Behind Brain Metastases Formation: Cellular and Molecular Alterations and Blood-Brain Barrier Disruption.

Breast cancer (BC) brain metastases is a life-threatening condition to which accounts the poor understanding of BC cells' (BCCs) extravasation into the brain, precluding the development of preventive strategies. Thus, we aimed to unravel the players involved in the interaction between BCCs and blood-brain barrier (BBB) endothelial cells underlying BBB alterations and the transendothelial migration of malignant cells. We used brain microvascular endothelial cells (BMECs) as a BBB in vitro model, under conditions mimicking shear stress to improve in vivo-like BBB features. Mixed cultures were performed by the addition of fluorescently labelled BCCs to distinguish individual cell populations. BCC-BMEC interaction compromised BBB integrity, as revealed by junctional proteins (ß-catenin and zonula occludens-1) disruption and caveolae (caveolin-1) increase, reflecting paracellular and transcellular hyperpermeability, respectively. Both BMECs and BCCs presented alterations in the expression pattern of connexin 43, suggesting the involvement of the gap junction protein. Myosin light chain kinase and phosphorylated myosin light chain were upregulated, revealing the involvement of the endothelial cytoskeleton in the extravasation process. ß4-Integrin and focal adhesion kinase were colocalised in malignant cells, reflecting molecular interaction. Moreover, BCCs exhibited invadopodia, attesting migratory properties. Collectively, hub players involved in BC brain metastases formation were unveiled, disclosing possible therapeutic targets for metastases prevention.

MicroRNAs and Extracellular Vesicles as Distinctive Biomarkers of Precocious and Advanced Stages of Breast Cancer Brain Metastases Development.

Triple negative breast cancer presents higher mortality and poorer survival rates than other breast cancer (BC) types, due to the proneness to brain metastases formation, which are usually diagnosed at advanced stages. Therefore, the discovery of BC brain metastases (BCBM) biomarkers appears pivotal for a timely intervention. With this work, we aimed to disclose microRNAs (miRNAs) and extracellular vesicles (EVs) in the circulation as biomarkers of BCBM formation. Using a BCBM animal model, we analyzed EVs in plasma by nanoparticle tracking analysis and ascertained their blood-brain barrier (BBB) origin by flow cytometry. We further evaluated circulating miRNAs by RT-qPCR and their brain expression by in situ hybridization. In parallel, a cellular model of BCBM formation, combining triple negative BC cells and BBB endothelial cells, was used to differentiate the origin of biomarkers. Established metastases were associated with an increased content of circulating EVs, particularly of BBB origin. Interestingly, deregulated miRNAs in the circulation were observed prior to BCBM detection, and their brain origin was suggested by matching alterations in brain parenchyma. In vitro studies indicated that miR-194-5p and miR-205-5p are expressed and released by BC cells, endothelial cells and during their interaction. These results highlight miRNAs and EVs as biomarkers of BCBM in early and advanced stages, respectively.

Small Molecule Fisetin Modulates Alpha-Synuclein Aggregation.

Phenolic compounds are thought to be important to prevent neurodegenerative diseases (ND). Parkinson's Disease (PD) is a neurodegenerative disorder known for its typical motor features, the deposition of α-synuclein (αsyn)-positive inclusions in the brain, and for concomitant cellular pathologies that include oxidative stress and neuroinflammation. Neuroprotective activity of fisetin, a dietary flavonoid, was evaluated against main hallmarks of PD in relevant cellular models. At physiologically relevant concentrations, fisetin protected SH-SY5Y cells against oxidative stress overtaken by tert-butyl hydroperoxide (t-BHP) and against methyl-4-phenylpyridinuim (MPP+)-induced toxicity in dopaminergic neurons, the differentiated Lund human Mesencephalic (LUHMES) cells. In this cellular model, fisetin promotes the increase of the levels of dopamine transporter. Remarkably, fisetin reduced the percentage of cells containing αsyn inclusions as well as their size and subcellular localization in a yeast model of αsyn aggregation. Overall, our data show that fisetin exerts modulatory activities toward common cellular pathologies present in PD; remarkably, it modulates αsyn aggregation, supporting the idea that diets rich in this compound may prove beneficial.

Breast Cancer Brain Metastases: Implementation and Characterization of a Mouse Model Relying on Malignant Cells Inoculation in the Carotid Artery.

