<p>The histologic subtypes of malignant glial neoplasms range from anaplastic astrocytoma to the most deadly World Health Organization (WHO) Grade IV glioblastoma (GBM), the most common primary brain tumor in adults. Over the past 40 years, only modest advancements in the treatment of GBM tumors have been reached. Current therapies are predominantly for palliative endpoints rather than curative, although some treatment modalities have been shown to extend survival in particular cases. Patients undergoing current standard of care therapy, including surgical resection, radiation therapy, and chemotherapy, have a median survival of 12-15 months, with less than 25% of patients surviving up to two years and fewer than 10% surviving up to five years. A variety of factors contribute to standard treatment failure, including highly invasive tumor grade at the time of diagnosis, the intrinsic resistance of glioma cells to radiation therapy, the frequent impracticality of maximal tumor resection of eloquent cortical structures, and the fragile intolerance of healthy brain for cytotoxic therapies. Treatment with immunotherapy is a potential answer to the aforementioned problems, as the immune system can be harnessed and educated to license rather potent antitumor responses in a highly specific and safe fashion. One of the most promising vehicles for immunotherapy is the use of dendritic cells, which are professional antigen-presenting cells that are highly effective in the processing of foreign antigens and the education of soon-to-be activated T cells against established tumors. The work outlined in this dissertation encompasses the potential of dendritic cell therapy, the current limitations of reaching full efficacy with this platform, and the recent efforts employed to overcome such barriers. This work spans the characterization and preclinical testing of utilizing protein antigens such as tetanus-diphtheria toxoid to pre-condition the injection site prior to dendritic cell vaccination against established tumors expressing tumor-specific antigens. </p><p>Chapter 1 comprises an overview of the current standard therapies for malignant brain tumors. Chapters 2 and 3 provide a review of immunotherapy for malignant gliomas in the setting of preclinical animal models and discuss issues relevant to the efficacy of dendritic cell vaccines for targeting of GBM. Chapters 4 provides the rationale, methodology, and results of research to improve the lymph node homing and immunogenicity of tumor antigen-specific dendritic cell vaccines in mouse models and in patients with newly diagnosed GBM. Chapter 5 delineates the interactions discovered through efforts in Chapter 4 that comprise protein antigen-specific CD4+ T cell responses to induced chemokines and how these interactions result in increased dendritic cell migration and antitumor responses. Lastly, Chapter 6 discusses the future utility of migration of DC vaccines as a surrogate for antitumor responses and clinical outcomes. </p><p>This dissertation comprises original research as well as figures and illustrations from previously published material used to exemplify distinct concepts in immunotherapy for cancer. These published examples were reproduced with permission in accordance with journal and publisher policies described in the Appendix. </p><p>In summary, this work 1) identifies inefficient lymph node homing of peripherally administered dendritic cells as one of the glaring barriers to effective dendritic cell immunotherapy, 2) provides answers to overcome this limitation with the use of readily available pre-conditioning recall antigens, 3) has opened up a new line of investigation for interaction between recall responses and host chemokines to activate immune responses against a separate antigen, and 4) provides future prospects of utilizing chemokines as adjuvants for additional immunotherapies targeting aggressive tumors. Together, these studies hold great promise to improve the responses in patients with GBM.</p> / Dissertation

This thesis is focused on neural stem cell (NSC) and glioma biology. I discuss how NSCs interact with extracellular matrix (ECM) proteins in the stem cell niche, and investigate the consequences of deregulated Platelet-derived growth factor (PDGF) signaling for embryonic NSCs in transgenic mice. Furthermore I present cell cultures of human glioblastoma multiforme (GBM) that models human disease, taking into account the heterogeneity of GBM. Finally, interactions between brain tumors and mast cells are studied using the glioma cultures. In paper I, the importance of NSC interactions with the ECM in the stem cell niche during development is discussed. Contacts between NSCs and the ECM in the subventricular zone (SVZ) are emerging as important regulatory mechanisms. We show that early postnatal neural stem and progenitor cells (NSPC) attach to collagen I, and that the adhesion is explained by higher expression of collagen receptor integrins compared to adult NSPC. Further, blood vessels in the SVZ express collagen I, indicating a possible functional relationship. Growth factors, e.g. PDGF, regulate NSC proliferation and differentiation. Aberrant activation of growth factor signaling pathways also plays a role in brain tumor formation. Paper II demonstrates that transgenic mice expressing PDGF-B at high levels in embryonic NSCs displayed mild neurological defects but no hyperplasia or brain tumors. This suggests that a high level of PDGF is not sufficient to induce brain tumors from NSCs without further mutations. Paper III presents a novel panel of human glioma stem cell (GSC) lines from GBM that display NSC markers in vitro and form secondary orthotopic tumors in vivo. GBM has recently been categorized in molecular subclasses and we demonstrate, for the first time, that these subclasses can be retained in vitro by stem cell culture conditions. We have thus generated models for research and drug development aiming at a focused treatment depending on GBM subtype. Interactions with the immune system are integral parts of tumorigenesis. Mast cells are found in glioma and in paper IV we demonstrate that the grade-dependent infiltration of mast cells is in part mediated by macrophage migration inhibitory factor and phosphorylation of STAT5.

Objetivo del trabajo: Explorar la relación entre probables factores de riesgo ambientales de cáncer cerebral (gliomas) en pacientes mayores de 16 años con diagnóstico de tumor primario de cerebro que asistieron al Policlínico Neuquén durante enero del 2006 hasta octubre del 2011.

