Gene therapy has received much attention the last decade as a method to correct a number of disorders arising from a defective gene. Gene therapy can be defined as the introduction of a functional genetic element into a cell for a therapeutic purpose. This is a very broad term and gene therapy can be applied to a wide range of diseases from genetic diseases such as cystic fibrosis to infectious diseases or even acquired genetic diseases such as cancers. Adenoviruses (Ad) are the second most common vector for gene therapy in clinical trials today, and these vectors are mostly based on serotype 2 or 5 (Ad2 and Ad5). It has been shown that Ad2 and Ad5 use a receptor that is often downregulated in malignant cells and they also suffer from shortcomings because of the high levels of pre-existing immunity against these serotypes in the society. Hence, new and improved vectors serving as alternatives to these serotypes need to be developed to make gene therapy a successful treatment option. The work presented herein is devoted to analyse what alternative adenovirus vectors could be used for treatment of glioma brain tumours. A number of different adenovirus serotypes were screened for their ability to infect human glioma tumour cells in vitro. Established cell lines as well as low-passage glioma cells from different donors were used. Adenovirus serotype 11p (Ad11p) proved to be a promising vector candidate because of its capacity to efficiently infect the glioma cells and its low prevalence in the society. The complete genome of this serotype was sequenced to further develop this as an alternative adenovirus vector. Furthermore, a number of cell lines were produced to generate E1 deleted Ad11p vectors. Other promising vector candidates were Ad16 and a chimpanzee adenovirus called CV23. Ad16 was the most efficient human serotype to infect human low-passage glioma cells and the prevalence for this serotype is also very low. The overall most efficient virus was surprisingly the non-human CV23 virus. This adenovirus has no prevalence in humans, but efficiently infects human cells in vitro. The first analysis was made on established glioma cell lines and was followed up by using low passage glioma cells from a number of different patients. The glioma cells were analysed when subjected to <20 passages (low passage) and then again at >40 passages (high passage). The cells at a higher passage number were significantly more permissive to Ad5 than the cells analysed at a low passage number. This could in part explain why some of the promising in vitro data for Ad5 have shown a limited success in vivo. In contrast, CV23 infected the low and high passage gliomas equally. This indicates that CV23 uses an internalisation mechanism subjected to less variation than the mechanism used by Ad5. We further characterised the receptor interaction of CV23 and found that none of the previously known primary receptors for adenoviruses were of importance for binding. We found that bovine serum albumin present in the growth medium was responsible for the high binding capacity to cells. Binding is a criterion for the first step of the infection, but not necessarily a good correlate to the infection capacity. CV23 infected human cells efficiently also in the absence of bovine serum albumin.

Glioblastoma multiforme (GBM) is the most common malignant central nervous system tumor. The ability of glioma cells to rapidly disperse and invade healthy brain tissue, coupled with their high resistance to chemotherapy and radiation have resulted in extremely poor prognoses among patients. In recent years, nitric oxide (NO) has been discovered to play a ubiquitous of role in human physiology and studies have shown that, at sufficient concentrations, NO is able to induce apoptosis as well as chemosensitization in tumor cells. This thesis discusses the synthesis and characterization of targeted NO donors for the treatment of GBM. Two glioma targeting biomolecules, Chlorotoxin (CTX) and VTWTPQAWFQWVGGGSKKKKK (VTW) were reacted with NO gas to synthesize NO donors. These NO donors, CTX-NO and VTW-NO, released NO for over 3 days and were able to induce cytotoxicity in a dose dependent manner in glioma cells. The biggest advantage, a result of the targeted delivery of NO, was that the NO donors did not have toxic effects on astrocytes and endothelial cells. To characterize the chemosensitizing effects of CTX-NO, cells were incubated with CTX-NO prior to exposure to temozolomide (TMZ) or carmustine (BCNU). These drugs are the most popular chemotherapeutics used in the treatment of GBM, but have only shown modest improvements in patient survival. Viability studies showed that CTX-NO selectively elicited chemosensitivity in glioma cells, whereas the chemosensitivty of astrocytes and endothelial cells remained unaffected. Further investigation showed that CTX-NO pretreatment decreased O6-methylguanine DNA methyltransferase (MGMT) and p53 levels, suggesting that a decrease in DNA repair ability may be the mechanism by which chemosensitivity is induced. Lastly, the effects of CTX-NO on glioma cell invasion and migration were studied using Boyden chamber and modified scratch assays. Non-toxic doses of CTX-NO decreased glioma cell invasion in a dose dependent manner. Studies quantifying matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) surface expression demonstrated that while MMP-2 expression was decreased by both CTX and CTX-NO, MMP-9 expression was decreased only by CTX-NO. Furthermore quantifying MMP-2 and MMP-9 activity levels showed that NO and CTX work synergistically to decrease the activity of the enzymes. These studies demonstrate that the decrease in glioma invasion resulting from CTX-NO treatment was partially a consequence of decreased levels of surface and activated MMP-2 and MMP-9. The work presented in this thesis describes a novel approach to treating GBM that can be modified to develop treatments for various other tumors. Furthermore this is the first study to develop glioma-targeting NO donors.

