Dan R. Laks

Neurosphere formation is an independent predictor of clinical outcome in malignant glioma.

Renewable neurosphere formation in culture is a defining characteristic of certain brain tumor initiating cells. This retrospective study was designed to assess the relationship among neurosphere formation in cultured human glioma, tumorigenic capacity, and patient clinical outcome. Tumor samples were cultured in neurosphere conditions from 32 patients with glioma, including a subpopulation of 15 patients with primary glioblastoma. A subsample of renewable neurosphere cultures was xenografted into mouse brain to determine if they were tumorigenic. Our study shows that both renewable neurosphere formation and tumorigenic capacity are significantly associated with clinical outcome measures. Renewable neurosphere formation in cultured human glioma significantly predicted an increased hazard of patient death and more rapid tumor progression. These results pertained to both the full population of glioma and the subpopulation of primary glioblastoma. Similarly, there was a significant hazard of progression for patients whose glioma had tumorigenic capacity. Multivariate analysis demonstrated that neurosphere formation remained a significant predictor of clinical outcome independent of Ki67 proliferation index. In addition, multivariate analysis of neurosphere formation, tumor grade and patient age, demonstrated that neurosphere formation was a robust, independent predictor of glioma tumor progression. Although the lengthy duration of this assay may preclude direct clinical application, these results exemplify how neurosphere culture serves as a clinically relevant model for the study of malignant glioma. Furthermore, this study suggests that the ability to propagate brain tumor stem cells in vitro is associated with clinical outcome. STEM CELLS 2009;27:980–987

A Microfluidic Platform for Systems Pathology: Multiparameter Single-Cell Signaling Measurements of Clinical Brain Tumor Specimens

The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform-microfluidic image cytometry (MIC)-capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine.

Siomycin A targets brain tumor stem cells partially through a MELK-mediated pathway

Glioblastoma multiforme (GBM) is a devastating disease, and the current therapies have only palliative effect. Evidence is mounting to indicate that brain tumor stem cells (BTSCs) are a minority of tumor cells that are responsible for cancer initiation, propagation, and maintenance. Therapies that fail to eradicate BTSCs may ultimately lead to regrowth of residual BTSCs. However, BTSCs are relatively resistant to the current treatments. Development of novel therapeutic strategies that effectively eradicate BTSC are, therefore, essential. In a previous study, we used patient-derived GBM sphere cells (stemlike GBM cells) to enrich for BTSC and identified maternal embryonic leucine-zipper kinase (MELK) as a key regulator of survival of stemlike GBM cells in vitro. Here, we demonstrate that a thiazole antibiotic, siomycin A, potently reduced MELK expression and inhibited tumor growth in vivo. Treatment of stemlike GBM cells with siomycin A resulted in arrested self-renewal, decreased invasion, and induced apoptosis but had little effect on growth of the nonstem cells of matched tumors or normal neural stem/progenitor cells. MELK overexpression partially rescued the phenotype of siomycin A-treated stemlike GBM cells. In vivo, siomycin A pretreatment abraded the sizes of stemlike GBM cell-derived tumors in immunodeficient mice. Treatment with siomycin A of mice harboring intracranial tumors significantly prolonged their survival period compared with the control mice. Together, this study may be the first model to partially target stemlike GBM cells through a MELK-mediated pathway with siomycin A to pave the way for effective treatment of GBM.

Maternal Inflammation Contributes to Brain Overgrowth and Autism-Associated Behaviors through Altered Redox Signaling in Stem and Progenitor Cells

Summary A period of mild brain overgrowth with an unknown etiology has been identified as one of the most common phenotypes in autism. Here, we test the hypothesis that maternal inflammation during critical periods of embryonic development can cause brain overgrowth and autism-associated behaviors as a result of altered neural stem cell function. Pregnant mice treated with low-dose lipopolysaccharide at embryonic day 9 had offspring with brain overgrowth, with a more pronounced effect in PTEN heterozygotes. Exposure to maternal inflammation also enhanced NADPH oxidase (NOX)-PI3K pathway signaling, stimulated the hyperproliferation of neural stem and progenitor cells, increased forebrain microglia, and produced abnormal autism-associated behaviors in affected pups. Our evidence supports the idea that a prenatal neuroinflammatory dysregulation in neural stem cell redox signaling can act in concert with underlying genetic susceptibilities to affect cellular responses to environmentally altered cellular levels of reactive oxygen species.

