Browsing by Subject "Glioblastoma"
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Item Comparative and molecular approaches to improve identification, classification, and therapeutic approaches to cancer(2013-01) Frantz, Aric M.A major area of contemporary research in cancer is focused on improving tumor classification into clinically relevant subgroups of disease. To achieve this, it is important to understand the molecular events that driver tumor heterogeneity both at the cellular level and at the tissue level. I initially tested the hypothesis that canine lymphoma is composed of a group of molecularly distinct entities with prognostic significance. The results show that canine lymphoma can be stratified into molecular subgroups that have prognostic value and can assist to guide therapy. Next, I tested the hypothesis that canine hemangiosarcoma (HSA) is organized hierarchically with a cancer stem cell (CSC)-like population of cells at the apex. The data show that variable numbers of CSC-like cells are invariably present in HSA. These CSC-like cells retain the capacity to differentiate into vascular, inflammatory, or adipogenic tissue, suggesting that their multipotency is a contributing factor to the observed heterogeneity in this disease. Finally, I tested the hypothesis that CSCs, or CSC-like cells from three histologically distinct types of canine cancer (HSA, osteosarcoma, and glioblastoma) share molecular and functional properties. Using a system that allowed me to eliminate tumor-specific culture conditions, I showed that despite extensive heterogeneity in CSC-like cells from these tumors, they all showed reduced activity of pathways associated with proliferation and development. In summary, my results confirm that cellular heterogeneity exists both within and among tumors. A better understanding of the mechanisms that drive this will improve patient stratification and guide efforts to develop rational, more effective therapies.Item Crosstalk Between Adhesion Molecules Influences Cell Traction and Migration(2023-07) Kelly, MarcusCell migration is the major driver of invasion and metastasis during cancer progression. For cells to migrate, they utilize the actin-myosin cytoskeleton and adhesion molecules, such as integrins and CD44, to generate traction forces in their environment. Whereas CD44 primarily binds to hyaluronic acid (HA), integrins primarily bind to extracellular matrix proteins (ECM) such as collagen. However, the role of CD44 under integrin-mediated conditions, and vice versa, is not well known. Here we used TFM to assess the functional mechanical relationship between integrins and CD44. Performing TFM on integrin-mediated adhesion conditions, i.e., collagen, we found that CD44KO U251 cells exerted more traction force than wild-type (WT) U251 cells. When using untreated WT and CD44-blocked WT, we observed comparable results with CD44KO cells again showing an increase in traction force on collagen gels. Conversely, in CD44-mediated adhesive conditions, integrin-blocked WT cells exerted higher traction force than untreated WT cells. Our data suggests that CD44 and integrins have a mutually antagonistic relationship where one receptor represses the other’s ability to generate traction force on its cognate substrate.Item Directing T Cells with Chemically Self-Assembled Nanorings as an Immunotherapy for Targeting Hematological Malignancies and Solid Tumors(2021-12) Mews, EllieEngineering cell-to-cell interactions has proven to be quite valuable due to the vast number of therapeutic applications that benefit from this technology. Although genetically engineering artificial receptors onto a patient’s cells has shown some success, preparation is costly, current applications are limited, and modifications are permanent. To address some of these concerns our lab has developed a non-genetic approach to facilitate selective cell-to-cell interactions with chemically self-assembling nanorings (CSANs). We have shown that functionalizing the CSAN construct with cancer antigen targeted protein scaffolds and a T-cell targeted single chain antibody fragment (?CD3 scFv) forms a mixture of bispecific nanorings that facilitate T cell interactions with the tumor. In this dissertation, the concept of directing T cell activity with bispecific CSANs is initially validated against CD19+ B cell lymphoma following the production and characterization of an ?CD19-DHFR2 fusion protein monomer. Previous work has demonstrated that the CSAN platform can also be used to target overexpressed tumor associated antigens on solid tumors; therefore, the remainder of this dissertation details the application of bispecific CSAN directed T cells against established brain tumors.Few therapeutic options are available for treating central nervous system (CNS) solid tumors, especially upon recurrence. Recent preclinical studies have shown promising results for eradicating various solid tumors by targeting the overexpressed immune checkpoint molecule, B7-H3. However, due to several therapy-related toxicities and reports of tumor escape, the full potential of targeting this pan-cancer antigen has yet to be realized. Here, we designed