Radiation treatment planning is a process through which a certain plan is devised in order to irradiate tumors or lesions to a prescribed dose without posing surrounding organs to the risk of receiving radiation. A plan comprises a series of shots at di erent positions with di erent shapes. The inverse planning approach which we propose utilizes certain optimization techniques and builds mathematical models to come up with the right location and shape, for each shot, automating the whole process. The models which we developed for PerfexionTM unit (Elekta, Stockholm, Sweden), in essence, have come to the assistance of oncologists in automatically locating isocentres and de ning sector durations. Sector duration optimization (SDO) and sector duration and isocentre location optimization (SDIO) are the two classes of these models. The SDO models, which are, in fact, variations of equivalent uniform dose optimization model, are solved by two nonlinear optimization techniques, namely Gradient Projection and our home-developed Interior Point Constraint Generation. In order to solve SDIO model, a commercial optimization solver has been employed. This study undertakes to solve the isocentre selection and sector duration optimization. Moreover, inverse planning is evaluated, using clinical data, throughout the study. The results show that automated inverse planning contributes to the quality of radiation treatment planning in an unprecedentedly optimal fashion, and signi cantly reduces computation time and treatment time.

Ionizing radiation induces specific proteins involved in DNA repair, cell death, inflammation and tissue injury. The radiation response of proteins within a uniform prostate cancer (PCa) radiotherapy (RT) population has been studied to a limited extent. In this thesis, the proteomic responses of patients undergoing curative RT for intermediate-risk PCa were determined for a panel of pro-inflammatory cytokines from pre-RT baseline to last treatment (39 vs. 33 fractions depending on whether the cohort received primary or post-prostatectomy RT respectively). Longitudinal proteomic research is feasible at our institution based on the study design presented herein. Interferon (IFN)-g and interleukin (IL)-6 were significantly increased during RT and these changes followed consistent patterns modeled by linear and quadratic functions respectively. Furthermore, acute gastrointestinal (GI) and genitourinary (GU) toxicities were significantly associated with IL-2 and IL-1 levels respectively during RT. Taken together this research supports cytokines as potential biomarkers of normal tissue radiation response.

Aggressive fibromatosis is a benign fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in patients with familial syndromes, such as familial adenomatous polyposis. Tumours are characterized by the stabilization of β-catenin and the activation of β-catenin-mediated transcription. Current treatment results are far from ideal, and recurrence rates are high. As a result, there remains a need for more effective therapeutic strategies. In this work, we demonstrate the effect of hedgehog signalling inhibition on aggressive fibromatosis tumour development and β-catenin modulation. We found that hedgehog inhibition decreased cell viability and proliferation as well as total β-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, following hedgehog inhibition in Apc+/Apc1638N aggressive fibromatosis mouse model, the number and volume of the tumours formed was reduced. Together, this work suggests that hedgehog signalling inhibitor agents are potential candidates to effectively manage aggressive fibromatosis.

Aggressive fibromatosis is a benign fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in patients with familial syndromes, such as familial adenomatous polyposis. Tumours are characterized by the stabilization of β-catenin and the activation of β-catenin-mediated transcription. Current treatment results are far from ideal, and recurrence rates are high. As a result, there remains a need for more effective therapeutic strategies. In this work, we demonstrate the effect of hedgehog signalling inhibition on aggressive fibromatosis tumour development and β-catenin modulation. We found that hedgehog inhibition decreased cell viability and proliferation as well as total β-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, following hedgehog inhibition in Apc+/Apc1638N aggressive fibromatosis mouse model, the number and volume of the tumours formed was reduced. Together, this work suggests that hedgehog signalling inhibitor agents are potential candidates to effectively manage aggressive fibromatosis.

