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Role of extracellular vesicles in brain metastases
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As metástases cerebrais são um dos principais impulsionadores da mortalidade por cancro. A sua iniciação requer que as células tumorais circulantes atravessem a barreira hematoencefálica (BHE) e remodelem o nicho neurovascular. As vesículas extracelulares derivadas de tumores, particularmente as pequenas vesículas extracelulares, são mediadores emergentes. Testámos se as vesículas extracelulares cancerígenas perturbam a estrutura/função da BHE e iniciam a neuroinflamação in vivo, e quais as características moleculares que acompanham esses efeitos. As vesículas extracelulares de linhas celulares de cancro humano (H1975 do pulmão; MDA-MB-231 e MDA-MB-231-BR4 da mama) foram isoladas e caracterizadas por análise de rastreio de nanopartículas, imunofenotipagem MACSPlex e Western blot. Os ensaios funcionais utilizaram peixe-zebra Tg(mpeg1:mCherry);Tg(fli1a:EGFP) com administração de vesículas extracelulares no rombencéfalo, dextrano de 10 kDa intravascular, imagiologia confocal e leituras quantitativas do recrutamento de macrófagos, extravasamento de dextrano, calibre interno dos vasos e intensidade de fluorescência endotelial. Os miRNAs candidatos nos cérebros de peixe-zebra após a exposição foram medidos por RT-qPCR. As vesículas extracelulares cancerígenas formaram uma população consistente de 100–200 nm (modal ~130 nm) enriquecida em tetraspaninas (CD9/CD63/CD81) com marcadores específicos da linhagem. A análise por Western blot confirmou a expressão de CD9 e Flotilina, enquanto o marcador endossomal Alix não foi detetado. In vivo, as vesículas extracelulares H1975 perturbaram a arquitetura da BHE e aumentaram o dextrano parenquimatoso em comparação com as vesículas extracelulares controlo SH-SY5Y. Quantitativamente, as vesículas extracelulares do cancro do pulmão aumentaram os macrófagos mpeg1⁺ em ~42% (média 36 vs 26; n=4/4), elevaram a MFI de dextrano extravascular/intravascular de 48,53% (n=2) para 85,61% (n=3), reduziram o calibre interno dos vasos em ~6,33% (9,33 para 8,74 μm; n=4/4) e diminuíram a fluorescência endotelial em ~17,03% (868.446 para 720.581 UA; n=4/4). O RT-qPCR detetou o miR-221 como o mais abundante (2^–dCt ~1,06), com miR-124 moderado, miR-129 intermédio, miR-146a baixo e miR-155 próximo do limiar de deteção; estes dados exploratórios (n=3; normalização de referência única; sem calibrador ΔΔCt) suportam apenas uma interpretação descritiva. Coletivamente, as vesículas extracelulares cancerígenas, exemplificadas pelas H1975, desencadeiam concomitantemente neuroinflamação e disfunção endotelial, rompendo a BHE e preparando um nicho pré-metastático. O modelo neurovascular de peixe-zebra fornece uma plataforma sensível e quantitativa para dissecar estas dinâmicas. O perfil molecular aponta superfícies ricas em tetraspaninas e a carga das vesículas extracelulares (ex: miR-221) como biomarcadores e alvos testáveis; é necessária validação em coortes maiores com estatísticas rigorosas e ómicas ortogonais.
