Cortical cell culture model for examining cancer extracellular vesicle dynamics and neuroinflammatory response.

Understanding the interactions of extracellular vesicles (EVs) with central nervous system (CNS) cells is important for identifying mechanisms of disease and developing EV-based therapies. Yet, such studies are typically limited to single cell assays that lack the ability to probe complex intercellular signaling, such as the processes involved in neuroinflammation. Critically, metastatic niche formation in the brain is hypothesized to be mediated in part by cancer-derived EVs, though details are poorly understood. Here we apply multi-cellular CNS mixed culture models to assess neuroinflammatory response for EVs isolated from brain-metastatic cancer cells as a proof-of-concept for the utility of this model. Using primary cortical tri-culture (neurons, astrocytes, microglia) and contrasting co-culture (neurons and astrocytes, without microglia), we assessed the cellular tropism and neuroinflammatory response of EVs from breast cancer cells (MDA-MB-231) to their brain-tropic variant (231-Br), and non-cancer control (HEK293T). EVs exhibited differential total uptake in neurons and astrocytes depending on EV source and whether microglia were included in culture. Furthermore, addition of tumor EVs in tri-cultures with microglia resulted in significantly higher production of cytokines compared to control. Taken together, these results suggest that EV fate and function may be modulated by complex intercellular communication. This study underscores the novelty and importance of employing a multicellular assay, offering a more comprehensive and physiologically relevant model to dissect the intricate dynamics of EVs in neuroinflammatory responses within the CNS, paving the way for nuanced understanding and therapeutic exploration.