Placental ABC Transporters: Biological Impact and Pharmaceutical Significance.

The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy.

Novel TCF4:TCF12 heterodimer inhibits glioblastoma growth.

TWIST1 (TW) is a pro-oncogenic basic helix-loop-helix (bHLH) transcription factor and promotes the hallmark features of malignancy (e.g., cell invasion, cancer cell stemness, and treatment resistance), which contribute to poor prognoses of glioblastoma (GBM). We previously reported that specific TW dimerization motifs regulate unique cellular phenotypes in GBM. For example, the TW:E12 heterodimer increases periostin (POSTN) expression and promotes cell invasion. TW dimer-specific transcriptional regulation requires binding to the regulatory E-box consensus sequences, but alternative bHLH dimers that balance TW dimer activity in regulating pro-oncogenic TW target genes are unknown. We leveraged the ENCODE DNase I hypersensitivity data to identify E-box sites and tethered TW:E12 and TW:TW proteins to validate dimer binding to E-boxes in vitro. Subsequently, TW knockdown revealed a novel TCF4:TCF12 bHLH dimer occupying the same TW E-box site that, when expressed as a tethered TCF4:TCF12 dimer, markedly repressed POSTN expression and extended animal survival. These observations support TCF4:TCF12 as a novel dimer with tumor-suppressor activity in GBM that functions in part through displacement of and/or competitive inhibition of pro-oncogenic TW dimers at E-box sites.

Aging neural progenitor cells have decreased mitochondrial content and lower oxidative metabolism.

Although neurogenesis occurs in discrete areas of the adult mammalian brain, neural progenitor cells (NPCs) produce fewer new neurons with age. To characterize the molecular changes that occur during aging, we performed a proteomic comparison between primary-cultured NPCs from the young adult and aged mouse forebrain. This analysis yielded changes in proteins necessary for cellular metabolism. Mitochondrial quantity and oxygen consumption rates decrease with aging, although mitochondrial DNA in aged NPCs does not have increased mutation rates. In addition, aged cells are resistant to the mitochondrial inhibitor rotenone and proliferate in response to lowered oxygen conditions. These results demonstrate that aging NPCs display an altered metabolic phenotype, characterized by a coordinated shift in protein expression, subcellular structure, and metabolic physiology.

Increased re-entry into cell cycle mitigates age-related neurogenic decline in the murine subventricular zone.

Although new neurons are produced in the subventricular zone (SVZ) of the adult mammalian brain, fewer functional neurons are produced with increasing age. The age-related decline in neurogenesis has been attributed to a decreased pool of neural progenitor cells (NPCs), an increased rate of cell death, and an inability to undergo neuronal differentiation and develop functional synapses. The time between mitotic events has also been hypothesized to increase with age, but this has not been directly investigated. Studying primary-cultured NPCs from the young adult and aged mouse forebrain, we observe that fewer aged cells are dividing at a given time; however, the mitotic cells in aged cultures divide more frequently than mitotic cells in young cultures during a 48-hour period of live-cell time-lapse imaging. Double-thymidine-analog labeling also demonstrates that fewer aged cells are dividing at a given time, but those that do divide are significantly more likely to re-enter the cell cycle within a day, both in vitro and in vivo. Meanwhile, we observed that cellular survival is impaired in aged cultures. Using our live-cell imaging data, we developed a mathematical model describing cell cycle kinetics to predict the growth curves of cells over time in vitro and the labeling index over time in vivo. Together, these data surprisingly suggest that progenitor cells remaining in the aged SVZ are highly proliferative.

Fast bound pool fraction imaging of the in vivo rat brain: association with myelin content and validation in the C6 glioma model.

Cross-relaxation imaging (CRI) is a quantitative magnetic resonance technique that measures the kinetic parameters of magnetization transfer between protons bound to water and protons bound to macromolecules. In this study, in vivo, four-parameter CRI of normal rat brains (N=5) at 3.0 T was first directly compared to histology. The bound pool fraction, f, was strongly associated with myelin density (Pearson's r=0.99, p<0.001). The correlation persisted in separate analyses of gray matter (GM; r=0.89, p=0.046) and white matter (WM; r=0.97, p=0.029). Subsequently, a new time-efficient approach for solely capturing the whole-brain parametric map of f was proposed, validated with histology, and used to estimate myelin density. Since the described approach for the rapid acquisition of f applied constraints to other CRI parameters, a theoretical analysis of error was performed. Estimates of f in normal and pathologic tissue were expected to have <10% error. A comparison of values for f obtained from the traditional four-parameter fit of CRI data versus the proposed rapid acquisition of f was within this expected margin for in vivo rat brain gliomas (N=4; mean±SE; 3.9±0.2% vs. 4.0±0.2%, respectively). In both whole-brain f maps and myelin density maps, replacement of normal GM and WM by proliferating and invading tumor cells could be readily identified. The rapid, whole-brain acquisition of the bound pool fraction may provide a reliable method for detection of glioma invasion in both GM and WM during animal and human imaging.

