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PDF File - (58K) - Secondary screen identified 168 GSC -effective compounds with selective inhibition profile

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types. Mol Cancer Ther; 10(10); 1818–28. ©2011 AACR.

PDF File - (32K) - GSC-enriched GBM samples form tumors after transplantation into immunodeficient mice

PDF File - (874K) - A - Representative images demonstrating tumor formation success in an animal transplanted with serum (I) and sphere (II) derived GBM146 Ki67 and Human Nuclei (HuNu) immunostaining shows that virtually all transplanted cells died off in the animal transplanted with serum derived cells while an infiltrating tumor mass is visible in the animal transplanted with sphere derived cells of the same GBM sample (i - injection site, V- ventricle, SVZ - subventricular zone). B - Immunohistological sections for Human Nuclei (HuNu) and Ki67 demonstrating good survival of transplanted cells in animals that were sacrificed on the same day as well as 48h after transplantation (IT- injection tract, ST-striatum).

PDF File - (67K) - Effects of OLDA, PALDA and NADA are not mediated via cannabinoid, vanilloid or dopamine receptors A - Exposure of GSC -enriched GBM 157 cells to CB1, CB2, TRPV1 receptor agonists or their combination, does not reproduce the phenotype seen after exposure to OLDA, PALDA or NADA. Graph showing ATP level expressed as % of DMSO treated controls. B - Exposure of OLDA, PALDA or NADA-pretreated GSC -enriched GBM 157 cells (at IC50 concentration) to antagonists of CB, TRPV and D receptors does not rescue the phenotype caused by OLDA, PALDA or NADA (B, C and D, respectively). The graphs are showing ATP levels expressed as % of the DMSO treated controls (100%). SE shown.

PDF File - (61K) - OLDA, Emetine, 5256360 and 5560509 decrease clonal sphere formation in low passage GBM312 Graphs show a concentration-dependent reduction of clonal neurosphere size, neurosphere number as well as total cell mass. Error bars represent SEM.

PDF File - (39K) - Limiting dilution transplantation of GBM157 cells treated with OLDA, 5256360 or 5560509 shows decreased tumor formation frequency

PDF File - (523K) - OLDA, Emetine, 5256360 and 5560509 inhibit proliferation of sphere cultured GBM312 cells, using CFSE (carboxyfluorescein diacetate, succinimidyl ester) cell tracing experiments Proliferative inhibition is graphically represented by an increase in fluorescence or a shift to the right, away from that of the control. A proliferation index (PI) is a numerical value generated for each sample that measures the total increase in cell number divided by the original parent number. PI's are expressed as a percent of control: Emetine (0.2�M)- 18.6%, OLDA (4�M)- 16.0%, Compound 33 (2�M)- 31%, and Compound 62 (2�M)- 24.3%.

PDF File - (40K) - Growth kinetics of serum and sphere derived cells in screening (sphere) media reveals only minor differences in the degree of growth/proliferation between sphere and serum derived cultures of the same cell type Graph shows results of an ATP-based proliferation assay as measured 3 days after cell plating. Error bars depict SEM.

PDF File - (55K) - Sensitivity of GBM cells to OLDA, PALDA and NADA changes as a function of time, for which the cells were cultured in GSC -enriching media GBM107 cells were exposed to GSC -enriching media for increasing amounts of time and were then screened for sensitivity to dopaminergic cannabinoid compounds. While screening in serum-containing media requires a higher compound concentration due to the protective effect of serum in general, serum-derived cells become more and more sensitive towards GSC -specific compounds over time after transfer into sphere media (A, B, C). Such a difference is not present using non-GSC selective chemotherapeutics (D) or non-GBM control cells (E, F). ('ser in ser': serum derived cells screened in serum media; 'ser in sph': serum derived cells screened in sphere media; '1d sph': serum derived cells were cultured in sphere media for one day before the screen, etc.)

Recent research suggests that mild traumatic brain injury (TBI) may exert deleterious effects on endogenous pain modulatory function, potentially underlying the elevated risk for persistent headaches following injury. Accumulating research also shows race differences in clinical and experimental pain, with African Americans (AA) generally reporting more severe pain, worse pain modulation, and greater pain sensitivity compared to Caucasians. However, race differences in pain-related outcomes following mild TBI have rarely been studied. The purpose of this study was to explore race differences in endogenous pain modulation, pain sensitivity, headache pain, and psychological factors among AA and Caucasian individuals with mild TBI in the first month following injury compared to healthy controls and across time. Patients with mild TBI were recruited from the local Emergency Department Trauma Centers. Sixty-three mild TBI participants (African Americans: n=23, Caucasians: n=40) enrolled in this study and completed study sessions at 1-2 weeks and 1-month post injury. Forty-one mild TBI-free control participants (African Americans: n=11, Caucasians: n=30), matched on age and sex, completed one study session. Assessments included a headache survey, Pain Catastrophizing Scale, CES-Depression Scale, and several quantitative sensory tests (QST) to measure endogenous pain modulatory function. The QST tests included conditioned pain modulation (CPM) to measure endogenous pain inhibitory function and temporal summation of pain and pressure pain thresholds (PPTs) of the head to measure pain sensitization and sensitivity. Two-way ANOVAs were used to determine whether the outcome measures differed as a function of race, mild TBI, and time. Mediation analysis was used to explore potential mediators for the race differences in headache pain intensity. The results showed that AA mild TBI participants reported significantly greater headache pain and pain catastrophizing and exhibited higher pain sensitivity and worse pain modulation on QST tests compared to Caucasian mild TBI participants. These same race differences were not observed among the healthy TBI-free control sample. The mediation analyses showed complete mediation for the relation between race and headache pain intensity by pain catastrophizing at 1-2 weeks and 1 month post injury. Overall, the results of this study suggest that African Americans compared to Caucasians are characterized by psychological and pain modulatory profiles following mild TBI that could increase the risk for the development of intense and persistent headaches following injury.

Background. Recent animal research suggests that mild traumatic brain injury (mTBI) facilitates abnormal endogenous modulation of pain, potentially underlying the increased risk for persistent headaches following injury. However, no human studies have directly assessed the functioning of endogenous facilitory and inhibitory systems in the early stages after an mTBI. Objective. The purpose of this exploratory study was to examine trigeminal sensitization and endogenous pain inhibitory capacity in mTBI patients in the acute stage of injury compared with matched controls. We also examined whether post-traumatic headache pain intensity within the mTBI sample was related to sensitization and pain inhibitory capacity. Methods. Twenty-four mTBI patients recruited from emergency departments and 21 age-, race-, and sex-matched controls completed one experimental session. During this session, participants completed quantitative sensory tests measuring trigeminal sensitization (pressure pain thresholds and temporal summation of pain in the head) and endogenous pain inhibition (conditioned pain modulation). Participants also completed validated questionnaires measuring headache pain, depression, anxiety, and pain catastrophizing. Results. The results revealed that the mTBI group exhibited significantly decreased pressure pain thresholds of the head and decreased pain inhibition on the conditioned pain modulation test compared with the control group. Furthermore, correlational analysis showed that the measures of trigeminal sensitization and depression were significantly associated with headache pain intensity within the mTBI group. Conclusions. In conclusion, mTBI patients may be at risk for maladaptive changes to the functioning of endogenous pain modulatory systems following head injury that could increase risk for post-traumatic headaches.