DEVELOPMENT AND CHARACTERIZATION OF A NEW GLIOBLASTOMA MULTIFORME MODEL FOR INVESTIGATION OF TUMOR CELL MOTILITY

GBM;organotypic;oncosphere;glioma;Chemical & Biomolecular Engineering

Binder, Zev A.Pardo-Villamizar, Carlos A. ;

Johns Hopkins University

关键词: GBM;    organotypic;    oncosphere;    glioma;    Chemical & Biomolecular Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39272/BINDER-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Glioblastoma multiforme (GBM) is the most common adult primary malignant brain tumor, with incidence peaking in the 5th and 6th decades of life.With a median survival of only 14.6 months despite aggressive surgery, radiotherapy and chemotherapy, this tumor strikes down patients in the middle of their productive years.The invasive capacity of GBM cells plays a key role in the devastation that they cause.At the time of diagnosis, tumor cells have already invaded away from the primary tumor mass, making curative resection an impossibility. Unfortunately, there is a paucity of clinical advancements in the treatments of GBMs, reflecting a translational gap between basic science and clinical application. One major contributor to this gap is the fact that the models currently used to study GBM invasion are markedly limited.In this work, I describe the development of a novel model that combines patient-derived suspension cell lines with human organotypic brain slices.This more pathophysiologically accurate model may serve as an alternative to the traditional models and may therefore address some of their limitations. GBM oncosphere cell lines are suspension cell lines that have demonstrated the ability to retain crucial genotypic and phenotypic characteristics of the primary brain tumors.Close collaboration with neurosurgeons at the Johns Hopkins Hospital allowed me to rapidly collect tissue specimens from brain tumor surgeries.Tissue samples were dissociated into single cell suspensions and then cultured. The use of specific culturing conditions and medium supplemented with growth factors allowed for the establishment of numerous suspension GBM oncosphere cell lines.Genetic and phenotypic characterization of these cell lines demonstrated close matches to the establishing, primary tumors.To address limitations in the culturing environment, I devised a method for using human organotypic brain slices as the scaffold for the study of tumor cell motility and invasion.Organotypic brain slices provide a physiologic environment with the appropriate extracellular matrix (ECM) and cell types for the GBM tumor cells to interact with, therefore resulting in more pathologically appropriate data.In addition to exposing GBM cells to physiologic ECM and cell types, organotypic brain slices immerse the tumor cells in a three dimensional environment, a crucial element found in pathology and animal models but missing in traditional two dimensional in vitro cell culture.Combination of the suspension GBM oncosphere cell lines and the physiologically-relevant environment of the human organotypic brain slices provides a model system with advantages taken from both in vitro and in vivo models.Similar to in vitro studies, organotypics can be studied in a dynamic setup.Using an incubated and humidified chamber on a confocal microscope, long-term imaging of tissue can be conducted.Manipulation of the tissue and implantation of tumor cells is relatively simple and can be performed precisely.Similar to in vivo work, organotypics provide a more complex and physiologically relevant environment for tumor cell growth.Organotypics can also be imaged for multiple days, whereas animals are unable to be kept under anesthesia for that long.To demonstrate the validity of the new model system, two cell surface proteins were knocked down and their effects on cell motility quantified. Integrins, cell-surface proteins, are an integral component of cell movement and were chosen because there has been recent clinical interest in integrins as new therapeutic targets for GBMs.In particular, cilengitide, a specific αvβ3 integrin inhibitor, recently completed phase III trials for primary GBMs.The second protein chosen for investigation was CD44, a cell surface, hyaluronic acid-binding protein.The main component of brain ECM is hyaluronic acid, which is not recognized by any known integrin combinations but does serve as a ligand for CD44.Integrin αv (ITGAV) depletion showed no significant effect on overall cell motility while CD44 depletion resulted in significantly decreased population-averaged instantaneous velocities.Cell behavior was also compared on Matrigel-coated plates to in human brain tissue as well as in mouse brains to in human brains.These results demonstrated the impact of the microenvironment on assay outcomes. The results presented in this work provide evidence that use of GBM oncosphere cell lines coupled with human organotypic brain slices is a viable model for study of GBM cell motility and invasion.Mechanistic insight into GBM invasion and integrin function stemming from the described work will lead to new scientific and clinical advancements. A greater understanding of the function of integrins and CD44 in cell motility and invasion will help elucidate the invasion process of GBMs.This will not only increase our basic science knowledge of these tumor cells but also provide new avenues for clinical treatment of GBMs.