Photodynamic therapy (PDT) derives its tumour selectivity from preferential photosensitizer accumulation and short light penetration in tissue. However, additional strategies are needed to improve the therapeutic index of PDT in oncological applications where light is delivered interstitially to large volumes (e.g. prostate), or when adjacent normal tissue is extremely sensitive (e.g. brain). Much research to improve PDT's selectivity is directed towards developing targeted photosensitizers. Here, I present two alternative strategies to improve PDT's selectivity, without compromising its efficacy. For interstitial delivery, I investigated whether customizable cylindrical diffusers can be used to deliver light doses that conform better to target geometries, specifically the prostate. Additionally, I examined whether the neuroprotectant erythropoietin, used as an adjuvant to PDT for brain tumours, can reduce the sensitivity of normal tissue, thereby improving treatment selectivity. To determine if tailored diffusers constitute an improvement over conventional ones, I introduce a novel optimization algorithm for treatment planning. I also analyze the sensitivity of the resulting plans to changes in the optical properties and diffuser placement. These results are contextualized by a mathematical formalism to characterize the light dose distributions arising from tailored diffusers. In parallel, I investigate the neuroprotective effects of erythropoietin in PDT of primary cortical neurons in culture and normal rat brain in vivo. I show that the most important parameter determining prostate coverage is the number of diffusers employed. Moreover, while tailored diffusers do offer an improvement over conventional ones, the improvement is likely masked by perturbations introduced by the uncertainties of light delivery. Although these results largely discard the use of tailored diffusers in prostate PDT, significant insight has been gained into PDT treatment planning, and tailored diffusers may still be advantageous in more complicated geometries. Additionally, I show that erythropoietin does not improve survival of PDT-treated neurons PDT, nor reduces the volume of necrosis in vivo, for the ranges of conditions and doses studied. To our knowledge, this is the first time this strategy has been tested in brain PDT and deserves to be investigated further, by using later time-points, functional outcomes, and other neuroprotectants.

Glioblastoma multiforme (GBM) is the most aggressive and most common primary brain tumor in adults accounting for 50-60% of primary brain tumors. The prognosis for patients with GBM remains poor and treatment is mainly palliative with a mean survival time of less than one year. Radiotherapy is used extensively in the management of glioblastoma either alone or in combination with surgery and/or chemotherapy. However, this tumor is one of the most resistant tumors to radiotherapy thus limiting the benefit of this form of treatment. <p>Studies have shown that malignant tumors have a high content of glutathione an antioxidant responsible for protecting the cells against damage from free radicals (mainly superoxide, hydroxyl and hydrogen peroxide). It is well established that glutathione, by neutralizing these free radicals plays a major role in radioresistance. Glioblastoma has relatively high levels of glutathione. In this study, by reducing the glutathione content of glioblastoma in a rat model, we were able to investigate the effect of this reduction in enhancing the effect of radiotherapy as a form of treatment for glioblastoma multiforme in a rat model. <p>By injecting L-Buthionine-SR-Sulfoximine (BSO) in to the tumor tissue, the glutathione content of the tumor was reduced by about 70% of its initial value. When administered into the tumors 2 hours prior to radiotherapy the animals so treated had a significantly longer median survival time compared with animals that received radiotherapy alone.

