Introduction: : Aggressive breast cancer subtypes, such as triple-negative breast cancer (TNBC), commonly metastasize to the brain. This poses a significant clinical burden since there are currently no effective treatments, leading to poor prognoses and a significant decline in quality of life in patients. Glial cells have been known to play a role in aiding tumor progression in brain cancer itself. However, the role of glial cells in metastatic colonization is not yet fully established. Recent studies extracellular vesicles (EVs) Recent studies show that extracellular vesicles (EVs), tiny particles that carry cancerous material to recipient cells and prime distant organs to promote metastasis. We hypothesize that EVs from TNBC prime the brain microenvironment through glial cells, enabling the brain to become a permissive niche for breast tumor cell colonization. Understanding mechanisms of breast tumor-glial cell crosstalk could lead to new strategies for early detection and ultimately improve the quality of life for patients at risk.
Materials and
Methods: : For the viability assay, astrocytes and microglia were individually seeded at 20,000 cells per well on poly-L-lysine-coated wells. Viability results were used to determine the media cocktail from which EVs would be derived during the 6-hour serum starvation. For co-culture experiments, astrocytes were initially seeded at a density of 40,000 cells per well in poly-L-lysine-coated 12-well plates using astrocyte media. After two days, the media were removed, and the wells were rinsed with phosphate-buffered saline and replaced with Neurobasal media supplemented with B27 minus vitamin A and N2 supplement. Microglia were then seeded at 40,000 cells per well and added to the astrocytes. For EV isolation, human TNBC cell lines, MDA-MB-231 and MDA-MB-468, were seeded in 6-well plates. Once monolayers were formed, the media were replaced with a 50:50 mixture of Neurobasal media and serum-free DMEM. After 6 hours of serum starvation, EVs were collected, centrifuged twice at 0.6 RCF for five minutes each, and 1.5 mL of the supernatant was added to the co-culture. To assess morphological changes, the glial cells were fixed with 4% paraformaldehyde, followed by an immunohistochemistry protocol. Astrocytes were stained using GFAP and microglia with Iba1, followed by secondary conjugated antibodies.
Results, Conclusions, and Discussions:: Preliminary viability data showed 90% survival for glial cells across all media conditions. Notably, we proceeded with a 50:50 ratio of neurobasal media and TNBC EVs in breast cancer media, with or without TNBC EVs, in 2D culture. Glial cells, specifically microglia, revealed a rod-shaped morphology, which has been associated with early signs of neuroinflammation observed in 2D. This morphological state has also been shown in several neurodegenerative diseases, including traumatic brain injury and Alzheimer's Disease, demonstrating that tumor EV exposure elicits a pathological response in microglia. Future work will include Luminex multiplex immunoassay analysis to quantify inflammatory cytokines and other secreted factors associated with pathological glial cell responses. Ultimately, this work will provide valuable insights into the impact of tumor-derived signals on glial cells in breast metastatic interactions.