Quantitative proteomic analysis of oligodendrogliomas with and without 1p/19q deletion.

Approximately 50-80% of oligodendrogliomas demonstrate a combined loss of chromosome 1p and 19q. Chromosome 1p/19q deletion, appearing early in tumorigenesis, is associated with improved clinical outcomes, including response to chemotherapy and radiation. Although many hypotheses have been proposed, the molecular mechanisms underlying improved clinical outcomes with 1p/19q deletion in oligodendrogliomas have not been characterized fully. To investigate the molecular differences between oligodendrogliomas, we employed an unbiased proteomic approach using microcapillary liquid chromatography mass spectrometry, along with a quantitative technique called isotope-coded affinity tags, on patient samples of grade II oligodendrogliomas. Following conventional biochemical separation of pooled tumor tissue from five samples of undeleted and 1p/19q deleted grade II oligodendrogliomas into nuclei-, mitochondria-, and cytosol-enriched fractions, relative changes in protein abundance were quantified. Among the 442 total proteins identified, 163 nonredundant proteins displayed significant changes in relative abundance in at least one of the three fractions between oligodendroglioma with and without 1p/19q deletion. Bioinformatic analyses of differentially regulated proteins supported the potential importance of metabolism and invasion/migration to the codeleted phenotype. A subset of altered proteins, including the pro-invasive extracellular matrix protein BCAN, was further validated by Western blotting as candidate markers for the more aggressive undeleted phenotype. These studies demonstrate the utility of proteomic analysis to identify candidate biological motifs and molecular mechanisms that drive differential malignancy related to 1p19q phenotypes. Future analysis of larger patient samples are warranted to further refine biomarker panels to predict biological behavior and assist in the identification of deleted gene products that define the 1p/19q phenotype.

TWIST1 promotes invasion through mesenchymal change in human glioblastoma.

BACKGROUND: Tumor cell invasion into adjacent normal brain is a mesenchymal feature of GBM and a major factor contributing to their dismal outcomes. Therefore, better understandings of mechanisms that promote mesenchymal change in GBM are of great clinical importance to address invasion. We previously showed that the bHLH transcription factor TWIST1 which orchestrates carcinoma metastasis through an epithelial mesenchymal transition (EMT) is upregulated in GBM and promotes invasion of the SF767 GBM cell line in vitro. RESULTS: To further define TWIST1 functions in GBM we tested the impact of TWIST1 over-expression on invasion in vivo and its impact on gene expression. We found that TWIST1 significantly increased SNB19 and T98G cell line invasion in orthotopic xenotransplants and increased expression of genes in functional categories associated with adhesion, extracellular matrix proteins, cell motility and locomotion, cell migration and actin cytoskeleton organization. Consistent with this TWIST1 reduced cell aggregation, promoted actin cytoskeletal re-organization and enhanced migration and adhesion to fibronectin substrates. Individual genes upregulated by TWIST1 known to promote EMT and/or GBM invasion included SNAI2, MMP2, HGF, FAP and FN1. Distinct from carcinoma EMT, TWIST1 did not generate an E- to N-cadherin "switch" in GBM cell lines. The clinical relevance of putative TWIST target genes SNAI2 and fibroblast activation protein alpha (FAP) identified in vitro was confirmed by their highly correlated expression with TWIST1 in 39 human tumors. The potential therapeutic importance of inhibiting TWIST1 was also shown through a decrease in cell invasion in vitro and growth of GBM stem cells. CONCLUSIONS: Together these studies demonstrated that TWIST1 enhances GBM invasion in concert with mesenchymal change not involving the canonical cadherin switch of carcinoma EMT. Given the recent recognition that mesenchymal change in GBMs is associated with increased malignancy, these findings support the potential therapeutic importance of strategies to subvert TWIST1-mediated mesenchymal change.

