Avaliação do potencial terapêutico de vesículas extracelulares derivadas de células-tronco da polpa dentária imatura humana no carcinoma papilífero de tireoide
Data
2024-11-12
Autores
Teixeira, Michelli Ramires 
Orientadores
Araldi, Rodrigo Pinheiro
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivos: O estudo teve como objetivo isolar e investigar os efeitos terapêuticos de vesículas extracelulares (EVs, do inglês extracellular vesicles) provenientes do meio condicionado de células-tronco da polpa dentária imatura humana (EVs-CTPDIh) em duas linhagens celulares de carcinoma papilífero da tireoide (CPT: BCPAP e TPC1). Métodos: As EVs-CTPDIh foram isoladas por ultracentrifugação e tiveram seu tamanho e concentração determinados através do rastreio de nanopartículas (NTA) e concentração proteica através do ensaio de ácido bicinconínico (BCA). As células de CPT foram tratadas com 50 µg/mL de proteína-relativa de EVs em todos os ensaios. A internalização das EVs pelas células BCPAP e TPC1 foi avaliada por microscopia de fluorescência, microscopia confocal e citometria de fluxo. As alterações na expressão gênica e celulares após o tratamento com as EVs foram realizadas através de: arrays de qPCR para avaliar mecanismos gerais do câncer e metástase; ensaio de viabilidade celular, proliferação, ciclo celular, metabolismo energético e motilidade celular. Resultados: as duas linhagens de CPT internalizaram eficientemente as EVs, de forma tempo-dependente. Todos os tratamentos com as EVs impactaram a expressão gênica das duas linhagens, porém poucas vias de sinalização foram reguladas. Entre estas vias, o tratamento por 72 h resultou na regulação positiva da via de sinalização Wnt nas células BCPAP. Apesar disso, não foram observadas alterações na proliferação celular. Nos ensaios de motilidade celular, houve a diminuição da invasão celular de ambas as linhagens de CPT após 120 h de tratamento. Os demais ensaios não apresentaram diferenças entre os grupos tratados e controle. Conclusões: Os resultados do estudo indicam que as EVs-CTPDIh têm a capacidade de reduzir a invasão das linhagens BCPAP e TPC1, sem induzir comportamentos pró-tumorais in vitro. Embora os efeitos observados não estabeleçam as EVs-CTPDIh como uma terapia isolada, a sua capacidade de serem internalizadas pelas células tumorais sugere um potencial para essas nanopartículas como uma plataforma de transporte de moléculas quimioterápicas, ampliando suas possíveis aplicações no tratamento do câncer.
Objectives: This study aimed to isolate and investigate the therapeutic effects of extracellular vesicles (EVs) from the conditioned medium of human immature dental pulp stem cells (hIDPSC-EVs) in two papillary thyroid carcinoma (PTC) cell lines (BCPAP and TPC1). Methods: EVs from hIDPSC were isolated by ultracentrifugation. EVs size and concentration were determined by nanoparticle tracking analysis (NTA), and protein concentration was determined by bicinchoninic acid (BSA) assay. An EV concentration of 50 µg/mL was used in all assays. The internalization of EVs by BCPAP and TPC1 cells was evaluated using fluorescence microscopy, confocal microscopy, and flow cytometry. Molecular and functional alterations in PTC cells after EV treatment were assessed using qPCR arrays for general cancer and metastasis mechanisms, and assays to assess cell viability, proliferation, cell cycle, energy metabolism, and cell motility. Results: Both PTC cell lines efficiently internalized the EVs in a time-dependent manner. Treatment with EVs affected gene expression in both cell lines, although few signaling pathways were regulated. Notably, 72-hour treatment resulted in upregulation of the Wnt signaling pathway in BCPAP cells. Despite these changes, no alterations in cell proliferation were observed. In the cell motility assays, there was a decrease in the invasion capacity of both PTC cell lines after 120 h of treatment. Other assays revealed no differences between the treated and control groups. Conclusions: The study results indicate that EV-hIDPSCs can reduce the invasion of BCPAP and TPC1 cell lines without inducing in vitro pro-tumoral behaviors. Although the observed effects do not establish hIDPSC-EVs as a standalone therapy, their ability to be internalized by tumor cells suggests the potential of these nanoparticles as a platform for delivering chemotherapeutic molecules, thereby expanding their potential applications in cancer treatment.
Objectives: This study aimed to isolate and investigate the therapeutic effects of extracellular vesicles (EVs) from the conditioned medium of human immature dental pulp stem cells (hIDPSC-EVs) in two papillary thyroid carcinoma (PTC) cell lines (BCPAP and TPC1). Methods: EVs from hIDPSC were isolated by ultracentrifugation. EVs size and concentration were determined by nanoparticle tracking analysis (NTA), and protein concentration was determined by bicinchoninic acid (BSA) assay. An EV concentration of 50 µg/mL was used in all assays. The internalization of EVs by BCPAP and TPC1 cells was evaluated using fluorescence microscopy, confocal microscopy, and flow cytometry. Molecular and functional alterations in PTC cells after EV treatment were assessed using qPCR arrays for general cancer and metastasis mechanisms, and assays to assess cell viability, proliferation, cell cycle, energy metabolism, and cell motility. Results: Both PTC cell lines efficiently internalized the EVs in a time-dependent manner. Treatment with EVs affected gene expression in both cell lines, although few signaling pathways were regulated. Notably, 72-hour treatment resulted in upregulation of the Wnt signaling pathway in BCPAP cells. Despite these changes, no alterations in cell proliferation were observed. In the cell motility assays, there was a decrease in the invasion capacity of both PTC cell lines after 120 h of treatment. Other assays revealed no differences between the treated and control groups. Conclusions: The study results indicate that EV-hIDPSCs can reduce the invasion of BCPAP and TPC1 cell lines without inducing in vitro pro-tumoral behaviors. Although the observed effects do not establish hIDPSC-EVs as a standalone therapy, their ability to be internalized by tumor cells suggests the potential of these nanoparticles as a platform for delivering chemotherapeutic molecules, thereby expanding their potential applications in cancer treatment.
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Citação
TEIXEIRA, Michelli Ramires. Avaliação do potencial terapêutico de vesículas extracelulares derivadas de células-tronco da polpa dentária imatura humana no carcinoma papilífero de tireoide. 2024. 111 f. Dissertação (Mestrado em Endocrinologia e Metabologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2024.