Brain metastases (BMs) are a major driver of cancer mortality. Their initiation requires circulating tumour cells to traverse the blood–brain barrier (BBB) and remodel the neurovascular niche. Tumour-derived extracellular vesicles (EVs), particularly small EVs (sEVs), are emerging mediators. We tested whether cancer sEVs perturb BBB structure/function and initiate neuroinflammation in vivo and which molecular features accompany these effects. EVs from human cancer lines (H1975 lung; MDA-MB-231 and MDA-MB-231-BR4 breast) were isolated and profiled by nanoparticle tracking analysis, MACSPlex immunophenotyping, and Western blot. Functional assays used Tg(mpeg1:mCherry);Tg(fli1a:EGFP) zebrafish with hindbrain EV delivery, intravascular 10-kDa dextran, confocal imaging, and quantitative readouts of macrophage recruitment, dextran leakage, vessel inner calibre, and endothelial fluorescence intensity. Candidate miRNAs in zebrafish brains after exposure were measured by RT-qPCR. Cancer sEVs formed a consistent 100–200-nm population (modal ~130 nm) enriched for tetraspanins (CD9/CD63/CD81) with lineage-specific markers. Western blot analysis confirmed the expression of CD9 and Flotillin, while the endosomal marker Alix was not detected. In vivo, H1975 EVs disrupted BBB architecture and increased parenchymal dextran versus control SH-SY5Y EVs. Quantitatively, lung-cancer EVs increased mpeg1⁺ macrophages by ~42% (mean 36 vs 26; n=4/4), elevated extravascular/intravascular dextran MFI from 48.53% (n=2) to 85.61% (n=3), reduced vessel inner calibre by ~6.33% (9.33 to 8.74 μm; n=4/4), and lowered endothelial fluorescence by ~17.03% (868,446 to 720,581 AU; n=4/4). RT-qPCR detected miR-221 as most abundant (2^–dCt ~1.06), with miR-124 moderate, miR-129 intermediate, miR-146a low, and miR-155 near the detection floor; these exploratory data (n=3; single-reference normalisation; no ΔΔCt calibrator) support descriptive interpretation only. Collectively, cancer sEVs, exemplified by H1975, concurrently trigger neuroinflammation and endothelial dysfunction, breaching the BBB and priming a pre-metastatic niche. The zebrafish neurovascular model provides a sensitive, quantitative platform to dissect these dynamics. Molecular profiling nominates tetraspanin-rich surfaces and EV cargo (e.g., miR-221) as testable biomarkers and targets; validation in larger cohorts with rigorous statistics and orthogonal omics is required.
Brain metastases (BMs) are a major driver of cancer mortality. Their initiation requires circulating tumour cells to traverse the blood–brain barrier (BBB) and remodel the neurovascular niche. Tumour-derived extracellular vesicles (EVs), particularly small EVs (sEVs), are emerging mediators. We tested whether cancer sEVs perturb BBB structure/function and initiate neuroinflammation in vivo and which molecular features accompany these effects. EVs from human cancer lines (H1975 lung; MDA-MB-231 and MDA-MB-231-BR4 breast) were isolated and profiled by nanoparticle tracking analysis, MACSPlex immunophenotyping, and Western blot. Functional assays used Tg(mpeg1:mCherry);Tg(fli1a:EGFP) zebrafish with hindbrain EV delivery, intravascular 10-kDa dextran, confocal imaging, and quantitative readouts of macrophage recruitment, dextran leakage, vessel inner calibre, and endothelial fluorescence intensity. Candidate miRNAs in zebrafish brains after exposure were measured by RT-qPCR. Cancer sEVs formed a consistent 100–200-nm population (modal ~130 nm) enriched for tetraspanins (CD9/CD63/CD81) with lineage-specific markers. Western blot analysis confirmed the expression of CD9 and Flotillin, while the endosomal marker Alix was not detected. In vivo, H1975 EVs disrupted BBB architecture and increased parenchymal dextran versus control SH-SY5Y EVs. Quantitatively, lung-cancer EVs increased mpeg1⁺ macrophages by ~42% (mean 36 vs 26; n=4/4), elevated extravascular/intravascular dextran MFI from 48.53% (n=2) to 85.61% (n=3), reduced vessel inner calibre by ~6.33% (9.33 to 8.74 μm; n=4/4), and lowered endothelial fluorescence by ~17.03% (868,446 to 720,581 AU; n=4/4). RT-qPCR detected miR-221 as most abundant (2^–dCt ~1.06), with miR-124 moderate, miR-129 intermediate, miR-146a low, and miR-155 near the detection floor; these exploratory data (n=3; single-reference normalisation; no ΔΔCt calibrator) support descriptive interpretation only. Collectively, cancer sEVs, exemplified by H1975, concurrently trigger neuroinflammation and endothelial dysfunction, breaching the BBB and priming a pre-metastatic niche. The zebrafish neurovascular model provides a sensitive, quantitative platform to dissect these dynamics. Molecular profiling nominates tetraspanin-rich surfaces and EV cargo (e.g., miR-221) as testable biomarkers and targets; validation in larger cohorts with rigorous statistics and orthogonal omics is required.
Descrição
Tese de mestrado, Ciências Biofarmacêuticas, 2025, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
204141451 Extracellular vesicles Vasculature Brain metastases Pre-metastatic niche and microRNAs Teses de mestrado - 2025