Photodynamic therapy (PDT) derives its tumour selectivity from preferential photosensitizer accumulation and short light penetration in tissue. However, additional strategies are needed to improve the therapeutic index of PDT in oncological applications where light is delivered interstitially to large volumes (e.g. prostate), or when adjacent normal tissue is extremely sensitive (e.g. brain). Much research to improve PDT's selectivity is directed towards developing targeted photosensitizers. Here, I present two alternative strategies to improve PDT's selectivity, without compromising its efficacy. For interstitial delivery, I investigated whether customizable cylindrical diffusers can be used to deliver light doses that conform better to target geometries, specifically the prostate. Additionally, I examined whether the neuroprotectant erythropoietin, used as an adjuvant to PDT for brain tumours, can reduce the sensitivity of normal tissue, thereby improving treatment selectivity. To determine if tailored diffusers constitute an improvement over conventional ones, I introduce a novel optimization algorithm for treatment planning. I also analyze the sensitivity of the resulting plans to changes in the optical properties and diffuser placement. These results are contextualized by a mathematical formalism to characterize the light dose distributions arising from tailored diffusers. In parallel, I investigate the neuroprotective effects of erythropoietin in PDT of primary cortical neurons in culture and normal rat brain in vivo. I show that the most important parameter determining prostate coverage is the number of diffusers employed. Moreover, while tailored diffusers do offer an improvement over conventional ones, the improvement is likely masked by perturbations introduced by the uncertainties of light delivery. Although these results largely discard the use of tailored diffusers in prostate PDT, significant insight has been gained into PDT treatment planning, and tailored diffusers may still be advantageous in more complicated geometries. Additionally, I show that erythropoietin does not improve survival of PDT-treated neurons PDT, nor reduces the volume of necrosis in vivo, for the ranges of conditions and doses studied. To our knowledge, this is the first time this strategy has been tested in brain PDT and deserves to be investigated further, by using later time-points, functional outcomes, and other neuroprotectants.

Glioblastoma multiforme (GBM), the most common and malignant brain tumor, is characterized by high molecular and cellular heterogeneity within and among tumors. Parameters such as invasive growth, infiltration of immune cells and endothelial proliferation contribute in a systemic manner to maintain the malignancy. Studies in this thesis show that the expression of Sox2 is correlated with Sox21 in human gliomas. We demonstrate that an upregulation of Sox21 induces loss of proliferation, apoptosis and differentiation in glioma cells in vitro and in vivo and seems to correlate with decreased Sox2 expression. Induced expression of Sox21 in vivo significantly reduces the tumor size and increase the survival extensively, suggesting that Sox21 can act as a tumor suppressor Our studies indicate that the balance of Sox21-Sox2 in glioma cells is decisive of either a proliferative or a non-proliferative state. Several TGFß family members have an important role in glioma development. TGFß promotes proliferation and tumorigenicity whereas BMPs mostly inhibit proliferation. We demonstrate that BMP7 can induce the transcription factor Snail in glioma cells and that this reduces the tumorigenicity with a concomitant increase in invasiveness. Thus, we have identified a mechanism to the double-edged sword of proliferation versus invasiveness in GBM, the latter contributing to relapse in patients. Experimental gliomas were induced with the Sleeping Beauty (SB) model in mice with different immunological status of their T cells. The tumors that developed were either GBMs or highly diffuse in their growth, reminiscent of gliomatosis cerebri (GC). GC is a highly uncommon form of glioma characterized by extensive infiltrative growth in large parts of the brain. It is an orphan disease and today there is practically a total lack of relevant experimental models. The SB system would constitute a novel experimental model to study the mechanisms behind the development of diffusely growing tumors like GC. The presence or absence of T cells did not affect tumor development. The work in this thesis demonstrates that the proliferative and the invasive capacities of glioma cells can be dissociated and that the SB model constitutes an excellent model to study the highly proliferative cells in GBMs versus the highly invasive cells in diffuse tumors like .GC.

Glioblastoma multiforme (GBM) is the most aggressive and most common primary brain tumor in adults accounting for 50-60% of primary brain tumors. The prognosis for patients with GBM remains poor and treatment is mainly palliative with a mean survival time of less than one year. Radiotherapy is used extensively in the management of glioblastoma either alone or in combination with surgery and/or chemotherapy. However, this tumor is one of the most resistant tumors to radiotherapy thus limiting the benefit of this form of treatment. <p>Studies have shown that malignant tumors have a high content of glutathione an antioxidant responsible for protecting the cells against damage from free radicals (mainly superoxide, hydroxyl and hydrogen peroxide). It is well established that glutathione, by neutralizing these free radicals plays a major role in radioresistance. Glioblastoma has relatively high levels of glutathione. In this study, by reducing the glutathione content of glioblastoma in a rat model, we were able to investigate the effect of this reduction in enhancing the effect of radiotherapy as a form of treatment for glioblastoma multiforme in a rat model. <p>By injecting L-Buthionine-SR-Sulfoximine (BSO) in to the tumor tissue, the glutathione content of the tumor was reduced by about 70% of its initial value. When administered into the tumors 2 hours prior to radiotherapy the animals so treated had a significantly longer median survival time compared with animals that received radiotherapy alone.

