The blood-brain barrier in brain metastasization of breast cancer : a source of biomarkers, a target for modulation, and an obstacle to overcome

• Main Author: Pereira, Joana
• Publication Date: 2024
• Language: eng
• Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
• Download full: http://hdl.handle.net/10400.5/98226
• topic: Abordagens terapêuticas preventivas e de tratamento; Barreira hematoencefálica; Biomarcadores circulantes; metástases encefálicas de cancro da mama; Sistemas dirigidos de veiculação de moléculas; Blood-brain barrier; breast cancer brain metastases; targeted-drug delivery systems; preventive and treatment therapeutic approaches; circulating biomarkers; Domínio/Área Científica::Ciências Naturais::Ciências Biológicas

As of 2020, breast cancer (BC) has become the leading cause of neoplastic disease worldwide, with an estimated 3 million new cases, and projections indicate that it will result in 1 million fatalities by 2040. An alarming concern of this disease is its metastatic stage, responsible for most BC deaths. Among all BC subtypes, triple-negative BC (TNBC) stands out as the most aggressive with a poor prognosis. It is also particularly susceptible to developing brain metastases. BC brain metastases (BCBM) leave patients with limited treatment options and extremely low survival rates, due to lack of a timely diagnose owing to the absence of biomarkers, while blood-brain barrier (BBB) restricts most drugs’ brain access. Thus, it is crucial to understand the mechanism involved in the interaction between BBB endothelial cells and breast cancer cells (BCCs) prior to brain metastases formation, as well as the identification of early biomarkers of this process. Additionally, new therapeutics addressing BCCs extravasation process and eradicate BCBM should be investigated. This thesis stands out due to its multifaceted approach, which includes the 1) investigation of the key molecules involved in the interaction between BBB endothelial cells and BCCs during BCBM development; 2) identification of circulating biomarkers indicative of BCBM development, with potential diagnostic and prognostic value, utilizing liquid biopsy; 3) uncover molecules capable of preserving the BBB during extravasation, aiming to impede the transendothelial migration (TEM) of BCCs as a preventive measure; 4) and explore novel treatment strategies, such as the co-administration of a cytotoxic drug and a small interfering RNA (siRNA) to abrogate the already established BCBM. Firstly, the players involved in TNBC cells (4T1 cell line) extravasation, as well as its temporal and spatial profile were disclosed in vivo and in vitro. Key mechanisms such as paracellular and transcellular BBB permeability, cell communication, cytoskeleton remodelling, proliferative and migratory BCC´s properties accompanied with invadopodium formation were revealed. Moreover, brain microvascular endotelial-derived extracellular vesicles and specific microRNAs (miR) were identified as biomarkers of advanced and precocious stages of BCBM, respectively. Secondly, given the altered BBB permeability upon BCCs contact, a preventive strategy was explored aiming the improvement of BBB’ properties. A high-throughput microscopy screening was performed on a library of new and already approved drugs to assess their ability to preserve BBB properties and prevent BCCs from adhering and migrating across the endothelium. Among the tested drugs, minocycline hydrochloride (MH) was the most promising in preventing BBB disruption and 4T1 cells migration, thus rising as a potential modulator of BBB integrity preservation. To minimize side effects and ensure specific BBB-delivery, a trojan horse drug carrier approach was employed targeting the transferrin receptor (TfR), which is highly expressed in brain microvascular endothelial cells, using the RI7217 (Ri7), an anti-TfR antibody. A transferrin receptor-targeted liposome (Lip) carrying MH (Ri7-MH-Lip) was developed and characterized. Ri7-MH-Lip disclosed increased cellular uptake via clathrin-dependent pathway, with no endothelial safety concerns. The Ri7-MH-Lip treatment not only reduced in vitro BCC-induced junctional protein disruption but also decreased endothelial matrix metalloproteinase 9 (MMP-9) overexpression. Additionally, it hampered BCCs’ adhesion to the endothelial monolayer via downregulation of endothelial vascular cell adhesion molecule 1. Ri7-MH-Lip also showed a safe and efficient outcome in vivo. No alteration in the enzymes associated with cardiac, renal, and hepatic toxicity was observed. Moreover, a reduction in BCBM was demonstrated, with the decrease of paracellular permeability and immune-cell infiltration. Furthermore, modulation of circulant miR-802-5p and miR-194-5p expression revealed their potential to be used as biomarkers of therapeutic response. Finally, given the persistent expression of platelet-derived growth factor subunit B (PDGF-B) in BCCs during the formation of brain metastases, coupled with the absence of specific therapies for this pathology, there is a pressing need for new therapeutic strategies. This thesis unveiled the potential of a BBB-permeant dual therapeutic strategy for the delivery of a cytotoxic molecule [salinomycin - SAL] and siRNA therapy [anti-PDGF-B - siPDGF-B] to induce BCBM eradication. Stable nucleic acid lipid particle (SNALP) for encapsulating siPDGF-B, as well as liposomes for incorporating SAL were developed and characterized. Both were conjugated with chlorotoxin (CTX) for to specifically target BCCs. The resulting formulations are denoted as CTX-siPDGF-B-SNALP and CTX-SAL-Lip, respectively. The co-administration of these systems in vitro inhibited BCCs’ proliferation (Ki-67) while the application of each formulation individually led to cell senescence in both cases. The formulations did not affect endothelial viability, although SAL liposomes impaired BBB integrity. In vivo, treatment with siPDGF-B lipid particles effectively reduced both the number of metastases and the levels of PDGF-B and Ki-67. In conclusion, this study has unveiled the cellular and molecular processes involved in BCBM formation, it has also revealed how preventive and therapeutic strategies influence the pathological events associated with disease development. Notably, miR-802 and miR-194 have emerged as valuable diagnostic and prognostic biomarkers. While this study is merely the tip of the iceberg, it introduces exciting possibilities. It suggests repurposing MH as a preventive therapeutic approach and utilizing siPDGF-B as treatment. These findings pave the way for the development of personalized pharmacological strategies to combat this illness.