CNC. IBILI

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Publications

Inoculated cell density as a determinant factor of the growth dynamics and metastatic efficiency of a breast cancer murine model

Publication . Gregório, Ana C.; Fonseca, Nuno A.; Moura, Vera; Lacerda, Manuela; Figueiredo, Paulo; Simões, Sérgio; Dias, Sérgio; Moreira, João Nuno

4T1 metastatic breast cancer model have been widely used to study stage IV human breast cancer. However, the frequent inoculation of a large number of cells, gives rise to fast growing tumors, as well as to a surprisingly low metastatic take rate. The present work aimed at establishing the conditions enabling high metastatic take rate of the triple-negative murine 4T1 syngeneic breast cancer model. An 87% 4T1 tumor incidence was observed when as few as 500 cancer cells were implanted. 4T1 cancer cells colonized primarily the lungs with 100% efficiency, and distant lesions were also commonly identified in the mesentery and pancreas. The drastic reduction of the number of inoculated cells resulted in increased tumor doubling times and decreased specific growth rates, following a Gompertzian tumor expansion. The established conditions for the 4T1 mouse model were further validated in a therapeutic study with peguilated liposomal doxorubicin, in clinical used in the setting of metastatic breast cancer. Inoculated cell density was proven to be a key methodological aspect towards the reproducible development of macrometastases in the 4T1 mouse model and a more reliable pre-clinical assessment of antimetastatic therapies.

2016Journal article

Open access

Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis

Publication . Ribeiro-Rodrigues, Teresa M.; Laundos, Tiago L.; Pereira-Carvalho, Rita; Batista-Almeida, Daniela; Pereira, Ricardo; Coelho-Santos, Vanessa; Silva, Ana P.; Fernandes, Rosa; Zuzarte, Monica; Enguita, Francisco J.; Costa, Marina C.; Pinto-do-Ó, Perpetua; Pinto, Marta T.; Gouveia, Pedro; Ferreira, Lino; Mason, Justin C.; Pereira, Paulo; Kwak, Brenda R.; Nascimento, Diana S.; Girão, Henrique

Aims: Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). Methods and results: Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. Conclusions: This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply.

2017Journal article

Open access

Sorting hidden patterns in nanoparticle performance for glioblastoma using machine learning algorithms

Publication . Basso, João; Mendes, Maria; Silva, Jessica; Cova, Tânia; Luque-Michel, Edurne; Jorge, Andreia F.; Grijalvo, Santiago; Gonçalves, Lídia; Eritja, Ramon; Blanco-Prieto, María J.; Almeida, António José; Pais, Alberto; Vitorino, Carla

Cationic compounds have been described to readily penetrate cell membranes. Assigning positive charge to nanosystems, e.g. lipid nanoparticles, has been identified as a key feature to promote electrostatic binding and design ligand-based constructs for tumour targeting. However, their intrinsic high cytotoxicity has hampered their biomedical application. This paper seeks to establish which cationic compounds and properties are compelling for interface modulation, in order to improve the design of tumour targeted nanoparticles against glioblastoma. How can intrinsic features (e.g. nature, structure, conformation) shape efficacy outcomes? In the quest for safer alternative cationic compounds, we evaluate the effects of two novel glycerol-based lipids, GLY1 and GLY2, on the architecture and performance of nanostructured lipid carriers (NLCs). These two molecules, composed of two alkylated chains and a glycerol backbone, differ only in their polar head and proved to be efficient in reversing the zeta potential of the nanosystems to positive values. The use of unsupervised and supervised machine learning (ML) techniques unraveled their structural similarities: in spite of their common backbone, GLY1 exhibited a better performance in increasing zeta potential and cytotoxicity, while decreasing particle size. Furthermore, NLCs containing GLY1 showed a favorable hemocompatible profile, as well as an improved uptake by tumour cells. Summing-up, GLY1 circumvents the intrinsic cytotoxicity of a common surfactant, CTAB, is effective at increasing glioblastoma uptake, and exhibits encouraging anticancer activity. Moreover, the use of ML is strongly incited for formulation design and optimization.

2021-01Journal article

Open access

Microglia dysfunction caused by the loss of Rhoa disrupts neuronal physiology and leads to neurodegeneration

Publication . Socodato, Renato; Portugal, Camila C.; Canedo, Teresa; Rodrigues, Artur; Almeida, Tiago O.; Henriques, Joana F.; Vaz, Sandra H.; Magalhães, João; Silva, Cátia M.; Baptista, Filipa I.; Alves, Renata L.; Coelho-Santos, Vanessa; Silva, Ana Paula; Paes de Carvalho, Roberto; Magalhães, Ana; Brakebusch, Cord; Sebastião, Ana M; Summavielle, Teresa; Ambrósio, António F.; Relvas, João B.

Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of β-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aβ oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.

2020Journal article

Open access