Intratumoral injection of hydrogel-embedded nanoparticles enhances retention in glioblastoma.

Intratumoral drug delivery is a promising approach for the treatment of glioblastoma multiforme (GBM). However, drug washout remains a major challenge in GBM therapy. Our strategy, aimed at reducing drug clearance and enhancing site-specific residence time, involves the local administration of a multi-component system comprised of nanoparticles (NPs) embedded within a thermosensitive hydrogel (HG). Herein, our objective was to examine the distribution of NPs and their cargo following intratumoral administration of this system in GBM. We hypothesized that the HG matrix, which undergoes rapid gelation upon increases in temperature, would contribute towards heightened site-specific retention and permanence of NPs in tumors. BODIPY-containing, infrared dye-labeled polymeric NPs embedded in a thermosensitive HG (HG-NPs) were fabricated and characterized. Retention and distribution dynamics were subsequently examined over time in orthotopic GBM-bearing mice. Results demonstrate that the HG-NPs system significantly improved site-specific, long-term retention of both NPs and BODIPY, with co-localization analyses showing that HG-NPs covered larger areas of the tumor and the peri-tumor region at later time points. Moreover, NPs released from the HG were shown to undergo uptake by surrounding GBM cells. Findings suggest that intratumoral delivery with HG-NPs has immense potential for GBM treatment, as well as other strategies where site-specific, long-term retention of therapeutic agents is warranted.

Quantitative analysis of mitotic Olig2 cells in adult human brain and gliomas: implications for glioma histogenesis and biology.

The capacity of adult human glial progenitor cells (AGPs), to proliferate and undergo multipotent differentiation, positions them as ideal candidate cells of origin for human gliomas. To investigate this potential role we identified AGPs as mitotically active Olig2 cells in nonneoplastic adult human brain and gliomas. We conservatively estimated that one in 5,000 human temporal lobe neocortical gray or subcortical white matter cells is mitotic. Extrapolating from a mean Olig2/Mib-1 labeling index (LI) of 52% and total cell number of 100 billion, we estimated the overall prevalence of mitotic Olig2 AGPs in nonneoplastic human brain parenchyma at 10 million. These data identify a large reservoir of Olig2 AGPs which could be potential targets for human gliomagenesis. The vast majority of mitotic cells in Grade II and Grade III gliomas of all histologic subtypes expressed Olig2 (mean LI 75%) but rarely S100B (LI 0.6%), identifying the Olig2 cell as a distinct contributor to the proliferating cell population of human gliomas of both oligodendroglial and astrocytic lineages. In the most malignant Grade IV glioma, or glioblastoma multiforme (GBM), the prevalence of Olig2/Mib-1 cells was significantly decreased (24.5%). The significantly lower Olig2/Mib-1 LI in GBMs suggests that a decrease in the prevalence of Olig2 cells to the total mitotic cell pool accompanies increasing malignancy. The novel framework provided by this quantitative and comparative analysis supports future studies to examine the histogenetic role of Olig2 AGPs in adult gliomas, their potential contribution to the tumor stroma and the molecular role of Olig2 in glioma pathogenesis.

TWIST1 Heterodimerization with E12 Requires Coordinated Protein Phosphorylation to Regulate Periostin Expression.

Diffuse invasion into adjacent brain matter by glioblastoma (GBM) is largely responsible for their dismal prognosis. Previously, we showed that the TWIST1 (TW) bHLH transcription factor and its regulated gene periostin (POSTN) promote invasive phenotypes of GBM cells. Since TW functional effects are regulated by phosphorylation and dimerization, we investigated how phosphorylation of serine 68 in TW regulates TW dimerization, POSTN expression, and invasion in glioma cells. Compared with wild-type TW, the hypophosphorylation mutant, TW(S68A), impaired TW heterodimerization with the E12 bHLH transcription factor and cell invasion in vitro but had no effect on TW homodimerization. Overexpression of TW:E12 forced dimerization constructs (FDCs) increased glioma cell invasion and upregulated pro-invasive proteins, including POSTN, in concert with cytoskeletal reorganization. By contrast, TW:TW homodimer FDCs inhibited POSTN expression and cell invasion in vitro. Further, phosphorylation of analogous PXSP phosphorylation sites in TW:E12 FDCs (TW S68 and E12 S139) coordinately regulated POSTN and PDGFRa mRNA expression. These results suggested that TW regulates pro-invasive phenotypes in part through coordinated phosphorylation events in TW and E12 that promote heterodimer formation and regulate downstream targets. This new mechanistic understanding provides potential therapeutic strategies to inhibit TW-POSTN signaling in GBM and other cancers.

