This Administrative Supplement to University of Idaho INBRE Program establishes an INBRE-COBRE research collaboration. The project, Cellular Crosstalk Between Glioma and Blood-brain Barrier Endothelia, brings together a neurobiologist and a cell physiologist to study glioblastoma multiforme, an aggressive brain cancer. The INBRE-Developmental Research Program Investigator, R. L. Daniels, and COBRE-project investigator, R.S. Beard will combine their talents and expertise. The partnership will enhance the quality of scientific work for both investigators and increase research opportunities for undergraduate students. Their long-term goal is to better understand the cellular and molecular basis of glioblastoma multiforme pathobiology. Basic knowledge will contribute to future effective therapies. The hypothesis to be tested is that tumor-induced hypoxia initiates a positive-feedback loop between glioma cells and blood-brain barrier endothelia that ultimately promotes tumor growth and migration. Strong preliminary data supports this idea. The Daniels laboratory will use their expertise in culturing glioma cells and measuring cell signaling to test if hypoxic blood- brain epithelia release enough ATP to induce glutamate discharge from recipient tumor cells. Conditioned media from blood-brain barrier endothelia grown for various times in normal or hypoxic conditions will be used (provided by the Beard laboratory). ATP in conditioned media will be measured with a colorimetric assay and the media tested in glioma cultures. Calcium signaling coupled to glutamate secretion will be measured with a ratiometric calcium probe and high-performance liquid chromatography. Finally, the role of the P2X7 receptor in the mechanism of this cellular crosstalk will be confirmed using a receptor agonist, siRNA silencing, and measuring receptor gene expression. The Beard laboratory will use their expertise in culturing blood-brain barrier endothelia and measuring cell signaling to test if glioma-derived glutamate directly induces barrier dysfunction. The contribution of glioma-derived glutamate to blood-brain barrier dysfunction by triggering the glutamate receptor, NMDAr, activation will be evaluated. The same cell culture models used in the Daniels laboratory will be used except that mechanistic endpoints will evaluate endothelia barrier disruption in the hypoxic microenvironment surrounding necrotic cores in glioblastoma multiforme tumors. Comparable endothelia-injury studies will include challenging barrier endothelia monolayers with 1) glutamate, 2) conditioned medium from ATP-stimulated glioma, 3) conditioned medium from glioma cells or astrocytes grown under hypoxic conditions, and 4) the direct effect on barrier dysfunction induced by co-culturing endothelia with glioma cells in hypoxic conditions using luminal/abluminal chambers of COL4-coated transwell inserts. The Daniels-Beard collaboration has strong institutional support and will use lDeA-built research core laboratories. Both the Biomolecular Research Core and the Biomedical Research Vivarium will be used in this project. Boise State University supports this research with Core access and technical assistance

Relevance This project melds two complementary INBRE and COBRE investigator projects in a collaborative effort to address important aspects in brain cancer development and migration. Each investigator brings unique molecular and physiological expertise to address the intercellular signaling between brain tumor cells and nascent blood-brain barrier endothelial cells that results in the spread of cancer cells. This collaboration will provide high-impact learning experiences for undergraduate students at two Idaho INBRE primarily undergraduate institutions.