Clinical outcome in pediatric glial and embryonal brain tumors correlates with in vitro multi‐passageable neurosphere formation

Cultured brain tumors can form neurospheres harboring tumorigenic cells with self renewal and differentiation capacities. Renewable neurosphere formation has clinical predictive value in adult malignant gliomas, yet its prognostic role for pediatric brain tumors is unknown.

Brain tumor stem cells as therapeutic targets in models of glioma.

At this time, brain tumor stem cells remain a controversial hypothesis while malignant brain tumors continue to present a dire prognosis of severe morbidity and mortality. Yet, brain tumor stem cells may represent an essential cellular target for glioma therapy as they are postulated to be the tumorigenic cells responsible for recurrence. Targeting oncogenic pathways that are essential to the survival and growth of brain tumor stem cells represents a promising area for developing therapeutics. However, due to the multiple oncogenic pathways involved in glioma, it is necessary to determine which pathways are the essential targets for therapy. Furthermore, research still needs to comprehend the morphogenic processes of cell populations involved in tumor formation. Here, we review research and discuss perspectives on models of glioma in order to delineate the current issues in defining brain tumor stem cells as therapeutic targets in models of glioma.

Asparagine Depletion Potentiates the Cytotoxic Effect of Chemotherapy against Brain Tumors

Targeting amino acid metabolism has therapeutic implications for aggressive brain tumors. Asparagine is an amino acid that is synthesized by normal cells. However, some cancer cells lack asparagine synthetase (ASNS), the key enzyme for asparagine synthesis. Asparaginase (ASNase) contributes to eradication of acute leukemia by decreasing asparagine levels in serum and cerebrospinal fluid. However, leukemic cells may become ASNase-resistant by upregulating ASNS. High expression of ASNS has also been associated with biologic aggressiveness of other cancers, including gliomas. Here, the impact of enzymatic depletion of asparagine on proliferation of brain tumor cells was determined. ASNase was used as monotherapy or in combination with conventional chemotherapeutic agents. Viability assays for ASNase-treated cells demonstrated significant growth reduction in multiple cell lines. This effect was reversed by glutamine in a dose-dependent manner—as expected, because glutamine is the main amino group donor for asparagine synthesis. ASNase treatment also reduced sphere formation by medulloblastoma and primary glioblastoma cells. ASNase-resistant glioblastoma cells exhibited elevated levels of ASNS mRNA. ASNase cotreatment significantly enhanced gemcitabine or etoposide cytotoxicity against glioblastoma cells. Xenograft tumors in vivo showed no significant response to ASNase monotherapy and little response to temozolomide alone. However, combinatorial therapy with ASNase and temozolomide resulted in significant growth suppression for an extended duration of time. Taken together, these findings indicate that amino acid depletion warrants further investigation as adjunctive therapy for brain tumors. Implications: Findings have potential impact for providing adjuvant means to enhance brain tumor chemotherapy. Mol Cancer Res; 12(5); 694–702. ©2014 AACR.

Large-scale assessment of the gliomasphere model system

BACKGROUND Gliomasphere cultures are widely utilized for the study of glioblastoma (GBM). However, this model system is not well characterized, and the utility of current classification methods is not clear. METHODS We used 71 gliomasphere cultures from 68 individuals. Using gene expression-based classification, we performed unsupervised clustering and associated gene expression with gliomasphere phenotypes and patient survival. RESULTS Some aspects of the gene expression-based classification method were robust because the gliomasphere cultures retained their classification over many passages, and IDH1 mutant gliomaspheres were all proneural. While gene expression of a subset of gliomasphere cultures was more like the parent tumor than any other tumor, gliomaspheres did not always harbor the same classification as their parent tumor. Classification was not associated with whether a sphere culture was derived from primary or recurrent GBM or associated with the presence of EGFR amplification or rearrangement. Unsupervised clustering of gliomasphere gene expression distinguished 2 general categories (mesenchymal and nonmesenchymal), while multidimensional scaling distinguished 3 main groups and a fourth minor group. Unbiased approaches revealed that PI3Kinase, protein kinase A, mTOR, ERK, Integrin, and beta-catenin pathways were associated with in vitro measures of proliferation and sphere formation. Associating gene expression with gliomasphere phenotypes and patient outcome, we identified genes not previously associated with GBM: PTGR1, which suppresses proliferation, and EFEMP2 and LGALS8, which promote cell proliferation. CONCLUSIONS This comprehensive assessment reveals advantages and limitations of using gliomaspheres to model GBM biology, and provides a novel strategy for selecting genes for future study.

