Browsing by Subject "Glioma"

Now showing 1 - 6 of 6
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    Improving delivery of molecularly targeted agents to glioma.
    (2011-06) Agarwal, Sagar Suresh
    Treatment of glioblastoma multiforme is at a crossroads. Promising new molecularly-targeted agents have failed to show any significant clinical benefit. Treatment is particularly challenging since the tumor resides in a tissue that is considered to be a pharmacological and immunological sanctuary due to the presence of the blood-brain barrier. Protective mechanisms at the blood-brain barrier (BBB), such as the endothelial tight junctions and drug efflux transporters, restrict the passage of most large and small molecules into the brain. Limited drug delivery to the tumor is a plausible explanation for the failure of molecularly-targeted therapy in glioma. If therapeutic agents do not reach their target, regardless of their potency, they cannot be effective. The objective of this work was to show that active efflux transporters at BBB restrict delivery of potent molecularly-targeted agents to their targets. More importantly, the aim was to demonstrate that the targets in question are in invasive tumor cells that are left behind after surgery and remain shielded behind an intact blood-brain barrier. The ultimate goal of this endeavor is to improve delivery of molecularly-targeted therapy to the tumor and show that this can translate to enhanced efficacy against this lethal disease. We show that brain distribution of the tyrosine kinase inhibitors, gefitinib, erlotinib and sorafenib, is restricted due to active efflux mediated by p-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP). We further demonstrate that delivery of these drugs to the brain increases dramatically when the two transporters are genetically absent or pharmacologically inhibited. Using a rat xenograft model and a spontaneous mouse model of glioma, we show that the BBB is heterogeneously disrupted in the brain. The blood-brain barrier is disrupted in the tumor core resulting in high tumoral concentrations of erlotinib and dasatinib. However, it is intact in areas immediately adjacent to the tumor, and therefore restricts drug delivery to these sites. Thus, clinical assessment of drug delivery when using drug concentrations in tumor core (the resected tissue) as a guide for the adequacy of drug delivery can be misleading. Furthermore, we show that increasing drug delivery to these areas, by genetic deletion or pharmacological inhibition of P-gp and BCRP, results in a remarkable enhancement in efficacy of the tyrosine kinase inhibitor, dasatinib. Finally, we show that efficacy of dasatinib increases dramatically in tumor bearing transgenic mice, that are deficient in P-gp and BCRP, and consequently, these mice survive for a significantly longer time compared to the wild-type mice. These observations underline that restricted delivery of molecularly-targeted agents to their targets can be a significant determinant of drug efficacy against glioma. In an invasive tumor, such as glioblastoma, it is important to realize that the target resides within the invasive glioma cells, that remain shielded by an intact blood-brain barrier, and evade chemotherapy. Overall, this work highlights the need to develop strategies to improve drug delivery to the invasive tumor in glioma and translate these strategies to the clinic.
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    Molecular Mechanisms Underlying the Failures of Therapeutics in the Treatment of Malignant Glioma
    (2016-05) Becker, Chani
    Glioblastoma multiforme (GBM) is a lethal cancer. Without treatment, patients diagnosed with this disease survive nine months. With the best therapeutics science has to offer, including surgical resection, radiation therapy, and temozolomide, patients survive only five more months. Despite numerous clinical trials, the vast majority of tested drugs fail to provide therapeutic benefit to patients. It was the intent of this thesis to characterize the molecular mechanisms that prevent or limit the efficacy of targeted agents against malignant glioma. This work specifically explores how the internal characteristics of the tumor including its invasiveness and genetic heterogeneity as well as external attributes of therapeutic agents including brain penetrance contributes to the chemotherapeutic failure in GBM. By clarifying the biological processes that constrain treatment of this disease, scientists can strategize the development of better therapeutics with greater likelihoods for clinical success. We compared the brain distribution, molecular targeting efficiency, and survival benefit of GDC-0980 and GNE-317, two PI3K/mTOR inhibitor analogues. We showed that GDC-0980 is liable for efflux by P-glycoprotein (Pgp) and Breast Cancer Resistance Protein (BCRP) at the blood-brain barrier (BBB) while GNE-317 remains relatively resistant to efflux. Because GNE-317 is more brain penetrant than GDC-0980, it showed greater accumulation in the brain and stronger ability to impede the activation of PI3K/mTOR pathways in the GL261 mouse glioma model. Unexpectedly, neither drug affected survival, an effect that underscores the challenges presented by the genetic heterogeneity associated with cancer and the consequences of inadequate target selection. We also sought to determine the influence of anti-angiogenic therapy (AAT) on the delivery and efficacy of concurrently administered targeted agents. Again, we used GDC-0980 and GNE-317 to determine whether susceptibility to efflux impacted these parameters. We demonstrated that the vascular endothelial growth factor (VEGF) monoclonal antibody, bevacizumab (Avastin) could decrease the brain distribution of GDC-0980, although not significantly, but had no effect on the brain accumulation of GNE-317. We further showed that while bevacizumab alone provided a survival benefit in patient-derived glioma xenograft models, this therapeutic benefit could only be enhanced with co-treatment of a brain-penetrant drug like GNE-317. Collectively, these data suggest that AAT-induced BBB normalization is more likely to limit the delivery of targeted agents that are subject to active efflux. Finally, we examined the therapeutic potential of targeting cancer stem cells (CSCs) through experiments with parthenolide and LC-1 in the GL261 mouse glioma model. Effectively killing CSCs is an important goal in brain tumor research because this cell population is thought to responsible for tumor growth and recurrence, and is known to be particularly resistant to chemotherapies. In vitro studies of parthenolide and LC-1 in multiple glioma cell lines demonstrated that both drugs exhibited similar cytotoxicity profiles and were able to induce total cell death. LC-1 was also shown to be brain penetrant and non-toxic after prolonged exposure, and produced a demonstrable delay in tumor growth and a significant survival benefit. For these reasons, glioma stem cells remain a compelling therapeutic target for future clinical therapies.