Effect of pregnancy on cytochrome P450 3a and P-glycoprotein expression and activity in the mouse: mechanisms, tissue specificity, and time course.

The plasma concentrations of orally administered anti-human immunodeficiency virus protease inhibitors are significantly reduced during human and mouse pregnancy. We have shown that in the mouse, at gestational day 19, this reduction is due to increased hepatic cytochrome P450 3a (Cyp3a) protein expression and activity. In the current study, we investigated the mechanisms by which Cyp3a activity is increased by pregnancy and the time course of change in expression of Cyp3a and P-glycoprotein (P-gp) in various tissues. We found that hepatic transcripts of Cyp3a16, Cyp3a41, and Cyp3a44 were significantly increased during pregnancy, whereas those of Cyp3a11 and Cyp3a25 were significantly decreased. This resulted in a net increase in Cyp3a protein expression and activity in the liver during pregnancy. The increase in Cyp3a41 and Cyp3a44 transcripts was positively correlated (p < 0.05) with hepatocyte nuclear factor 6 and estrogen receptor-alpha transcripts. The pregnancy-related factors that transcriptionally activated mouse Cyp3a isoforms also activated the human CYP3A4 promoter in pregnant CYP3A4-promoter-luciferase transgenic (CYP3A4-tg) mice. In contrast, intestinal Cyp3a protein expression was not significantly affected by pregnancy. No change in P-gp protein expression was observed in the liver or kidney during pregnancy, although a significant decrease was observed in the placenta. Because hepatic CYP3A activity also seems to be induced during human pregnancy, the mouse (including CYP3A4-tg mouse) seems to be an excellent animal model to determine the molecular mechanisms for such an induction.

Comparative genomics of susceptibility to mammary carcinogenesis among inbred rat strains: role of reduced prolactin signaling in resistance of the Copenhagen strain.

To elucidate the molecular basis for differential susceptibilities to mammary carcinogenesis, we compared the transcriptomes of normal mammary glands from pubescent female rats of the resistant Copenhagen (Cop) strain with those of the susceptible Fischer 344 (F344), August x Copenhagen Irish (ACI), Buffalo/N (Buf/N), Wistar-Furth (WF) strains and F1 (Cop x F344) progeny (F1). Gene expression profiles in mammary tissue within each rat strain were remarkably similar, indicating that gene expression was determined by genetic background. We next identified the subset of genes that were differentially expressed in all susceptible strains relative to the resistant Cop strain. Among these, the messenger RNAs encoding prolactin (Prl) and its cell surface receptor were significantly elevated in all susceptible strains. The expression levels of several Prl-regulated genes were also significantly elevated, indicating the presence of increased Prl signaling in mammary glands of all susceptible strains. Pathway analysis of gene expression profiles further identified the Prl-activated Jak/STAT-signaling pathway among the pathways that most distinguished sensitive rat strains from the resistant Cop rat. To test the hypothesis that reduced levels of the Prl signaling in mammary tissue partially contributed to the genetic resistance to mammary carcinogenesis, we used the neuroleptic drug, perphenazine, to transiently elevate serum Prl levels in the Cop strain. Whereas Cop rats are resistant to N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis, approximately 5% of pubescent Cop females treated with perphenazine and NMU exposure developed mammary adenocarcinomas with latencies comparable with those of sensitive strains. Together, these finding indicated that in the rat, the molecular mechanisms underlying genetic susceptibility to mammary carcinogenesis include de-regulation of Prl signaling.

Dickkopf-1 mediated tumor suppression in human breast carcinoma cells.