Both tissue microenvironment and genetic changes are involved in development of cancer. We employed the Friend murine leukemia virus (F-MuLV)- induced erythroleukemia model to study the role of these parameters in induction of malignancy. The tissue microenvironment is composed of non-cellular and cellular components. In regards to the non-cellular part, we previously reported that vascular endothelial growth factor (VEGF), in combination with macrophage chemoattractant protein-5, contributes to leukemia progression in F-MuLV- infected mice. To study the influence of constitutively elevated VEGF levels on the progression of erythroleukemia, we inoculated VEGF hi/+ mice, which are heterozygous for a VEGF “hypermorphic” allele, with F-MuLV. Unexpectedly, a significant delay in erythroleukemia was observed in these mice when compared with wild-type controls. The VEGF hi/+ mice exhibited a higher natural killer (NK) cell activity, elevated B cells, and a decrease in T-cell number. Furthermore, higher erythroid progenitors (i.e. CD34+, CD36+, and TER119+ cells) were evident in the bone marrow, spleen, and peripheral blood of these mice. Also, the CFU-E levels were significantly elevated in VEGF hi/+ bone marrow cultures. We propose that a compensatory erythropoietic response combined with increased NK cell activity account for the extended survival of erythroleukemic, VEGF hi/+mice. In regards to the cellular component of tissue microenvironment we studied the role of B cells in response to F-MuLV. To test the hypothesis that virus- neutralizing antibodies are involved in providing sterilizing immunity to F-MuLV we inoculated adult female mice with F-MuLV so that their newborns are provided with anti-viral antibodies. F-MuLV challenge of these newborns did not lead to induction of erythroleukemia. Conversely, mice from a control group (newborns whose mother had not received viral inoculation) contracted erythroleukemia upon F-MuLV challenge, as shown by hepatosplenomegaly, anemia, and emergence of leukemic cells in the spleen. These results indicated the importance of anti-viral antibodies in immunity to F-MuLV and suggested that anti-F-MuLV antibodies were generated in mothers, transferred to their offspring and protected them from viral challenge. The key genetic event upon F-MuLV infection is viral integration at the Fli-1 locus. We set to identify F-MuLV integration sites in SCID mice following two observations that these mice show a delay in induction of leukemia and also they do not exhibit viral integration at the Fli-1 locus. We hypothesized that development of leukemia in these mice is due to F-MuLV integration at a region other than the Fli-1 locus. Using a GenomeWalking approach we identified a total of 15 viral integration sites in F-MuLV-infected SCID mice, with eight of them interrupting the following genes: Mex3d, Fam125b, Prdm16, Rhoq, Ahdc1, Zc3h4, Msh3, and Hcls1. Using PCR to amplify the virus- host DNA junction fragment we found that one of the viral insertion sites (chromosome 10; position 20,942,825) occurs with a frequency of 35 % and therefore is considered as a common integration site.

Ionizing radiation induces specific proteins involved in DNA repair, cell death, inflammation and tissue injury. The radiation response of proteins within a uniform prostate cancer (PCa) radiotherapy (RT) population has been studied to a limited extent. In this thesis, the proteomic responses of patients undergoing curative RT for intermediate-risk PCa were determined for a panel of pro-inflammatory cytokines from pre-RT baseline to last treatment (39 vs. 33 fractions depending on whether the cohort received primary or post-prostatectomy RT respectively). Longitudinal proteomic research is feasible at our institution based on the study design presented herein. Interferon (IFN)-g and interleukin (IL)-6 were significantly increased during RT and these changes followed consistent patterns modeled by linear and quadratic functions respectively. Furthermore, acute gastrointestinal (GI) and genitourinary (GU) toxicities were significantly associated with IL-2 and IL-1 levels respectively during RT. Taken together this research supports cytokines as potential biomarkers of normal tissue radiation response.

Aberrant activation of the canonical Wnt signaling pathway accounts for the vast majority of colorectal cancers. The Rac1 GTPase is overexpressed in colon cancer, and its splice variant, Rac1b, is preferentially expressed in colon tumours. Rac1 and Rac1b have both been previously shown to crosstalk with the canonical Wnt signaling pathway in colon cancer; however, the specific means by which this crosstalk occurs were unclear. This study examines the molecular mechanisms of Rac1/1b in the cooperation with canonical Wnt signaling in colon cancer. In a colon cancer cell line with dysregulated Wnt signaling, the constitutively active Rac1 mutant, V12Rac1, was observed to transcriptionally upregulate the expression of a gene set associated with cellular migration. Further, V12Rac1-mediated promotion of cell migration was dependent on its nuclear localization. Previous work in our lab has shown a Rac1-specific activator, Tiam1, is present in the nucleus at the promoter of Wnt target genes upon Wnt3a stimulation; and that exogenous introduction of Tiam1 increased the expression of a Wnt-responsive reporter (TopFlash). Given the importance of nuclear localization of Rac1 in the promotion of tumourigenic processes, we demonstrated that knockdown of endogenous Tiam1 reduced TopFlash expression, proving reverse specificity and strengthening the evidence of a nuclear role for Rac1. Since some functional differences exist between Rac1 and Rac1b, we also examined Rac1b for transcriptional targets following induction, and identified the RhoA effector, ROCK2, which has been previously associated with cell migration. ROCK2 demonstrated a positive correlation with Rac1b transcript expression in primary colon tumours as compared to matched normal tissue specimens. Interestingly, the observed induction in ROCK2 transcript did not translate into a detectable change in protein expression or kinase activity. Like Rac1, Rac1b also promotes cellular motility, which is dependent on nuclear localization. Cell migration can be negatively regulated by E-cadherin. Following Rac1b knockdown in HT29 cells, we show that Rac1b might contribute to motility through upregulation of the E-cadherin-repressor, Slug. Taken together, we provide greater insight into the mechanistic roles of Rac1 and Rac1b in transcriptionally regulating target genes to promote cellular processes, such as cell migration, in colon cancer with dysregulated canonical Wnt signaling.

