1 SCIENTIFIC REPORTS | 7: 15593 | DOI:10.1038/s41598-017-15849-w www.nature.com/scientificreports 5-Aminolevulinic Acid Guided Sampling of Glioblastoma Microenvironments Identifes Pro- Survival Signaling at Infltrative Margins James L. Ross 1,4,7 , Lee A. D. Cooper 2,5,6,7,8 , Jun Kong 2,5,6,8 , David Gutman 3,5,6 , Merete Williams 1 , Carol Tucker-Burden 1 , Myles R. McCrary 6,8 , Alexandros Bouras 9 , Milota Kaluzova 4 , William D. Dunn Jr. 2 , Duc Duong 3 , Constantinos G. Hadjipanayis 9 & Daniel J. Brat 1,2,5,6 Glioblastoma (GBM) contains diverse microenvironments with uneven distributions of oncogenic alterations and signaling networks. The difusely infltrative properties of GBM result in residual tumor at neurosurgical resection margins, representing the source of relapse in nearly all cases and suggesting that therapeutic eforts should be focused there. To identify signaling networks and potential druggable targets across tumor microenvironments (TMEs), we utilized 5-ALA fuorescence- guided neurosurgical resection and sampling, followed by proteomic analysis of specifc TMEs. Reverse phase protein array (RPPA) was performed on 205 proteins isolated from the tumor margin, tumor bulk, and perinecrotic regions of 13 previously untreated, clinically-annotated and genetically-defned high grade gliomas. Diferential protein and pathway signatures were established and then validated using western blotting, immunohistochemistry, and comparable TCGA RPPA datasets. We identifed 37 proteins diferentially expressed across high-grade glioma TMEs. We demonstrate that tumor margins were characterized by pro-survival and anti-apoptotic proteins, whereas perinecrotic regions were enriched for pro-coagulant and DNA damage response proteins. In both our patient cohort and TCGA cases, the data suggest that TMEs possess distinct protein expression profles that are biologically and therapeutically relevant. Glioblastoma (GBM) is a highly aggressive primary brain tumor characterized by dismal patient outcomes, with median overall survival of 11–15 months 1 . Despite maximal safe resection, adjuvant chemotherapy and radiation, nearly all patients eventually relapse. Te degree of maximal tumor resection is correlated with patient prognosis, yet distinguishing between neoplastic and healthy tissue at resection margins remains a challenge, and the dif- fusely infltrative nature of the disease precludes complete resection 2,3 . In almost all cases, GBM recurrence occurs near the site of previous resection, suggesting that therapeutic targeting eforts should focus on this region 4,5 . GBM has three dominant tumor microenvironments (TMEs) that include the perinecrotic region (PN), bulk tumor (BT), and the infltrative tumor margin (TM). Te perinecrotic zone is characterized by severe hypoxia and contains a hypercellular zone of pseudopalisading cells that are enriched for glioma stem cells 6,7 . Bulk tumor is characterized by densely packed sheets of infltrating glioma cells with high mitotic activity, while tumor margins are considered the intersection of infltrating tumor and normal brain. Troughout this micro-environmental 1 Departments of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, Georgia. 2 Biomedical Informatics, Emory University, Atlanta, GA 30322, Georgia. 3 Neurology, Emory University, Atlanta, GA 30322, Georgia. 4 Pediatrics, Emory University, Atlanta, GA 30322, Georgia. 5 Winship Cancer Institute, Emory University, Atlanta, GA 30322, Georgia. 6 Emory University School of Medicine, Emory University, Atlanta, GA 30322, Georgia. 7 Emory University Graduate Program in Cancer Biology, Atlanta, GA 30322, Georgia. 8 Biomedical Engineering, Emory University/Georgia Institute of Technology, Atlanta, GA 30322, Georgia. 9 Department of Neurosurgery, Icahn School of Medicine at Mount Sinai, New York, NY, 10003, USA. Correspondence and requests for materials should be addressed to C.G.H. (email: Constantinos.Hadjipanayis@mountsinai.org) or D.J.B. (email: dbrat@emory.edu) Received: 12 September 2017 Accepted: 31 October 2017 Published: xx xx xxxx OPEN