and characterized bispecific CSANs that target the T cell receptor, CD3ε, and tumor associated antigen, B7-H3. Two different B7-H3 targeted proteins were incorporated into the CSAN scaffold, a single chain variable fragment (scFv) derived from the 8H9 monoclonal antibody and an affibody that was affinity matured via yeast display technology. We show that both B7-H3 targeted protein scaffolds form bispecific nanorings with the ?CD3 monomer to increase T cell infiltration and facilitate selective cytotoxicity of B7-H3+ medulloblastoma cells. Additionally, ?B7-H3-?CD3 CSANs directed robust T cell responses against preclinical models of established medulloblastoma and glioblastoma tumors. Furthermore, the combination of ?EGFR-?CD3 CSANs and ?B7-H3-?CD3 CSANs further improved the anti-tumor immune response in these models, suggesting therapeutic synergism between EGFR and B7-H3. Intraperitoneal (IP) injections of ?B7-H3-?CD3 bispecific CSANs were found to effectively cross the blood-tumor barrier into the brain and elicit significant anti-tumor T cell activity intracranially as well as systemically in an orthotopic medulloblastoma model. Moreover, following treatment with ?B7-H3-?CD3 CSANs, intratumoral CD4+ and CD8+ T cells were found to primarily have a central memory phenotype that displayed significant levels of characteristic activation markers. Importantly, we demonstrate that the bispecific CSAN directed T cell cytotoxicity is not dependent on MHC class I interactions with target cells, suggesting that downregulation of MHCI expression as an immune evasion mechanism would not affect CSAN directed anti-tumor activity. Furthermore, due to the modularity of the nanorings, non-specific T cell activation against the ONS 2303 medulloblastoma cell line can be reduced by tuning the valency of the ?CD3 targeted monomer in the oligomerized CSAN. Collectively, these results demonstrate the ability of our multi-valent, bispecific CSANs to direct potent anti-tumor T cell responses and indicate its potential utility as an alternative or complementary therapy for immune cell targeting of B7-H3+ brain tumors.Item Immunological benefits of a novel polycaprolactone-polyorthoester-based therapeutic vaccine in a mouse model of glioma(2014-08) Grinnen, Karen LynnCancer immunotherapy has led to significant improvement in the survival of patients with previously untreatable malignancies. The use of therapeutic vaccines is a promising form of immunotherapy, but their efficacy remains ambiguous. Much of the difficulty in identifying the optimal formulation and delivery is related to the complicated nature of the immune response, where it is uncertain which aspects would be most effective in destroying cancer cells. In this thesis, a novel polymeric delivery system, involving poly (caprolactone)-co-poly (ortho ester) [PCL-POE], was used to deliver tumor antigens and adjuvants in a controlled manner. We hypothesized that persistent release of tumor antigens from the biodegradable polymer would result in an increase in the number and persistence of anti-tumor lymphocytes in the effector state. To test this hypothesis, vaccines were administered to mice and the time dependent immunological response was evaluated. The polymeric delivery system resulted in an in vitro release profile characterized by a burst release of both antigen and adjuvant followed, in both cases, by a much slower phase of release. We also observed that the slow release provided by the PCL-POE polymer stimulated prolonged maturation of dendritic cells, activation and persistence of anti-OVA antibodies and antigen-specific T cells following a single vaccination. The vaccine system was also tested in a mouse model of glioblastoma multiforme (GBM). We observed a significant, potentially translatable increase in overall survival.Item Improving delivery of molecularly targeted agents to glioma.(2011-06) Agarwal, Sagar SureshTreatment 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.Item Improving The Delivery Of Novel Molecularly-Targeted Therapies For The Treatment Of Primary And Metastatic Brain Tumors(2019-01) Gampa, GauthamTumors in the brain are challenging to diagnose and are associated with poor survival outcomes. Brain tumors are difficult to treat, in part, due to restricted drug delivery across the blood-brain barrier (BBB). Although the BBB is compromised in some regions of brain tumors, the degree of disruption is not uniform and certain tumor locations have a functionally intact BBB. A critical component of BBB that restricts entry of therapeutics into brain is active efflux. The objective of this work is to examine brain distribution of novel molecularly-targeted therapies, including evaluation of influence of P-gp and Bcrp-mediated efflux at the BBB, assessment of spatial heterogeneity in drug distribution to brain tumors, and comparison of unbound (active) drug exposures with in vitro efficacy. Ispinesib is a KIF11 inhibitor that inhibits both tumor proliferation and invasion in glioblastoma (GBM). We demonstrate that ispinesib has a limited brain delivery due to efflux by P-gp and Bcrp, and ispinesib delivery is heterogeneous to areas within a tumor