Both tissue microenvironment and genetic changes are involved in development of cancer. We employed the Friend murine leukemia virus (F-MuLV)- induced erythroleukemia model to study the role of these parameters in induction of malignancy. The tissue microenvironment is composed of non-cellular and cellular components. In regards to the non-cellular part, we previously reported that vascular endothelial growth factor (VEGF), in combination with macrophage chemoattractant protein-5, contributes to leukemia progression in F-MuLV- infected mice. To study the influence of constitutively elevated VEGF levels on the progression of erythroleukemia, we inoculated VEGF hi/+ mice, which are heterozygous for a VEGF “hypermorphic” allele, with F-MuLV. Unexpectedly, a significant delay in erythroleukemia was observed in these mice when compared with wild-type controls. The VEGF hi/+ mice exhibited a higher natural killer (NK) cell activity, elevated B cells, and a decrease in T-cell number. Furthermore, higher erythroid progenitors (i.e. CD34+, CD36+, and TER119+ cells) were evident in the bone marrow, spleen, and peripheral blood of these mice. Also, the CFU-E levels were significantly elevated in VEGF hi/+ bone marrow cultures. We propose that a compensatory erythropoietic response combined with increased NK cell activity account for the extended survival of erythroleukemic, VEGF hi/+mice. In regards to the cellular component of tissue microenvironment we studied the role of B cells in response to F-MuLV. To test the hypothesis that virus- neutralizing antibodies are involved in providing sterilizing immunity to F-MuLV we inoculated adult female mice with F-MuLV so that their newborns are provided with anti-viral antibodies. F-MuLV challenge of these newborns did not lead to induction of erythroleukemia. Conversely, mice from a control group (newborns whose mother had not received viral inoculation) contracted erythroleukemia upon F-MuLV challenge, as shown by hepatosplenomegaly, anemia, and emergence of leukemic cells in the spleen. These results indicated the importance of anti-viral antibodies in immunity to F-MuLV and suggested that anti-F-MuLV antibodies were generated in mothers, transferred to their offspring and protected them from viral challenge. The key genetic event upon F-MuLV infection is viral integration at the Fli-1 locus. We set to identify F-MuLV integration sites in SCID mice following two observations that these mice show a delay in induction of leukemia and also they do not exhibit viral integration at the Fli-1 locus. We hypothesized that development of leukemia in these mice is due to F-MuLV integration at a region other than the Fli-1 locus. Using a GenomeWalking approach we identified a total of 15 viral integration sites in F-MuLV-infected SCID mice, with eight of them interrupting the following genes: Mex3d, Fam125b, Prdm16, Rhoq, Ahdc1, Zc3h4, Msh3, and Hcls1. Using PCR to amplify the virus- host DNA junction fragment we found that one of the viral insertion sites (chromosome 10; position 20,942,825) occurs with a frequency of 35 % and therefore is considered as a common integration site.

Low Dose Metronomic (LDM) chemotherapy, combined with VEGF pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, in three pediatric extracranial solid tumors mouse models. We also investigated the effect of prolonged combination therapy with the combination on tumor behavior in a neuroblastoma mouse xenograft model. In-vitro dose-response study of topotecan and pazopanib was conducted on several cell lines. In-vivo antitumor efficacies of drugs, as single agents and combination, were tested in immunodeficient mice models. For studying the mechanisms of resistance to our therapy, a time-response study (28, 56 and 80 days) was conducted in SK-N-BE(2) xenografts model, treated in same way as earlier. In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, the combination of topotecan and pazopanib demonstrated significant anti-tumor activity compared to the respective single agents in all models. Reductions in the levels of viable Circulating Endothelial Progenitors and/or Circulating Endothelial Cells and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. However, the combination also caused significantly higher myelotoxicity than single agents. Pharmacokinetic study did not reveal any interaction between the two co-administered drugs. In the time-response study, we found that only combination treated animals survived till 80 days. However, tumors in these animals started growing gradually after 50 days. Unlike single agents, all three durations of combination treatment significantly lowered tumor microvessel densities, compared to the control. However, tumors treated with the combination for 56 and 80 days had higher pericyte coverage. The combination increased the hypoxia, angiogenic expression and proliferative index and caused metabolic reprogramming of tumor cells. We conclude that the combination of LDM topotecan and pazopanib has superior efficacy than either single agents, which is attributed to superior antiangiogenic activity. However, prolonged treatment with the combination can have additive myelotoxicity and may encounter adaptive resistance associated with metabolic reprogramming and increased proliferation of the tumor cells.

Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.