[show abstract][hide abstract] ABSTRACT: The impact of pressure waves on cells may provide several possible applications in biology and medicine including the direct killing of tumors, drug delivery or gene transfection. In this study we characterize the physical properties of mechanical pressure waves generated by a nanosecond laser pulse in a setup with well-defined cell culture conditions. To systematically characterize the system on the relevant length and time scales (micrometers and nanoseconds) we use photon Doppler velocimetry (PDV) and obtain velocity profiles of the cell culture vessel at the passage of the pressure wave. These profiles serve as input for numerical pressure wave simulations that help to further quantify the pressure conditions on the cellular length scale. On the biological level we demonstrate killing of glioblastoma cells and quantify experimentally the pressure threshold for cell destruction.
[show abstract][hide abstract] ABSTRACT: The cellular reprogramming factor LIN28A promotes tumorigenicity in cancers arising outside the central nervous system, but its role in brain tumors is unknown. We detected LIN28A protein in a subset of human gliomas observed higher expression in glioblastoma (GBM) than in lower grade tumors. Knockdown of LIN28A using lentiviral shRNA in GBM cell lines inhibited their invasion, growth and clonogenicity. Expression of LIN28A in GBM cell lines increased the number and size of orthotopic xenograft tumors. LIN28A expression also enhanced the invasiveness of GBM cells in vitro and in vivo. Increasing LIN28A was associated with down-regulation of tumor suppressing microRNAs let-7b and let-7g and up-regulation of the chromatin modifying protein HMGA2. The increase in tumor cell aggressiveness in vivo and in vitro was accompanied by an upregulation of pro-invasive gene expression, including SNAI1. To further investigate the oncogenic potential of LIN28A, we infected hNSC with lentiviruses encoding LIN28A together with dominant negative R248W-TP53, constitutively active KRAS and hTERT. Resulting subclones proliferated at an increased rate and formed invasive GBM-like tumors in orthotopic xenografts in immunodeficient mice. Similar to LIN28A-transduced GBM neurosphere lines, hNSC-derived tumor cells showed increased expression of HMGA2. Taken together, these data suggest a role for LIN28A in high grade gliomas and illustrate an HMGA2-associated, pro-invasive program that can be activated in GBM by LIN28A-mediated suppression of let-7 microRNAs.
[show abstract][hide abstract] ABSTRACT: Purpose: The concept of transplantation of neuronal cells to treat Huntington's and Parkinson's diseases is based on the proven principle that dopaminergic and GABA-ergic progenitor neurons (from the human developing ventral mesencephalon and whole ganglionic eminence) can survive, differentiate and functionally integrate into an allogenic host brain. However, several donor and host-specific variables play a major role in the safety and outcome of this procedure. In this paper, we seek to summarize an updated neural transplant transplantation protocol, based on our institutional experience and many years of collaboration with other neurotransplantation centers. Methods: We present a detailed clinical neurotransplantation protocol for Parkinson's (PD) and Huntington's (HD) diseases with special emphasis in understanding the anatomical relationships of the human fetal tissue that are relevant for selection of the desired cells population. Results: Two detailed step-wise neurotransplantation protocols are presented, outlining strategies facilitating the avoidance of possible procedure-related complications. Conclusions: In this paper we delineated some crucial technical factors enabling the execution of a safe and effective neural transplant trial. The protocols presented here might contribute to further development of the experimental clinical neurotransplantation towards a routine therapeutic procedure.
[show abstract][hide abstract] ABSTRACT: Tumor dissemination and metastatic behavior account for the vast majority of cancer associated mortality. Epithelial tumors achieve this progressive state via epithelial-to-mesenchymal transition (EMT); however, the importance of this process in the neuroepithelial context is currently very controversially discussed. The review describes the current research status concerning EMT-like changes in malignant gliomas including the role of TWIST1, ZEB1/ZEB2 and SNAIl1/SNAIl2 as inducers for cell-invasiveness in GBMs. Furthermore, WNT/β-catenin signaling with its key-component FRIZZLED4 activating an EMT-like program in malignant gliomas and its relationship to the stem-like phenotype as well as discoveries on micro-RNA-level regulating the EMT-like process are discussed.
