Mechanisms of Metastatic Spread of Breast Cancer to the Brain

Breast cancer is the most common cancer in females. Triple-negative breast cancer (TNBC), in which the tumour cells lack estrogen and progesterone receptors, and the HER2 protein, accounts for approximately 15% of all breast cancers and tends to have a younger onset and higher metastasis risk compared with other breast cancer subtypes. It also has the highest rate of brain metastasis and this metastasis location, in particular, is associated with poor patient outcomes, severely impacting quality of life and life expectancy. Breast cancer metastasis to the brain requires tumour cells, released from the primary tumour, to travel to the brain via the circulatory system, attach to and then pass through (extravasate) the endothelial cell (EC) layer of the cerebral capillary wall. These tumour cells then interact with astrocytes, the main supporting cells within the brain. The cerebral ECs are a key component of the blood brain barrier (BBB), a specialised and relatively impermeable blood vessel wall. Although this structure should protect the brain from tumour cell invasion, the TNBC cells still possess the ability to cross the BBB. The first structure that circulating tumour cells interact with on the BBB, is the cerebral endothelial surface layer or “glycocalyx” that covers the luminal surface of all blood vessels. Glycocalyx disruption has been reported in tumour progression, but it is not fully understood how circulating TNBC cells interact with, and alter, the cerebral capillary glycocalyx and then attach to the ECs of the BBB in order to extravasate. Thus, the first aim of this thesis was to explore, using an in vitro model, the mechanism involved in this process. The second main aim was to explore the interaction between the TNBC cells and astrocytes, to examine whether the cross-talk between the two could induce astrocyte changes that could further promote metastatic growth. A better understanding of these processes could highlight future therapeutic targets. All experiments were carried out using human cerebral microvascular ECs (hCMEC/D3) to model the BBB in vitro, human adult primary astrocytes and the breast cancer cell lines, MDA-MB-231 and MCF-7 cells, which represented aggressive TNBC cells and less aggressive hormone positive breast cancer cells 3 respectively. The effects of the cancer cell secretome (as conditioned medium or selected secreted proteins) on the hCMEC/D3 glycocalyx was studied using cell-based fluorescence assays to quantify surface levels of three key glycocalyx components (sialic acid, [SA], chondroitin sulfate [CS] and heparin sulfate [HS]. Tumour cell attachment to an hCMEC/D3 monolayer was studied under low shear stress levels to mimic in vivo conditions. Interrogation of the tumour proteins secreted into the conditioned medium was performed using commercial Proteome Profiler kits and ELISAs. The impact of tumour secreted proteins on astrocyte properties i.e. a quiescent vs an activated (potentially tumour supporting) phenotype was assessed using immunohistochemistry and flow cytometry to quantify expression of key markers of astrocyte activation (glial fibrillary acidic protein, nestin and sphingosine-1-phosphate receptor 3. The key findings presented were that: (1) TNBC cells secrete a range of factors that can degrade the cerebral endothelial glycocalyx, as assessed by a reduction in surface levels of SA, HS, and CS. These factors include but are probably not limited to the key identified secreted pro-metastatic proteins, IL8, AXL and uPAR; (2) the reduction in glycocalyx levels caused by both the entire secretome and IL8, AXL and uPAR individually, was reflected by an increase in the adhesion of TNBCs to a cerebral EC monolayer under flow conditions. This interaction may be partially, but not fully dependent on the endothelial adhesion molecule E-selectin; (3) IL8, AXL and uPAR secreted from the TNBCs all induced astrocyte activation, a process that is thought to initially be a protective response to brain invasion by tumour cells but can actually ultimately contribute to the growth of the metastatic tumour. These findings identified key TNBC secreted pro-metastatic proteins (IL8, AXL, and uPAR) that may contribute to the ability of these aggressive breast cancer cells to cross the BBB and establish a secondary tumour in the brain. They also highlight the potential of using specific inhibitors of these proteins as a therapeutic approach to target the extravasation stage of metastasis to prevent or reduce the formation of brain tumours.<p></p>

Author dc:creator

• Rahmaneh Moosavi (21040598)

• Uncategorised value

Statement dc:rights

• All rights reserved
• Open Access after 2027-07-31

Identifier

10779/exe.31223152.v1

OAI identifier oai:identifier

oai:figshare.com:article/31223152