MODELLING IMMUNE CELL INTERACTIONS WITH AN IN VITRO BLOOD BRAIN BARRIER-GLIOMA MODEL

Abstract

AIMS The presence of the blood brain barrier (BBB) may reduce disposition of novel therapies to the brain. Cell-cell interactions in the glioma microenvironment also influence tumour progression. There is a need for an in vitro BBB-glioma model to mimic the complex tumour microenvironment (TME) for prediction of patient outcomes. A dynamic BBB model, that incorporates an immune microenvironment and glioblastoma spheroids, will be used to test delivery and efficacy of novel therapies. METHODS The BBB model consisted of primary derived brain endothelial cells (HBMEC), astrocytes (HA) and pericytes (HBVP) grown on 2D transwell or 3D inserts. Glioblastoma spheroids were grown from patient-derived cells. An inflammatory immune microenvironment was created using conditioned media from microglia (BV2, HMC3 and IM) activated with LPS. RESULTS Confirmation of the presence of a physical cell barrier was measured by TEER, exclusion of varying molecular weights of FitC-dextran and the expression of tight junction proteins ZO-1 and Claudin-1. Initial validation studies illustrated a stronger BBB was created with co-cultures compared to an endothelial monolayer. The presence of an inflammatory immune microenvironment was confirmed by measuring an increased release of IL-6, IL-1β, and TNF-α from microglial cells stimulated with LPS compared to no LPS control. When the condition media from activated microglial cells was added to the BBB, there was a further increased detection of IL-6, IL-1β, and TNF-α. CONCLUSION This study confirmed that all BBB and microglia cells could produce a measurable inflammatory environment upon activation. The next stage is to introduce patient derived glioma spheroids to the BBB-microglia model to measure how the tumour creates an inflammatory microenvironment and how the inflammatory markers IL-6, IL-1β, and TNF-α respond. Ultimately this will provide a more realistic model of the TME so glioma biology and drug response can be determined.