Shahrzad Jalali

GBM's multifaceted landscape: highlighting regional and microenvironmental heterogeneity

Gliomas are a heterogeneous group of tumors that show variable proliferative potential, invasiveness, aggressiveness, histological grading, and clinical behavior. In this review, we focus on glioblastoma multiforme (GBM), a grade IV glioma, which is the most common and malignant of primary adult brain tumors. Research over the past several decades has revealed the existence of extensive cellular, molecular, genetic, epigenetic, and metabolic heterogeneity among tumors of the same grade and even within individual tumors. Evaluation of different tumor types has shown that tumors with advanced grade and clinical aggressiveness also display enhanced molecular, cellular, and microenvironmental heterogeneity. From a therapeutic standpoint, this heterogeneity is a major clinical hurdle for devising effective therapeutic strategies for patients and challenges personalized medicine. In this review, we will highlight key aspects of GBM heterogeneity, directing special attention to regional heterogeneity, hypoxia, genomic heterogeneity, tumor-specific metabolic reprogramming, neovascularization or angiogenesis, and stromal immune cells. We will further discuss the clinical implications of GBM heterogeneity in the context of therapy.

VEGF Regulates Region-Specific Localization of Perivascular Bone Marrow–Derived Cells in Glioblastoma

Glioblastoma multiforme (GBM) is characterized by a pathogenic vasculature that drives aggressive local invasion. Recent work suggests that GBM cells recruit bone marrow-derived progenitor cells (BMDC) to facilitate recurrence after radiotherapy, but how this may be achieved is unclear. In this study, we established the spatiotemporal and regional contributions of perivascular BMDCs (pBMDC) to GBM development. We found an increased recruitment of BMDCs to GBM in response to tumor growth and following radiotherapy. However, in this study, BMDCs did not differentiate into endothelial cells directly but rather provided a perivascular support role. The pBMDCs were shown to associate with tumor vasculature in a highly region-dependent manner, with central vasculature requiring minimal pBMDC support. Region-dependent association of pBMDC was regulated by VEGF. In the absence of VEGF, following radiotherapy or antiangiogenic therapy, we documented an increase in Ang2 that regulated recruitment of pBMDCs to maintain the vulnerable central vasculature. Together, our results strongly suggested that targeting pBMDC influx along with radiation or antiangiogenic therapy would be critical to prevent vascular recurrence of GBM.

Exploring Therapeutic Targets in Schwannoma Through Integrative Analysis

Vestibular schwannomas (VS) are common benign tumors of the vestibular nerve and spinal schwannomas (SS) arise form nerves within the spinal canal. SS are the most common intradural extramedullary spinal tumors, representing 30% of such lesions and both VS and SS both cause significant morbidity. The current treatment strategies for VS and SS include surgery or radiation with each treatment option having associated complications and side effects. The transcriptional landscape of schwannoma remains largely unknown. We first performed gene expression profiling of 49 VS schwannomas and seven normal control vestibular nerves to identify tumor-specific gene expression patterns. We identified over 2000 differentially expressed genes between control and schwannoma with network analysis uncovering proliferation and anti-apoptotic pathways previously not implicated in VS. Furthermore, using several distinct clustering technologies, we could not reproducibly identify distinct VS subtypes or significant differences between sporadic and germline NF2 associated schwannomas suggesting that VS comprises of a highly similar entity. We next performed a transcript analysis comparing VS to SS. Surprisingly; we identified few differential transcripts demonstrating that schwannoma in general is a homogenous entity. Current studies are focused on copy number, DNA methylation profiling and genome wide sequencing analysis. Preliminary data suggests that NF2 is the major driver of schwannoma as previously implicated. Interestingly, we identified several novel and recurrent point mutations of NF2 in exon 1 of both VS and SS, which may lead to loss of function. Our most recurrent activated pathway in schwannoma was overexpression of PI3K/AKT/mTOR signaling pathway, which is directly druggable and evaluated this pathway for therapeutic targeting. Testing compounds BEZ235 and PKI-587, both novel dual inhibitors of PI3K and mTOR, attenuated tumor growth in a pre-clinical cell line model of schwannoma (HEI-293). Our In vitro findings demonstrated that pharmacological inhibition of the PI3K/AKT/mTOR pathway with next generation compounds lead to decreased cell viability, and increased cell death in VS, SS but not normal schwann cells. Future work is testing these compounds in vivo using relevant cell lines and primary cultures of VS/SS. Our findings implicate aberrant activation of the PI3K/AKT/mTOR pathway as a molecular mechanism of pathogenesis in VS and suggest inhibition of this pathway as a potential treatment strategy.

AI-02HYPOXIA AS A MEASURE OF FUNCTIONAL STATUS OF GLIOBLASTOMA VASCULATURE

INTRODUCTION: Glioblastoma (GBMs) exhibit distinct histopathological features including microvascular hyperplasia and heterogenous hypoxia within the tumor microenvironment. Newly formed vessels are often leaky/dysfunctional with intravascular thrombosis and potentially contribute to the hypoxic/necrotic foci seen throughout GBMs. By analyzing orthotopic xenograft models of GBM, we have established the correlation between tumor blood vessel and oxygen diffusion gradient. Additionally, we have established the effect of anti-angiogenic therapies (AATx) such as VEGF-Trap on this correlation. METHOD: Patient derived GSCs and established GBM cell line (U87) with or without VEGF-Trap were injected orthotopically in forebrains of NOD/SCID mice. Following 3-4 weeks of cell implantation, the mice were sacrificed and brains were harvested and imbedded in paraffin blocks. The sections were then stained for CD31 (endothelial marker) and CAIX (hypoxia marker). Images were scanned and quantified; using ImageJ (http://imagej.nih.gov/ij/), measurements include distance from tumor blood vessels to nearest hypoxic foci, total percent hypoxia, and cell proliferation. RESULTS: In literature it has been shown that any functional micro-vessel in normal tissue allows oxygen diffusion up to 100 µm radius. Using this as a guideline, we have defined the functional state of microvessels in our orthoptic tumor. We found that approximately 13% of the tumor blood vessels were dysfunctional, as their distance from immediate hypoxic cells was less than 20 µm. Upon treatment with AATx, we observed significant decrease in numbers of functional tumor blood vessels and cell prolliferation with concomitant increase in hypoxic areas within the tumor. CONCLUSIONS: AATx results in a significant decrease in the number of functional blood vessels - as measured by their proximity to the nearest hypoxic cells - and cell proliferation. We have provided a quantitative measure - distinct histopathological features including hypoxic and necrotic foci, microvascular hyperplasia and increased cell proliferation - for what was previously describe in qualitive terms in literature.