The invasive region of glioblastoma defined by 5ALA guided surgery has an altered cancer stem cell marker profile compared to central tumour

Glioblastoma, a WHO grade IV astrocytoma, is a highly aggressive and heterogeneous tumour that infiltrates deeply into surrounding brain parenchyma, making complete surgical resection impossible. Despite chemo-radiotherapy, the residual cell population within brain parenchyma post-surgery causes inevitable recurrence. Previously, the tumour core has been the focus of research and the basis for targeted therapeutic regimes, which have failed to improve survival in clinical trials. Here, we focus on the invasive margin as defined by the region with 5-aminolevulinic acid (5ALA) (GliolanTM) fluorescence at surgery beyond the T1 enhancing region on magnetic resonance imaging (MRI). This area is hypothesized to constitute unique microenvironmental pressures, and consequently be molecularly distinct to tumour core and enhancing rim regions. We conducted hematoxylin and eosin (H&E), array real time polymerase chain reaction (PCR), and immunohistochemistry staining on various intra-tumour regions of glioblastoma to determine molecular heterogeneity between regions. We analyzed 73 tumour samples from 21 patients and compared cellular density, cell proliferation, and the degree of vascularity. There is a statistically significant difference between the core, invasive margin and other regions for cell density (p < 0.001), cell proliferation (p = 0.029), and vascularity (p = 0.007). Aldehyde dehydrogenase 1 (ALDH1) and Nestin immunohistochemistry were used as a measure of stem-like properties, showing significantly decreased Nestin expression (p < 0.0001) in the invasive margin. Array PCR of the core, rim, and invasive regions showed significantly increased fibroblast growth factor (FGF) and ALDH1 expression in the invasive zone, with elevated hypoxia inducing factor 1-alpha (HIF1 alpha) in the rim region, adjacent to the hypoxic core. The influence of varying microenvironments in the intra-tumour regions is a major key to understanding intra-tumour heterogeneity. This study confirms the distinct molecular composition of the heterogeneous invasive margin and cautions against purported therapy strategies that target candidate glioblastoma stem-like genes that are predominantly expressed in the tumour core. Full characterization of tumour cells in the invasive margin is critical, as these cells may more closely resemble the residual cell population responsible for tumour recurrence. Their unique nature should be considered when developing targeted agents for residual glioblastoma multiforme (GBM)

Metabolism based isolation of invasive glioblastoma cells with specific gene signatures and tumorigenic potential

BackgroundGlioblastoma (GBM) is a highly aggressive brain tumor with rapid subclonal diversification, harboring molecular abnormalities that vary temporo-spatially, a contributor to therapy resistance. Fluorescence guided neurosurgical resection utilizes administration of 5-aminolevulinic acid (5ALA) generating individually fluorescent tumor cells within a background population of non-neoplastic cells in the invasive tumor region. The aim of the study was to specifically isolate and interrogate the invasive GBM cell population using a novel 5ALA based method.MethodsWe have isolated the critical invasive GBM cell population by developing 5ALA-based metabolic fluorescence activated cell sorting. This allows purification and study of invasive cells from GBM without an overwhelming background “normal brain” signal to confound data. The population was studied using RNAseq, rtPCR and immunohistochemistry, with gene targets functionally interrogated on proliferation and migration assays using siRNA knockdown and known drug inhibitors.ResultsRNAseq analysis identifies specific genes such as SERPINE1 which is highly expressed in invasive GBM cells but at low levels in the surrounding normal brain parenchyma. siRNA knockdown and pharmacological inhibition with specific inhibitors of SERPINE1 reduced the capacity of GBM cells to invade in an in vitro assay. Rodent xenografts of 5ALA positive cells were established and serially transplanted, confirming tumorigenicity of the fluorescent patient derived cells but not the 5ALA negative cells.ConclusionsIdentification of unique molecular features in the invasive GBM population offer hope for developing more efficacious targeted therapies compared to targeting the tumor core and for isolating tumor sub-populations based upon intrinsic metabolic properties

Genome-Wide Expression and Anti-Proliferative Effects of Electric Field Therapy on Pediatric and Adult Brain Tumors

The lack of treatment options for high-grade brain tumors has led to searches for alternative therapeutic modalities. Electrical field therapy is one such area. The Optune™ system is an FDA-approved novel device that delivers continuous alternating electric fields (tumor treating fields— TTFields) to the patient for the treatment of primary and recurrent Glioblastoma multiforme (GBM). Various mechanisms have been proposed to explain the effects of TTFields and other electrical therapies. Here, we present the first study of genome-wide expression of electrotherapy (delivered via TTFields or Deep Brain Stimulation (DBS)) on brain tumor cell lines. The effects of electric fields were assessed through gene expression arrays and combinational effects with chemotherapies. We observed that both DBS and TTFields significantly affected brain tumor cell line viability, with DBS promoting G0-phase accumulation and TTFields promoting G2-phase accumulation. Both treatments may be used to augment the efficacy of chemotherapy in vitro. Genome-wide expression assessment demonstrated significant overlap between the different electrical treatments, suggesting novel interactions with mitochondrial functioning and promoting endoplasmic reticulum stress. We demonstrate the in vitro efficacy of electric fields against adult and pediatric high-grade brain tumors and elucidate potential mechanisms of action for future study

Overall survival in malignant glioma is significantly prolonged by neurosurgical delivery of etoposide and temozolomide from a thermo-responsive biodegradable paste