Dickkopf-1 (DKK-1) is a secreted inhibitor of the Wnt signaling pathway. We previously identified DKK-1 as a candidate tumor suppressor and demonstrated that ectopic expression of the DKK-1 suppressed the tumorigenicity of HeLa cells in vitro and in vivo. Since suppression of tumorigenicity of HeLa cells by DKK-1 overexpression was not mediated by effects on beta-catenin dependent transcription, we hypothesized that DKK-1 might also inhibit tumorigenicity of breast carcinoma cell lines lacking an activated canonical Wnt pathway. In the present study we show that ectopic expression of DKK-1 in various breast cancer cell lines resulted in a change in the cell phenotype, increased sensitivity to apoptosis, inhibition of anchorage independent growth in vitro, and suppression of tumorigenicity in vivo. Consistent with known effects of DKK-1 on the canonical Wnt signaling pathway, ectopic expression of DKK-1 in breast carcinoma cells was associated with increased phosphorylation and degradation of beta-catenin. However, none of the breast tumor cells used in this study showed detectable levels of beta-catenin dependent activation of TCF/Lef promoter activity measured by reporter constructs. Consistent with the results of these transient transfection assays, we were unable to demonstrate the expected beta-catenin dependent, TCF/Lef mediated inhibition of cyclin D1 and c-myc gene transcription in breast cells overexpressing DKK-1. However, we found that cells with DKK-1 overexpression have increased activity of CamKII pathway. Overexpression of the constitutively active form of CamKII (T286D) resulted in inhibition of breast cancer cell tumorigenicity. Thus, our study supports the hypothesis that DKK-1 mediated tumor suppressor effect is independent of beta-catenin dependent transcription and identified the CamKII pathway that contributes into DKK-1 signaling.

Targeting TWIST1 through loss of function inhibits tumorigenicity of human glioblastoma.

TWIST1 (TW) is a bHLH transcription factor (TF) and master regulator of the epithelial-to-mesenchymal transition (EMT). In vitro, TW promotes mesenchymal change, invasion, and self-renewal in glioblastoma (GBM) cells. However, the potential therapeutic relevance of TW has not been established through loss-of-function studies in human GBM cell xenograft models. The effects of TW loss of function (gene editing and knockdown) on inhibition of tumorigenicity of U87MG and GBM4 glioma stem cells were tested in orthotopic xenograft models and conditional knockdown in established flank xenograft tumors. RNAseq and the analysis of tumors investigated putative TW-associated mechanisms. Multiple bioinformatic tools revealed significant alteration of ECM, membrane receptors, signaling transduction kinases, and cytoskeleton dynamics leading to identification of PI3K/AKT signaling. We experimentally show alteration of AKT activity and periostin (POSTN) expression in vivo and/or in vitro. For the first time, we show that effect of TW knockout inhibits AKT activity in U87MG cells in vivo independent of PTEN mutation. The clinical relevance of TW and candidate mechanisms was established by analysis of the TCGA and ENCODE databases. TW expression was associated with decreased patient survival and LASSO regression analysis identified POSTN as one of top targets of TW in human GBM. While we previously demonstrated the role of TW in promoting EMT and invasion of glioma cells, these studies provide direct experimental evidence supporting protumorigenic role of TW independent of invasion in vivo and the therapeutic relevance of targeting TW in human GBM. Further, the role of TW driving POSTN expression and AKT signaling suggests actionable targets, which could be leveraged to mitigate the oncogenic effects of TW in GBM.

Twist1 mediated regulation of glioma tumorigenicity is dependent on mode of mouse neural progenitor transformation.

Twist1 is a master regulator of epithelial mesenchymal transition and carcinoma metastasis. Twist1 has also been associated with increased malignancy of human glioma. However, the impact of inhibiting Twist1 on tumorigenicity has not been characterized in glioma models in the context of different oncogenic transformation paradigms. Here we used an orthotopic mouse glioma model of transplanted transformed neural progenitor cells (NPCs) to demonstrate the effects of Twist1 loss of function on tumorigenicity. Decreased tumorigenicity was observed after shRNA mediated Twist knockdown in HPV E6/7 Ha-RasV12 transformed NPCs and Cre mediated Twist1 deletion in Twist1 fl/fl NPCs transformed by p53 knockdown and Ha-RasV12 expression. By contrast, Twist1 deletion had no effect on tumorigenicity of NPCs transformed by co-expression of Akt and Ha-RasV12. We demonstrated a dramatic off-target effect of Twist1 deletion with constitutive Cre expression, which was completely reversed when Twist1 deletion was achieved by transient administration of recombinant Cre protein. Together these findings demonstrate that the function of Twist1 in these models is highly dependent on specific oncogenic contexts of NPC transformation. Therefore, the driver mutational context in which Twist1 functions may need to be taken into account when evaluating mechanisms of action and developing therapeutic approaches to target Twist1 in human gliomas.

Periostin is a novel therapeutic target that predicts and regulates glioma malignancy.