[show abstract][hide abstract] ABSTRACT: In addition to intrinsic regulatory mechanisms, brain tumor stemlike cells (BTSCs), a small subpopulation of malignant glial tumor-derived cells, are influenced by environmental factors. Previous reports showed that lowering oxygen tension induced an increase of BTSCs expressing CD133 and other stem cell-related genes and more pronounced clonogenic capacity in vitro. We investigated the mechanisms responsible for hypoxia-dependent induction of CD133-positive BTSCs in glioblastomas. We confirmed that cultures exposed to lowered oxygen levels showed a severalfold increase of CD133-positive BTSCs. Both the increase of CD133-positive cells and deceleration of the growth kinetics were reversible after transfer to normoxic conditions. Exposure to hypoxia induced BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3)-dependent apoptosis preferentially in CD133-negative cells. In contrast, CD133-positive cells proved to be more resistant to hypoxia-induced programmed cell death. Application of the demethylating agent 5'-azacitidine resulted in an increase of BNIP3 expression levels in CD133-positive cells. Thus, epigenetic modifications led to their better survival in lowered oxygen tension. Moreover, the, hypoxia-induced increase of CD133-positive cells was inhibited after 5'-azacitidine treatment. These results suggest the possible efficacy of a novel therapy for glioblastoma focused on eradication of BTSCs by modifications of epigenetic regulation of gene expression.
Journal of Neuropathology and Experimental Neurology 11/2012; · 4.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Here we show that activation of the canonical WNT/β-catenin pathway increases the expression of stem cell genes and promotes the migratory and invasive capacity of glioblastoma. Modulation of WNT signaling alters the expression of epithelial-to-mesenchymal transition activators, suggesting a role of this process in the regulation of glioma motility. Using immunohistochemistry in patient-derived glioblastoma samples we showed higher numbers of cells with intranuclear signal for β-catenin in the infiltrating edge of tumor compared to central tumor parenchyma. These findings suggest that canonical WNT/β-catenin pathway is a critical regulator of GBM invasion and may represent a potential therapeutic target.
Cancer letters 05/2012; 325(1):42-53. · 4.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glioblastoma multiforme (GBM), as many other solid tumours, contains a subpopulation of cells termed cancer stem-like cells responsible for the initiation and propagation of tumour growth. However, a unique immunophenotype/surface antigen composition for the clear identification of brain tumour stem cells (BTSC) has not yet been found. Here we report a novel code of cell surface markers for the identification of different cell subpopulations in neurospheres derived from a GBM with a primitive neuroectodermal tumour (PNET)-like component (GBM-PNET). These subgroups differ in their CD133/CD15 expression pattern and resemble cells with different stem-like genotype and developmental pathway activation levels. Strikingly, clonogenic analysis of cultures differentially expressing the investigated markers enabled the identification of distinct subpopulations of cells endowed with stem cell characteristics. High clonogenicity could be found in CD133-/CD15- and CD133+/CD15+ but not in CD133-/CD15+ cells. Moreover, cell subpopulations with pronounced clonogenic growth were characterized by high expression of stem cell-related genes. Interestingly, these observations were unique for GBM-PNET and differed from ordinary GBM cultures derived from tumours lacking a PNET component. This work elucidates the complex molecular heterogeneity of in vitro propagated glioblastoma-derived cells and potentially contributes to the development of novel diagnostic modalities aiming at the identification of the brain tumour stem-like cell population in a subgroup of GBMs.
Folia neuropathologica / Association of Polish Neuropathologists and Medical Research Centre, Polish Academy of Sciences. 01/2012; 50(4):357-68.
[show abstract][hide abstract] ABSTRACT: Susceptibility differences among tissues were recently used for highlighting complementary contrast in MRI different from the conventional T(1), T(2), or spin density contrasts. This method, based on the signal phase, previously showed improved image contrast of human or rodent neuroarchitecture in vivo, although direct MR phase imaging of cellular architecture was not available until recently. In this study, we present for the first time the ability of microcoil-based phase MRI to resolve the structure of human glioma neurospheres at significantly improved resolutions (10 × 10 μm(2)) with direct optical image correlation. The manganese chloride property to function as a T(1) contrast agent enabled a closer examination of cell physiology with MRI. Specifically the temporal changes of manganese chloride uptake, retention and release time within and from individual clusters were assessed. The optimal manganese chloride concentration for improved MR signal enhancement was determined while keeping the cellular viability unaffected. The presented results demonstrate the possibilities to reveal structural and functional observation of living glioblastoma human-derived cells. This was achieved through the combination of highly sensitive microcoils, high magnetic field, and methods designed to maximize contrast to noise ratio. The presented approach may provide a powerful multimodal tool that merges structural and functional information of submilimeter biological samples.
Magnetic Resonance in Medicine 11/2011; 68(1):86-97. · 3.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain.
The constitutively active BRAF(V600E) allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16(INK4a) levels in pilocytic astrocytoma.
BRAF(V600E) expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAF(V600E)-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16(INK4a) whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16(INK4a) immunoreactivity in the majority of cases, but patients with tumors negative for p16(INK4a) had significantly shorter overall survival.
BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16(INK4a) expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.
Clinical Cancer Research 06/2011; 17(11):3590-9. · 7.84 Impact Factor