ESS - CQB - Posters apresentados em eventos científicos
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Browsing ESS - CQB - Posters apresentados em eventos científicos by Author "Aguiar, Gonçalo"
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- Development of a stable melanoma dual reporter cell line expressing Luciferase and GFPPublication . Aguiar, Gonçalo; Torres, Sílvia; Prudêncio, Cristina; Soares, Raquel; Coelho, Pedro; Prudêncio, Cristina; Coelho, PedroMelanoma is the most aggressive and lethal form of skin cancer, with a high risk of metastatic spread. Obesity is recognized as a risk factor for various types of cancer. However, regarding melanoma, this association remains controversial. Obesity might act as a double-edged sword in melanoma, promoting primary tumour growth but at the same time limiting metastatic spread - the "obesity paradox”. Herein, we aimed to create a stable murine B16F10 melanoma cell line expressing both firefly luciferase (Luc) and green fluorescent protein (GFP), which will later be engrafted into diet induced-obesity animal model for future in vivo studies. B16F10-Luc-GFP cells were generated by transfection with premade lentiviral particles, featuring a construct with Luc and GFP under a cytomegalovirus promoter and mediated by a F2A element. The antibiotic selection marker (puromycin) is expressed under a Rous sarcoma virus promoter. Afterwards, the transfected cells were selected with 1 μg/ml of puromycin. The clones with the highest levels of GFP-positive cells and GFP fluorescence were purified by two rounds of cell sorting and submitted to fluorescence and bioluminescence quantification, morphology, injury, BrdU incorporation, 7-AAD, and PI cell cycle assays and compared to the parental cell line. B16F10-Luc-GFP were successfully generated, and both GFP fluorescence and D-luciferin bioluminescence are present and proportional to cell density. As expected, the parental cell line didn’t display GFP or Luc activities. Moreover, transduced cells exhibit similar morphology, motility, proliferation, viability, and cell cycle progression as B16F10 cells. Conclusions: Altogether, the future engraftment of B16F10-LucGFP in obese mice, will improve melanoma research models, enabling the in vivo and ex vivo visualization of primary tumours and metastasis, providing a better understanding of the underlying molecular mechanisms, to clarify the “obesity paradox” in melanoma.
- Effect of umbilical cord mesenchymal stem cells secretome in melanomaPublication . Accioly, Gustavo; Aguiar, Gonçalo; Areal, Lara; Costa, Pedro; Coelho, Pedro; Gomes, AndreiaMelanoma of the skin is one of the most prominent and fastest growing malignancies. More than 300 thousand diagnosed cases and 57 thousand deaths in 2020 worldwide and roughly 517 thousand cases of were registered during the 2015-2020 period. Melanoma is generally regarded as an aggressive and unpredictable cancer whose conventional therapies, such as local excision, chemotherapy and immunotherapy, have encountered difficulties to prevent larger scale-tumours and metastasis, as well as overcome recurrence and development of drug resistance. Stem cellbased therapies have been studied as interesting therapeutical approaches for cancer whenever conventional therapy fails to impede its progression. That is owing to the anti-proliferative and immunomodulatory capacity of some SC, being one of the major examples, Mesenchymal Stem Cells. Due to its high abundance, well defined extraction and expansion protocols as well as documented anti-tumorigenic characteristics, Umbilical Cord derived Mesenchymal Stem Cells (UC-MSC) have been observed as a promising candidate for Melanoma treatment, specially through acellular therapy using its secretome. MSC secretome is defined as the set of MSCs-derived bioactive factors available extracellularly and is responsible for the major therapeutic effects of MSCs, namely in oncological pathologies. In this study we hypothesize the ability of UC-MSC’s secretome to inhibit Melanoma growth in vitro. UC-MSC secretome, in the form of conditioned medium (CM), was obtained by extraction from selected umbilical cords and expansion of while murine melanoma cell line B16-F10 culture was established. After treating melanoma cells with different concentrations of CM (100%, 50% and 25%), common cancer hallmarks such as cell viability, motility, colony formation and cell interactions were assessed through MTT, Wound Healing and Colony formation and Hanging-Drop assays, respectively. General analysis of viability and motility showed no statistically significant difference between treated and control groups as well as no concentration-dependent effect whereas formation of cellular aggregates follows an inhibition trend on the treated groups. These results put into perspective the effect of secretome of UC-MSCs. Moreover, further larger scale studies are needed for deeper understanding of MSC secretome mechanisms of action, therefore enabling their use in acellular therapies against melanoma in the future.