Mercury rising: response to the EPA assessment of mercury exposure

In November 2013, the United States EPA (Environmental Protection Agency) released a study (EPA 2013) of the NHANES (National Health and Nutrition Examination Survey) dataset claiming that mercury concentrations were decreasing in the human population over time. In fact, the opposite conclusion is supported by their data. The unreported rise in blood inorganic mercury levels (IHg) indicates a rise of chronic mercury exposure in the population over time. Mercurial by nature, chronic exposure to this potent neurotoxin is difficult to measure. The EPA chose the ill suited organic form of mercury, methyl mercury (MeHg), as their biomarker of exposure. Although MeHg is the primary form of mercury in fish, it is well documented that MeHg is only a fleeting occurrence in the blood after ingestion, with a half-life of around 2 months in blood. Therefore, MeHg is more a measure of recent exposure than it is of long term, chronic exposure to mercury. In numerous studies of organic mercury exposure, MeHg levels vanish from blood and tissue after cessation of exposure. My metaphor is that using MeHg as a biomarker for Hg exposure is like looking at the gas tank in order to assay how far a person has driven. MeHg is merely a biomarker of recent exposure, as a gas tank is only indicative of how long ago a person filled up their tank. In contrast, blood inorganic mercury (IHg) levels serve as a better biomarker for long-term exposure to mercury. IHg, as a biomarker, is akin to looking at the engine oil in order to determine how far a person has driven; it may signify how much a person has accumulated exposure. Methyl mercury is converted to IHg in the body tissue and subsequently IHg is deposited in the tissue for years. Long term deposition of IHg in tissues of the body was detected by Sallsten et al. in (1993) when they showed that mercury was retained by the body after chronic exposure (Sallsten et al. 1993). In Vahter et al. (1994) they demonstrated that organic mercury, MeHg, demethylates into inorganic mercury, IHg, and is detected in the blood (Vahter et al. 1994). Furthermore, inorganic mercury in the blood, in one instance, remained elevated even after cessation of exposure while organic mercury was always eliminated. Therefore, IHg served as a better bioindicator of chronic exposure because it alone could remain in the blood long after cessation of exposure. Moreover, the one primate that developed liver damage after exposure to organic mercury had elevated IHg but not MeHg. This indicates that IHg is a better bioindicator of the toxic effects of mercury exposure. Some MeHg, Organic Mercury, will naturally exit the body through elimination, while some MeHg will biotransform into IHG, Inorganic Mercury. Blood organic mercury levels are transitory; either it is on its way out of the body or on its way to becoming IHG. D. R. Laks (&) Department of Biological Chemistry, UCLA, 379 Neuroscience Research Bldg, Suite 379, 635 Charles E. Young Drive South, Los Angeles, CA 90095-7332, USA e-mail: dlaks@mednet.ucla.edu

Luteinizing hormone provides a causal mechanism for mercury associated disease

Previous studies have demonstrated that the pituitary is a main target for inorganic mercury (I-Hg) deposition and accumulation within the brain. My recent study of the US population (1999-2006) has uncovered a significant, inverse relationship between chronic mercury exposure and levels of luteinizing hormone (LH). This association with LH signifies more than its presumed role as bioindicator for pituitary neurosecretion and function. LH is the only hormone with a rare and well characterized, high affinity binding site for mercury. On its catalytic beta subunit, LH has the structure to preferentially bind inorganic mercury almost irreversibly, and, by that manner, accumulate the neurotoxic element. Thus, it is likely that LH is an early and significant target of chronic mercury exposure. Moreover, due to the role of LH in immune-modulation and neurogenesis, I present LH as a central candidate to elucidate a causal mechanism for chronic mercury exposure and associated disease.