Brain Slice Culture Demonstrates Expedited Cell Traficking in the Presence of Fractalkine in a Medulloblastoma Tumor Microenvironment Model

Abstract

Abstract 20% of childhood brain tumors are medulloblastomas (MB), and the five-year survival rate can be as low as 60%. The interaction between MB and native immune cells are not well understood, and identifying methods of crosstalk could produce better outcomes for patients. Fractalkine (FKN) is a ligand for the receptor CX3CR1 that plays an inhibitory role in the brain’s immune system by keeping microglia quiescent. Normally, FKN is surface bound and expressed by neurons and astrocytes, but under inflammatory conditions FKN is secreted to serve as a chemoattractant. Preliminary work in our lab shows that FKN is produced by MB and may act as a decoy to maintain microglial quiescence leading to immune escape. Therefore we investigated FKN signaling and microglial kinetics ex vivo utilizing organotypic brain slice culture coupled with real time 2-photon laser scanning microscopy. To differentiate microglia from MB, CX3CR1+/GFP or CX3CR1GFP/GFP knock-in mice were sacrificed, and the brain was removed and sectioned. Slices were collected and cultured on inserts in an incubation chamber during imaging. To determine baseline activity, CX3CR1+/GFP and CX3CR1GFP/GFP brain slices were imaged over 48 hours. MB tumor cells, labelled with SNARF-1, were added to the cultures and imaged over the same time course. Images were processed to differentiate the motility of microglia and tumors. We found that microglia from CX3CR1+/GFP moved slower than microglia from CX3CR1GFP/GFP at baseline and in the presence of tumor. Furthermore, we found that MB moved faster in CX3CR1GFP/GFP slices, suggesting that fractalkine plays a role in the migration of MB.