Breast cancer (BC) brain metastases (BCBM) is a severe condition frequently occurring in the triple-negative subtype. The study of BCBM pathogenesis and treatment has been hampered by the difficulty in establishing a reliable animal model that faithfully recapitulates the preferential formation of brain metastases. The injection of BC cells in the carotid artery of mice has been proposed but the procedure is challenging, with the metastatic pattern being scarcely characterized. In this work, we thoroughly describe an improved procedure, highlighting the tricks and challenges of the process, and providing a characterization of the brain and peripheral metastatic pattern at the cellular and molecular level. Triple-negative BC (4T1) cells were inoculated in the common carotid artery of BALB/c mice. Brains and peripheral organs were harvested at 7-14 days for the histological characterization of the metastases' pattern and the immunofluorescence analysis of specific markers. With our surgical procedure, both mouse death and procedure-associated weight loss were negligible. Brain metastases mostly occurred in the hippocampus, while sparse peripheral lesions were only detected in the lungs. Brain-colonizing BC cells presented proliferative (Ki-67) and epithelial (pan-cytokeratin and tomato lectin) features, which account for metastases' establishment. The presented surgical approach constitutes an important and reliable tool for BCBM studies.

A Drug Screening Reveals Minocycline Hydrochloride as a Therapeutic Option to Prevent Breast Cancer Cells Extravasation across the Blood-Brain Barrier.

Among breast cancer (BC) patients, 15-25% develop BC brain metastases (BCBM), a severe condition due to the limited therapeutic options, which points to the need for preventive strategies. We aimed to find a drug able to boost blood-brain barrier (BBB) properties and prevent BC cells (BCCs) extravasation, among PI3K, HSP90, and EGFR inhibitors and approved drugs. We used BCCs (4T1) and BBB endothelial cells (b.End5) to identify molecules with toxicity to 4T1 cells and safe for b.End5 cells. Moreover, we used those cells in mixed cultures to perform a high-throughput microscopy screening of drugs' ability to ameliorate BBB properties and prevent BCCs adhesion and migration across the endothelium, as well as to analyse miRNAs expression and release profiles. KW-2478, buparlisib, and minocycline hydrochloride (MH) promoted maximal expression of the junctional protein ß-catenin and induced 4T1 cells nucleus changes. Buparlisib and MH further decreased 4T1 adhesion. MH was the most promising in preventing 4T1 migration and BBB disruption, tumour and endothelial cytoskeleton-associated proteins modifications, and miRNA deregulation. Our data revealed MH's ability to improve BBB properties, while compromising BCCs viability and interaction with BBB endothelial cells, besides restoring miRNAs' homeostasis, paving the way for MH repurposing for BCBM prevention.

Circulating (Poly)phenol Metabolites: Neuroprotection in a 3D Cell Model of Parkinson's Disease.

SCOPE: Diets rich in (poly)phenols have been associated with positive effects on neurodegenerative disorders, such as Parkinson's disease (PD). Several low-molecular weight (poly)phenol metabolites (LMWPM) are found in the plasma after consumption of (poly)phenol-rich food. It is expected that LMWPM, upon reaching the brain, may have beneficial effects against both oxidative stress and neuroinflammation, and possibly attenuate cell death mechanisms relate to the loss of dopaminergic neurons in PD. METHODS AND RESULTS: This study investigates the neuroprotective potential of two blood-brain barrier permeant LMWPM, catechol-O-sulfate (cat-sulf), and pyrogallol-O-sulfate (pyr-sulf), in a human 3D cell model of PD. Neurospheroids were generated from LUHMES neuronal precursor cells and challenged by 1-methyl-4-phenylpyridinium (MPP+ ) to induce neuronal stress. LMWPM pretreatments were differently neuroprotective towards MPP+ insult, presenting distinct effects on the neuronal transcriptome. Particularly, cat-sulf pretreatment appeared to boost counter-regulatory defense mechanisms (preconditioning). When MPP+ is applied, both LMWPM positively modulated glutathione metabolism and heat-shock response, as also favorably shifting the balance of pro/anti-apoptotic proteins. CONCLUSIONS: Our findings point to the potential of LMWPM to trigger molecular mechanisms that help dopaminergic neurons to cope with a subsequent toxic insult. They are promising molecules to be further explored in the context of preventing and attenuating parkinsonian neurodegeneration.

Exploring the Benefits of Cellular Models to Uncover Bioactive Polyphenols for Neurodegeneration.

Our society is currently experiencing increased lifespan; one of the top causes for the high incidence of neurodegenerative disorders. The lack of effective treatments delaying or blocking disease progression has encouraged the active search for novel therapies. Many evidences support the protective role of phytochemicals in the prevention of neurodegenerative diseases, particularly (poly)phenols. In this review, we described the use of cellular-based models of neurodegenerative diseases and the benefits of their use as potent tools in the search for bioactive molecules, particularly (poly)phenols. Studies to assess the biological activity of (poly)phenols involve experimentation with in vitro and in vivo systems. In vitro systems are a useful tool as a first approach to test the underlined molecular mechanisms of candidate molecules. They can provide valuable information about biological activity, which can be then used to design animal and human intervention studies.