Glioblastoma, grade IV glioma, is the one of the deadliest cancers, with a median survival of only 12-15 months despite aggressive treatment including surgery, chemotherapy and radiation. One hallmark of glioblastoma is the morphological and functional abnormalization of tumor blood vessels. The molecular mechanisms involved in this process and their functional and pathological implications are not yet fully understood. Indentification of molecular mechanisms that underlie vascular abnormalization in GBM is necessary to develop efficient treatment regimens for normalizing vascular function. By analyzing the RNA-content of laser microdissected vessels from human biobank specimens using affymetrix microarray analysis, we found that the abnormal glioblastoma vessels have a distinct gene expression signature. We found 95 genes which were differentially expressed in grade IV glioma vessels as compared to vessels in low grade tumors and control brain. 78 of which were up-regulated while 17 were down-regulated. Many of these genes are regulated by VEGFA or TGFβ signaling. In addition, we show a significant increase in Smad signaling complexes in the vasculature of human glioblastoma in situ, suggesting that TGFβ signaling may play important role in vessel abnormalization. CD93 is a single-pass transmembrane glycoprotein, which we found to be up-regulated in high grade glioma. Vascular expression of CD93 correlates to tumor grade in human glioma. Moreover, high grade glioma patients with high CD93 expression in the vasculature are associate with poor prognosis. We found that knocking down CD93 in endothelial cells with siRNA clearly impaired endothelial cell adhesion, migration and tube formation due to defects in cytoskeletal rearrangement. In addition, tumor growth was severely delayed in the CD93-/- mice. Pleiotrophin, a multi-functional heparin-binding growth factor, promotes glioma growth in several ways. Here, we identify pleiotrophin as a driver of vascular abnormalization in glioma. We found that high pleiotrophin expression correlates with poor survival of patients with astrocytomas. Pleiotrophin overexpression in orthotopic GL261 gliomas increases microvessel density, enhances tumour growth and decreases survival. Vessels in pleiotrophin-expressing gliomas are poorly perfused and display a high degree of abnormality, coinciding with elevated levels of vascular endothelial growth factor (VEGF) deposited in direct proximity to the vasculature. In addition to its role in vessel abnormalization, pleiotrophin enhanced PDGF-B-induced gliomagenesis. Taken together, our results indicate that PTN has an important role in glioma initiation and establishment of the characteristic abnormal tumor vasculature in glioblastoma, identifying PTN as a potential target for therapy.

<p>Glioma development and progression are driven by complex genetic alterations, including point mutations and gain or loss of genomic copy number, as well as epigenetic aberrations, including DNA methylation and histone modifications. However, the molecular mechanisms underlying the causes and effects of these alterations are poorly understood, and improved treatments are greatly needed. Here, we report a comprehensive evaluation of the recurrent genomic alterations in gliomas and further dissect the molecular effects of the most frequently-occurring genomic events. First, we performed a multifaceted genomic analysis to identify genes targeted by copy number alteration in glioblastoma, the most aggressive malignant glioma. We identify EGFR negative regulator, <italic>ERRFI1</italic>, as a glioblastoma-targeted gene within the minimal region of deletion in 1p36.23. Furthermore, we demonstrate that Aurora-A kinase substrate, <italic>TACC3</italic>, displays gain of copy number on 4p16.3 and is overexpressed in a grade-specific pattern. Next, using a gene targeting approach, we knocked-in a single copy of the most frequently observed point mutation in gliomas, <italic>IDH1<super>R132H/WT</super></italic>, into a human cancer cell line. We show that heterozygous expression of the <italic>IDH1<super>R132H</super></italic> allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Together, these data provide insight on genetic and epigenetic alterations which drive human gliomas.</p> / Dissertation

According to the cancer stem cell hypothesis a subpopulation of cells within a tumour has the capacity to sustain its growth. These cells are termed cancer stem cells, and are most simply defined as the cells within a primary tumour that can self-renew, differentiate and regenerate a phenocopy of that cancer when transplanted in vivo. Cancer stem cells have now been prospectively identified from numerous human tumours and are actively sought in many cancer types, both clinical and experimental. The cancer stem cell hypothesis remains controversial, with evidence both supporting and challenging its existence in human tumours and in animal models of disease. Here we prospectively identify and study brain cancer stem cells in clinically representative mouse models of the medulloblastoma and glioma. Cancer stem cells from both mouse brain tumour types are prospectively enriched by fluorescent activated cell sorting freshly dissociated cells for the surface antigen CD15, display a neural precursor phenotype, exhibit the hallmark stem cell characteristics of self-renewal and multilineage differentiation, and regenerate a phenocopy of the original tumour after orthotopic transplantation. Additionally, novel mouse medulloblastoma and glioma cancer stem cell lines were established and studied in vitro as adherent cultures in the same serum-free media conditions that support the growth of normal neural stem cells. When mouse and human glioma stem cell lines were compared, many novel molecular mediators of the tumour phenotype were identified, as were chemical compounds that selectively inhibit their growth. Our results have important implications regarding the cancer stem cell hypothesis, the mechanisms that drive brain tumour stem cell growth and the therapeutic strategies that may prove effective for the treatment of glioma and medulloblastoma.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Neurology and Neurosurgery. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 105-122).