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    Overcoming Obstacles to Glioma Immunotherapy
    (2014-04) Litterman, Adam
    Glioma is a type of malignant tumor of the non-neuronal cells of the central nervous system, the glia. These tumors are the most common malignant tumors of the central nervous system. The most aggressive and most prevalent of these, glioblastoma multiforme (GBM) is a deadly disease with a grim prognosis, with median survival at diagnosis of less than a year and a half. Standard treatment with irradiation and the DNA alkylating drug temozolomide yields incremental improvement in survival over irradiation alone but better therapies remain needed. Immune therapies are an emerging class of therapies that have shown great promise in the treatment of hematopoietic malignancies and solid tumors. These therapies harness the capability of the immune system to target and kill large numbers of tumor cells specifically, and it is has been suggested that most or all durable responses to treatment of solid tumors involve generation of an anti-tumor immune response. Several anecdotal reports of dramatic responses in GBM patients after receiving cancer vaccines (a type of immune therapy) suggest that immune therapies for glioma could yield substantial increases in survival of patients with these tumors. However, the overall record of vaccines for the treatment of this disease has been marked by failure, and substantial barriers remain to the implementation of other types of immune therapies in glioma patients. Several mechanisms by which tumors in general, and brain tumors in particular, evade the activity of the immune system have been outlined. These include accumulation of immune suppressive cell types, tumor intrinsic changes that directly suppress the activity of infiltrating immune cells, and brain specific mechanisms of immune privilege. While these mechanisms are doubtless operative in many cases, accumulating evidence from clinical trials of adoptive transfer of T cells demonstrate that the accumulation of sufficient numbers of tumor-specific T lymphocytes at the tumor site can result in an overwhelming anti-tumor immune response and associated durable clinical responses. Therefore, my research over the past several years has focused on clinically relevant mechanisms in glioma patients that present obstacles to the development of a robust T cell mediated anti-tumor immune response. In this thesis, I outline experiments performed to understand and develop strategies for overcoming two obstacles to expanding large numbers of tumor specific cytolytic T lymphocytes in glioma patients: the anti-proliferative effect of the alkylating drug temozolomide on in vivo T cell expansion by cancer vaccination, and the differentiated phenotype of ex vivo expanded T cells for adoptive immunotherapy that is associated with diminished proliferative potential in vivo. A focus in these experiments is the targeting of tumors with T cells that are specific for antigenic determinants derived from tumor-specific mutations. Engineered T cell responses targeting individual patient-specific mutations may someday lead to significant improvements in the efficacy of immune therapy for glioma, and ultimately to improved outcomes for patients with these malignancies.