<p>Malignant gliomas are highly lethal cancers characterized by florid angiogenesis. Glioma stem cells (GSCs), enriched through CD133 (Prominin1) selection, are highly tumorigenic and therapy resistance. However, the mechanism through which GSCs promote tumor growth was largely unknown. As we noticed that tumors derived from GSCs contain widespread tumor angiogenesis, necrosis, and hemorrhage, we examined thepotential of GSCs to support tumor angiogenesis. We measured the expression of a panel of angiogenic factors secreted by GSCs. In comparison with matched non-GSC populations, GSCs consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, GSC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-GSC glioma cell-conditioned medium. The proangiogenic effects of GSCs on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from GSCs but limited efficacy against xenografts derived from a matched non-GSC population. As hypoxia is a key regulator of angiogenesis, I further examined hypoxic responses in GSCs to determine the molecular mechanisms underlying their angiogenic drive. I demonstrated that multiple hypoxia response genes, including the hypoxia-inducible factors (HIFs)-1a and -2a(EPAS-1) were differentially expressed in GSCs in comparison to non-stem glioma cells and normal neural progenitors. GSCs preferentially induced HIF2a; and HIF2a-regulated genes under hypoxia in comparison to non-stem glioma cells. In contrast, neural progenitor/stem cells did not induce HIF2a in response to hypoxia suggesting that the HIF2a hypoxic response is not a general stem cell response. Targeting HIF1a or HIF2a in GSCs using short hairpin RNA (shRNA) inhibited neurosphere formation efficiency, indicating a requirement for HIFs in cancer stem cell self-renewal. HIF1a and HIF2a were also necessary for VEGF expression in GSCs, but HIF2a was not required in matched non-stem glioma cells. In vivo experiments determined that knockdown of HIFs significantly attenuated the tumorigenic capacity of GSCs and increased survival of immunocompromised mice. Together, our work provides the first evidence that that GSCs can be a crucial source of key angiogenic factors in cancers due to their differential hypoxia responses. It also suggests that anti-angiogenic therapies can be designed to target GSC-specific molecular mechanisms of neoangiogenesis, including the expression and/or activity of HIF2a.</p> / Dissertation

<p>Human cancer is a leading cause of morbidity and mortality in the developed world. Contrary to the classical model in which tumors are homogeneously composed of malignant cells, accumulating evidence suggests that subpopulations of highly malignant cells play a dominant role in tumor initiation and growth. These cells have the capacity for prolonged self-renewal and they efficiently generate tumors that phenotypically resemble the parental tumor in transplantation assays. Such characteristics are reminiscent of normal stem cells, and these potently tumorigenic cells have therefore been called cancer stem cells (CSCs). Importantly, studies have shown that CSCs are central mediators of therapeutic resistance, tumor angiogenesis, and metastatic or invasive potential. In the case of malignant glioma, poor patient survival and the paucity of effective therapeutic advances have been attributed to inherent CSC growth potential and treatment resistance, respectively. For this reason, there is great interest in elucidating the molecular features of CSCs, with the ultimate hope of developing CSC-directed therapies.</p><p>Given the overlap between the highly malignant characteristics exhibited by CSCs and those promoted by the PI3K/AKT pathway, we hypothesized that AKT activity within CSCs could represent a reasonable therapeutic target for CSC-directed therapies. Indeed, a pharmacological inhibitor of AKT preferentially targeted glioma CSCs versus non-CSCs and was associated with increased apoptosis and impaired tumorigenesis. These data suggest that interventions targeting AKT could effectively target glioma CSCs. </p><p>Quite distinct from the PI3K/AKT pathway, we hypothesized that the pro-survival and pro-growth features of nitric oxide (NO) might also operate in glioma CSCs. Our experiments found that glioma CSCs produced more NO than non-CSCs, which is attributed to inducible nitric oxide synthase (iNOS) expression and activity within the CSCs. Interference with iNOS activity or expression, as well as selective NO consumption, attenuated CSC growth and tumorigenicity. The mechanism behind iNOS-mediated survival appears to involve, at least in part, suppression of the cell cycle inhibitor CDA1. iNOS inhibition decreased glioma growth in murine xenografts and human expression studies demonstrate an inverse correlation between iNOS expression and patient survival.</p><p>To more fully evaluate the biological effects of NO in CSCs, we designed a novel strategy to consume NO within mammalian cells through heterologous expression of E. coli flavohemoglobin (FlavoHb). This enzyme is a highly specific NO dioxygenase which converts NO to inert nitrate several orders of magnitude faster than iNOS synthesizes NO. Expression of FlavoHb in mammalian cells is therefore a novel and functional tool to interrogate the role of NO in cellular stress and signaling. </p><p>In summary, this doctoral thesis focuses on several molecular characteristics that define malignant CSCs and describes a novel strategy for studying NO, which is one of the CSC-specific molecular effectors.</p> / Dissertation

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