in a GBM model. Furthermore, predicted unbound-concentrations in brain were less than in vitro cytotoxic concentrations, suggesting that delivery may limit in vivo efficacy. Also, pharmacological inhibition of efflux transport (elacridar co-administration) improves brain delivery of ispinesib, and future studies will evaluate if enhanced delivery will improve efficacy. CCT196969, LY3009120 and MLN2480 are panRAF inhibitors with minimal paradoxical activation of MAPK pathway and may overcome resistance observed with BRAF inhibitor therapy in melanoma. MEK inhibition is used in combination with BRAF inhibitors to delay resistance. E6201 is a potent MEK inhibitor with a unique macrocyclic structure. While brain distribution of panRAF inhibitors is limited by efflux, E6201 has a favorable brain distribution profile and interacts minimally with P-gp and Bcrp. The delivery of E6201 is variable to regions of tumor in an intracranial melanoma model. However, predicted unbound-concentrations in brain achieve levels higher than in vitro cytotoxic concentrations for LY3009120 and E6201, suggesting possible efficacy in melanoma brain metastases. Future studies evaluating in vivo efficacy in preclinical models will reveal the utility of selected compounds in brain tumor treatment, and if improved delivery translates to superior efficacy.Item Molecular Mechanisms Underlying the Failures of Therapeutics in the Treatment of Malignant Glioma(2016-05) Becker, ChaniGlioblastoma 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.Item Permeability, binding and distributional kinetics of Ponatinib, a multi-kinase inhibitor: implications for the treatment of brain tumors(2018-01) Laramy, JaniceGlioblastoma (GBM) is the most common malignant brain tumor and one of the unmet medical needs. Among over 1,000 GBM clinical trials testing molecularly-targeted agents, no single agent has demonstrated drastic improvement in patient survival, in part due to manifold drug delivery challenges to the brain tumor. Advances in genomic and proteomic technologies have identified numerous oncogenic targets, such as mutated or amplified receptor tyrosine kinase pathways, which have enabled proteomic-guided drug selection for the treatment of GBM. Despite these advances, drug discovery and development for the treatment of GBM is still complexed by the challenges that are unique to the brain tumor that resides behind the blood-brain barrier (BBB), a formidable barrier for reaching therapeutic drug concentration in the brain tumor. Many molecularly-targeted drugs that have been examined for the treatment of GBM are a substrate of two highly expressed BBB efflux transporters, P-glycoprotein (P-gp) and Breast cancer resistance protein (Bcrp). This dissertation examined the multiple drug delivery challenges, including central nervous system (CNS) penetration, binding, and distributional kinetics and the implications on drug efficacy and/or toxicity, for a tyrosine kinase inhibitor (ponatinib) that can serve as a case example.Item Physical Determinants of Glioma Cell Migration and Disease Progression(2015-09) Klank, RebeccaGlioblastoma (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.Item Unraveling the Signaling Pathways of the Cd200 Activation Receptor Family and Their Implications in Regulating Antitumor Response in Glioblastoma(2020-05) Ampudia Mesias, ElisabetGlioblastoma multiforme (GBM) is the most aggressive and incurable primary brain tumor with a current median overall survival of approximately 14 months. Immune checkpoint-based therapy has demonstrated successes in solid tumors including melanoma and lung cancer increasing overall survival, however, it has not been successful in combating Central Nervous System (CNS) tumors. Our studies seek to establish a successful checkpoint inhibitor-based immunotherapy model for treating GBM, and our central hypothesis is, synthetic ligands modulate CD200 activation receptors (CD200ARs) overriding the inhibitory effect mediated by CD200 binding to CD200IR. The CRISPR/Cas9 system was used to generate different murine raw264.7 macrophages (MØs) cell lines expressing different combinations or a single CD200 receptor. The resultant cell lines were stimulated with the synthetic ligand, and the effects of this binding were studied. The main achievements of this research were to demonstrate that CD200ARs stimulated by synthetic peptide-binding couples with DAP10, and stimulates downstream activation of phosphatidylinositol 3-kinase, Vav1, cJUN, and ERK1/2. Second, CD200ARs form complexes (CD200ARs 2&3) to interact with the peptide ligands to optimize the biological function of macrophages. Third, the signals initiated by CD200ARs/DAP10 induce cytokine secretion and immune activation that results in tumor control. Our research reveals the signaling pathway of the CD200 immune checkpoint that leads to activation rather than suppression of immune cells and improves the response of GBM to vaccine-based immunotherapy.Item Zika Virus Oncolytic and Tumor Vaccine Adjuvant Immunotherapy Treatment of Murine Glioblastoma GL261(2017-12) Sipe, ChristopherGlioblastomas (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.