BACKGROUND: Periostin is a secreted matricellular protein critical for epithelial-mesenchymal transition and carcinoma metastasis. In glioblastoma, it is highly upregulated compared with normal brain, and existing reports indicate potential prognostic and functional importance in glioma. However, the clinical implications of periostin expression and function related to its therapeutic potential have not been fully explored. METHODS: Periostin expression levels and patterns were examined in human glioma cells and tissues by quantitative real-time PCR and immunohistochemistry and correlated with glioma grade, type, recurrence, and survival. Functional assays determined the impact of altering periostin expression and function on cell invasion, migration, adhesion, and glioma stem cell activity and tumorigenicity. The prognostic and functional relevance of periostin and its associated genes were analyzed using the TCGA and REMBRANDT databases and paired recurrent glioma samples. RESULTS: Periostin expression levels correlated directly with tumor grade and recurrence, and inversely with survival, in all grades of adult human glioma. Stromal deposition of periostin was detected only in grade IV gliomas. Secreted periostin promoted glioma cell invasion and adhesion, and periostin knockdown markedly impaired survival of xenografted glioma stem cells. Interactions with αvß3 and αvß5 integrins promoted adhesion and migration, and periostin abrogated cytotoxicity of the αvß3/ß5 specific inhibitor cilengitide. Periostin-associated gene signatures, predominated by matrix and secreted proteins, corresponded to patient prognosis and functional motifs related to increased malignancy. CONCLUSION: Periostin is a robust marker of glioma malignancy and potential tumor recurrence. Abrogation of glioma stem cell tumorigenicity after periostin inhibition provides support for exploring the therapeutic impact of targeting periostin.

Alterations in gene expression induced in day-9 mouse embryos exposed to hyperthermia (HS) or 4-hydroperoxycyclophosphamide (4CP): analysis using cDNA microarrays.

Teratogen-induced alterations in gene expression play an important role in the genesis of malformations in animals. The recent development of DNA microarrays now offers the opportunity to monitor global changes in gene expression and therefore the potential to obtain significant new information concerning both normal and abnormal development. RNA was isolated from day-9 mouse embryos at 1 and 5 h after exposure to hyperthermia (HS) or 4-hydroperoxycyclophosphamide (4CP) and compared to RNA isolated from concurrent controls using mouse cDNA microarrays. Cy5/Cy3 intensity data were extracted using Spot-on Image software and then normalized using the statistical software program R/maanova. Differentially expressed genes were identified using a linear mixed-effects model and p values derived from t-test statistics. Approximately 9000 genes show statistically significant alterations in expression in day-9 mouse embryos exposed to HS or 4CP. HS and 4CP also induce alterations in the expression of distinct sets of genes, e.g., DNA replication/repair, cell cycle, signal transduction, and transcription-related genes. As expected, a variety of heat shock genes are upregulated by HS but not 4CP. Among genes whose expression is altered by both HS and 4CP, cluster analysis identified three p53 target genes (Cyclin G1, Gtse1, and Mdm2), and follow up studies confirmed that p53 is activated in embryos exposed to these two teratogens. In addition, cluster analyses also revealed that HS but not 4CP induces the downregulation of genes encoding key enzymes in the cholesterol biosynthesis pathway. Thus, our microarray data have identified one potentially important pathway (p53) common to both HS- and 4CP-induced teratogenesis and another pathway (cholesterol biosynthesis) potentially important, but specific to HS-induced teratogenesis.

Parallel microfluidic chemosensitivity testing on individual slice cultures.

There is a critical unmet need to tailor chemotherapies to individual patients. Personalized approaches could lower treatment toxicity, improve the patient's quality of life, and ultimately reduce mortality. However, existing models of drug activity (based on tumor cells in culture or animal models) cannot accurately predict how drugs act in patients in time to inform the best possible treatment. Here we demonstrate a microfluidic device that integrates live slice cultures with an intuitive multiwell platform that allows for exposing the slices to multiple compounds at once or in sequence. We demonstrate the response of live mouse brain slices to a range of drug doses in parallel. Drug response is measured by imaging of markers for cell apoptosis and for cell death. The platform has the potential to allow for identifying the subset of therapies of greatest potential value to individual patients, on a timescale rapid enough to guide therapeutic decision-making.

Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes.

BACKGROUND: The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors. RESULTS: We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations. CONCLUSIONS: Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.