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Projeto de investigação
Simplified chip prototyping for improved screening of gene-silencing therapeutics using 3D cell models.
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Epidermal growth factor alters silica nanoparticle uptake and improves gold-nanoparticle-mediated gene silencing in A549 cells
Publication . Susnik, Eva; Bazzoni, Amelie; Taladriz-Blanco, Patricia; Balog, Sandor; Moreno-Echeverri, Aura Maria; Glaubitz, Christina; Brito Oliveira, Beatriz; Ferreira, Daniela; Viana Baptista, Pedro; Petri-Fink, Alke; Rothen-Rutishauser, Barbara; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; Frontiers Media
Introduction: Delivery of therapeutic nanoparticles (NPs) to cancer cells represents a promising approach for biomedical applications. A key challenge for nanotechnology translation from the bench to the bedside is the low amount of administered NPs dose that effectively enters target cells. To improve NPs delivery, several studies proposed NPs conjugation with ligands, which specifically deliver NPs to target cells via receptor binding. One such example is epidermal growth factor (EGF), a peptide involved in cell signaling pathways that control cell division by binding to epidermal growth factor receptor (EGFR). However, very few studies assessed the influence of EGF present in the cell environment, on the cellular uptake of NPs. Methods: We tested if the stimulation of EGFR-expressing lung carcinomacells A549 with EGF affects the uptake of 59 nm and 422 nm silica (SiO2) NPs. Additionally, we investigated whether the uptake enhancement can be achieved with gold NPs, suitable to downregulate the expression of cancer oncogene c-MYC. Results: Our findings show that EGF binding to its receptor results in receptor autophosphorylation and initiate signaling pathways, leading to enhanced endocytosis of 59 nm SiO2 NPs, but not 422 nm SiO2 NPs. Additionally, we demonstrated an enhanced gold (Au) NPs endocytosis and subsequently a higher downregulation of c-MYC. Discussion: These findings contribute to a better understanding of NPs uptake in the presence of EGF and that is a promising approach for improved NPs delivery.
Breaking the mold
Publication . Cordeiro, Sandra; Oliveira, Beatriz B.; Valente, Ruben; Ferreira, Daniela; Luz, André; Baptista, Pedro V.; Fernandes, Alexandra R.; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; Frontiers Media
Despite extensive efforts to unravel tumor behavior and develop anticancer therapies, most treatments fail when advanced to clinical trials. The main challenge in cancer research has been the absence of predictive cancer models, accurately mimicking the tumoral processes and response to treatments. The tumor microenvironment (TME) shows several human-specific physical and chemical properties, which cannot be fully recapitulated by the conventional 2D cell cultures or the in vivo animal models. These limitations have driven the development of novel in vitro cancer models, that get one step closer to the typical features of in vivo systems while showing better species relevance. This review introduces the main considerations required for developing and exploiting tumor spheroids and organoids as cancer models. We also detailed their applications in drug screening and personalized medicine. Further, we show the transition of these models into novel microfluidic platforms, for improved control over physiological parameters and high-throughput screening. 3D culture models have provided key insights into tumor biology, more closely resembling the in vivo TME and tumor characteristics, while enabling the development of more reliable and precise anticancer therapies.
Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples
Publication . Oliveira, Beatriz B.; Costa, Beatriz; Morão, Bárbara; Faias, Sandra; Veigas, Bruno; Pereira, Lucília Pebre; Albuquerque, Cristina; Maio, Rui; Cravo, Marília; Fernandes, Alexandra R.; Baptista, Pedro Viana; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Springer
The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor’s temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes. Graphical Abstract: [Figure not available: see fulltext.]
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
OE
Número da atribuição
2020.07660.BD