, both in vitro and in vivo. Compared with controls, DAC/IFN-I-treated melanoma cells exhibited reduced cell growth, augmented apoptosis, and diminished migration. Moreover, IFN-I and DAC synergized to suppress the growth of three-dimensional human melanoma spheroids, altering tumor architecture. These direct antitumor effects correlated with induction of the IFN-stimulated gene Mx1. In vivo, DAC/IFN-I significantly reduced melanoma growth via stimulation of adaptive immunity, promoting tumor-infiltrating CD8+ T cells while inhibiting the homing of immunosuppressive CD11b+ myeloid cells and regulatory T cells. Accordingly, exposure of human melanoma cells to DAC/IFN-I induced the recruitment of immune cells toward the tumor in a Matrigel (Corning Life Sciences, Kennebunkport, ME)-based microfluidic device. Our findings underscore a beneficial effect of DAC plus IFN-I combined treatment against melanoma through both direct and immune-mediated anti-tumor effects.