Purpose: High-grade glioma (HGG) treatment is limited by the inability of otherwise potentially efficacious drugs to penetrate the blood brain barrier. We evaluate the unique intra-cavity delivery mode and translational potential of a blend of poly(DL-lactic acid-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) paste combining temozolomide and etoposide to treat surgically resected HGG. Experimental Design: To prolong stability of temozolomide pro-drug, combined in vitro drug release was quantitatively assessed from low pH-based PLGA/PEG using advanced analytical methods. In vitro cytotoxicity was measured against a panel of HGG cell lines and patient-derived cultures using metabolic assays. In vivo safety and efficacy was evaluated using orthotopic 9L gliosarcoma allografts, previously utilized pre-clinically to develop Gliadel®. Results: Combined etoposide and temozolomide in vitro release (22 and 7 days respectively) was achieved from a lactic acid-based PLGA/PEG paste, used to enhance stability of temozolomide prodrug. HGG cells from central-enhanced regions were more sensitive to each compound relative to primary lines derived from the HGG invasive margin. Both drugs retained cytotoxic capability upon release from PLGA/PEG. In vivo studies revealed a significant overall survival benefit in post-surgery 9L orthotopic gliosarcomas treated with intra-cavity delivered PLGA/PEG/temozolomide/etoposide and enhanced with adjuvant radiotherapy. Long-term survivorship was observed in over half the animals with histological confirmation of disease-free brain. Conclusions: The significant survival benefit of intra-cavity chemotherapy demonstrates clinical applicability of PLGA/PEG paste-mediated delivery of temozolomide and etoposide adjuvant to radiotherapy. PLGA/PEG paste offers a future platform for combination delivery of molecular targeted compounds

The molecular landscape of medulloblastoma in teenagers and young adults

SIMPLE SUMMARY: Medulloblastoma is a malignant primary brain tumour that commonly occurs in children but also occurs in teenagers and young adults (TYA, aged 13–24 years) frequently enough to warrant attention. While remarkable progress has been made with paediatric tumours, TYAs with medulloblastoma represents an understudied population, lacking dedicated studies. The study aims to comprehensively characterise medulloblastoma in TYAs using data obtained from genomic technologies. It highlights that TYA medulloblastoma constitutes heterogenous molecular subgroups with distinct epigenetic and transcriptomic characteristics. Additionally, the prognostic gene signature-based score stratifies TYA patients into low- and high-risk groups and associates significantly with the outcomes. These results demonstrate unique molecular characteristics of TYA medulloblastoma that may contribute to the clinical differences at presentation between TYAs and other age groups. A better understanding of the TYA-specific biology might impact the treatment of those patients in the future. ABSTRACT: Medulloblastoma (MB) is a childhood malignant brain tumour but also occurs in teenagers and young adults (TYA). Considering that MB is heterogeneous, this study aimed to define the molecular landscape of MBs in TYAs. We collated more than 2000 MB samples that included 287 TYA patients (13–24 years). We performed computational analyses consisting of genome-wide methylation and transcriptomic profiles and developed a prognostics model for the TYAs with MB. We identified that TYAs predominantly comprised of Group 4 (40%) and Sonic Hedgehog (SHH)-activated (33%) tumours, with Wingless-type (WNT, 17%) and Group 3 (10%) being less common. TYAs with SHH tumours displayed significantly more gene expression alterations, whereas no gene was detected in the Group 4 tumours. Across MB subgroups, we identified unique and shared sets of TYA-specific differentially methylated probes and DNA-binding motifs. Finally, a 22-gene signature stratified TYA patients into high- and low-risk groups, and the prognostic significance of these risk groups persisted in multivariable regression models (P = 0.001). This study is an important step toward delineating the molecular landscape of TYAs with MB. The emergence of novel genes and pathways may provide a basis for improved clinical management of TYA with MB

NIS mediates iodide uptake in the female reproductive tract and is a poor prognostic factor in ovarian cancer

et al.[Context]: The sodium iodide symporter (NIS) mediates active transport of iodide into the thyroid and the lactating mammary glands and is highly expressed in thyroid and breast carcinomas. NIS is clinically very relevant because it allows the treatment with radioiodine of thyroid cancer patients. [Objective]: In this study we wanted to explore whether NIS is expressed in the ovary and in ovarian cancer. [Methods/Patients]: Methods included NIS and paired box 8 expression and function in ovarian cancer patients and rats by immunochemistry, immunoblot, RT-PCR, and iodide uptake. [Results]: Here we demonstrate for the first time that NIS is expressed in the ovary and fallopian tube and actively accumulates significant levels of radioiodide in vivo. In a large survey of menstruating women receiving radioiodide for medical purposes, 15% showed significant uptake in the normal reproductive tract. Ovarian NIS activity is influenced by the estrous cycle stage in rats, being upregulated during peak levels of estrogens occurring immediately before the ovulation. We unveil that the regulatory mechanism underlying this phenomenon is based on the functional cooperation of estrogen receptor-α and paired box 8. We also show that NIS is highly expressed in ovarian cancer, predicting a poor prognosis in these patients. [Conclusions]: These results provide the basis that will help minimize the impact of therapeutic doses of radioiodide on gonadal function. We also suggest that NIS is a new ovarian cancer marker, opening a door for the use of radioiodide in the diagnosis and treatment of ovarian cancer patients. Copyright © 2014 by the Endocrine Society.This work was supported by Grants PS09-1387 and PI12-01201 from the Instituto de Salud Carlos III and Grant FSEEN (to A.D.l.V.); Grant BFU2010-16025 from the Direccion General de Ciencia y Tecnologia (to P.S.), Grant S2011/BMD-2328 Tironet from the Comunidad de Madrid (to P.S. and G.R.-E.); Grant RETICC RD12/0036/0030 from the Instituto de Salud Carlos III (to A.D.l.V. and P.S.). S.G.L. was supported in part by a JAE-Doc, an Intramural Consejo Superior de Investigaciones Científicas Project Grant 2011-20E101 and a Grant S2011/ BMD-2328 Tironet postdoctoral contract.Peer Reviewe