Modeling the multicellular blood-brain barrier with a PDX-derived glioblastoma microenvironment: comparing human and mouse biochip systems for tumor heterogeneity and drug response

Glioblastoma (GBM) is a highly malignant primary brain tumor whose therapeutic management is hindered by its invasive behavior and the restrictive properties of the blood-brain barrier (BBB). The BBB tightly regulates molecular transport between the circulatory system and the brain, limiting penetration of therapeutic agents. Widely used 2D in vitro systems offer little predictive value, as they lack key physiological features such as barrier integrity, multicellular complexity, and flow-dependent influences. As treatment efficacy depends on both, BBB permeability and direct tumor response, there is a critical need for advanced in vitro platforms that more accurately reproduce BBB function to support more reliable preclinical evaluation. To build a physiologically relevant BBB model, we assembled mouse or human brain capillary endothelial cells together with primary astrocytes and pericytes and tested their barrier formation both in static transwell systems and in a microfluidic chip platform. Different combinations and sources of BBB building cells were compared for junctional organization and barrier tightness. The addition of flow further reinforced barrier structure through shear-dependent junctional remodeling. These human and mouse BBB-on-chip models were then combined with patient-derived xenograft (PDX) GBM cell lines, generating a set of mouse PDX/GBM-on-chip and human GBM-on-chip models, that allow evaluation of tumor-heterogeneity under a human or mouse barrier. Initially, we compared treatment responses across conventional 2D cultures, static BBB constructs, the flow-based microfluidic model, and in vivo assays. Although 2D and in vivo studies demonstrated sensitivity towards cobimetinib, the compound was ineffective at inhibiting growth in the flow-integrated BBB chip. This is consistent with its inability to penetrate an intact barrier. In contrast, the BBB-permeable agent afatinib effectively reduced GBM growth in vivo and within the biochip, indicating that the chip reliably reflects whether therapeutic activity depends on BBB penetration. In summary, GBM-on-chip systems unite critical vascular barrier characteristics with GBM co-culture, offering a translational framework to examine tumor-barrier dynamics and determine compound passage across the BBB, thus more effectively connecting in vitro assays with PDX and human outcomes.