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    Physical Determinants of Glioma Cell Migration and Disease Progression
    (2015-09) Klank, Rebecca
    Glioblastoma (GBM) is a highly aggressive brain cancer (generally, “glioma”) with poor patient prognosis, even with current standard treatments. In order to rationally develop novel treatments that can significantly extend patient survival, we must first understand at a basic scientific level how the disease progresses. GBM is thought to be fatal due to highly invasive cells that migrate beyond the visible bulk tumor and lead to tumor recurrence after therapeutic intervention. Therefore, we sought to investigate what makes GBM cells invasive at the single-cell level (Chapter 1). Using a genetically induced mouse glioma model and confocal imaging of intact tumor-containing brain slices, we found that, consistent with previous biophysical models, glioma cell migration is biphasic with respect to the concentration of the transmembrane cell adhesion molecule CD44. By contrast, cell proliferation is independent of CD44 level. Additionally, mouse model and human patient survival are also biphasic with respect to CD44 level, with poorest prognosis occurring at intermediate CD44 levels. Thus, migration and survival are both biphasic and are anti-correlated to each other, suggesting that CD44-dependent migration directly affects survival outcomes. We next investigated how these single-cell behaviors impact overall tumor growth and progression (Chapter 2). Noticing that previous models of GBM migration use parameter values for migration rate (defined by a diffusion coefficient, also known as a random motility coefficeint) that are much higher than our measurements of single-cell migration behavior in Chapter 1, a Brownian dynamics (BD) approach was used to simulate single-cell growth, proliferation, and migration, and compare model assumptions. These studies showed that employing the physically-based assumption that tumor cells occupy volume, an assumption not captured in current reaction-diffusion (RD) simulations, resulted in increased tumor spreading behavior with the same input parameters. Specifically, non-overlapping cells can enter a jammed regime where interior cells are subdiffusive, and peripheral cells become biased outward and superdiffusive in a quasi-ballistic expansion. Thus, we show that, when we account for volume conservation, the relatively low values of diffusion coefficient, such as what was measured in Chapter 1, can generate fast progressing tumors that are similar to RD simulations which use diffusion coefficients much greater than what is observed experimentally for single migrating cells. Therefore, we suggest that cellular jamming behavior contributes to the fast spreading of GBM tumors, and that subsequent simulations of GBM growth should incorporate this assumption so that models are physically grounded and achieve consistency between single-cell behavior and bulk tumor progression. Overall, these studies demonstrate the potential importance of fundamental physical effects in driving tumor progression generally, and glioblastoma specifically.
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    A spontaneous murine model for the study of CD44 In glioma progression.
    (2012-09) Decker, Stacy Ann
    Mouse models of malignant glioma that accurately recapitulate the genetic and phenotypic heterogeneity are essential for advancing brain tumor therapeutics. We have developed a novel model using the Sleeping Beauty transposable element to achieve chromosomal integration of human oncogenes into endogenous brain cells of any mouse strain. The phenotype of these genetically engineered brain tumors is influenced by the combination of oncogenes delivered, but many of the pathological features of malignant human glioma are present in the majority of cases. At least five different genes can be cotransfected simultaneously, including reporters that allow measurement of tumor progression. The flexibility of this model enabled studies on the role of the microenvironment in brain tumor development. The data presented here demonstrate that glioma cells make an abundance of CD44 and HA starting early in tumor development and continue to make both receptor and ligand as gliomas progress to highly invasive disease. We show that CD44-/- tumors progressed significantly slower than CD44+/+ tumors, but that expression of full length CD44 within tumors restores the CD44-/- tumor progression. In addition, we show that CD44 loss of function caused severe impairment in single cell motility. The data presented here suggest that HA-engaged CD44 mediates tumor progression by facilitating glioma cell invasion. These studies highlight the therapeutic potential for targeting the infiltrative glioma population through antagonists of the HA-CD44 complex.
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    Zika Virus Oncolytic and Tumor Vaccine Adjuvant Immunotherapy Treatment of Murine Glioblastoma GL261
    (2017-12) Sipe, Christopher
    Glioblastomas (GBMs) are highly aggressive brain tumors with a five-year survival rate of <5% upon diagnosis [1]. Current treatments have become routine, with little improvement in survival the last ten years which has opened the door for experimentation with oncolytic and immunotherapies. The Zika virus (ZIKV) is a relatively asymptomatic Flavivirus which infects and kills fetal neural stem cells (fNSCs) [45], neural progenitor cells (NPCs) [46], and induced pluripotent stem cell (iPSC) derived neurospheres [47] through apoptosis and autophagy. Current literature implicates Tyro3, Axl, TIM-1, and DC-SIGN as putative ZIKV entry receptors [51]. Here, an in vitro characterization of murine GBM cell line GL261 was carried out examining the presence of these putative receptors as well as their susceptibility to viral infection. The presence of Tyro3 and Axl RNA was confirmed by qRT-PCR and RNA-Seq although their role in ZIKV infection is still undetermined. GL261 cells were found to potentially become infected by ZIKV as shown by viral RNA presence in cells although a Plaque Forming Assay (PFA) was mostly negative indicating viral replication and cell death may not be occurring. After implanting GL261 tumors into mice and treating different groups with varying concentrations of ZIKV, it was found that ZIKV did not improve survival, potentially confirming the results found through the PFA. Treatment of tumor implanted mice with an irradiated tumor vaccine previously infected with ZIKV, GM-CSF, and ZIKV directly into the tumor site did dramatically improve overall survival. The working hypothesis of increased survival is that of a powerful immune response as shown by these effects disappearing after using SCID mice with no immune system. Confirmation is currently ongoing through additional experimentation.