Tumors are composed not only of malignant cells, but also of various types of normal cells, including vascular cells and infiltrating immune cells, which drive tumor development and progression. The tumor vasculature is abnormal and dysfunctional due to sustained tumor angiogenesis driven by high levels of pro-angiogenic factors. Proteins differentially expressed in tumor vessels affect vascular function and the tumor microenvironment and may serve as targets for therapy. The tumor is also a site of sustained chronic inflammation. The recruitment and activation of inflammatory cells significantly influence tumor progression and regression. Targeting molecules regulating tumor angiogenesis and inflammation in the tumor microenvironment is therefore a promising strategy for the treatment of cancer. This thesis is aiming to understand and investigate the molecular regulation of these two processes in tumors. αB-crystallin is a heat shock protein previously proposed as a target for cancer therapy due to its role in increasing survival of tumor cells and enhancing tumor angiogenesis. In this thesis, we demonstrate a novel role of αB-crystallin in limiting expansion of CD11b+Gr1+ immature myeloid cells in pathological conditions, including tumor development. In addition, we show that αB-crystallin regulates leukocyte recruitment by promoting expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin during TNF-α-induced endothelial activation. Therefore, targeting of αB-crystallin may influence tumor inflammation by regulating immature myeloid cell expansion and leukocyte recruitment. Abnormal, dysfunctional vessels are characteristic of glioblastomas, which are aggressive malignant brain tumors. We have identified the orphan G-protein coupled receptor ELTD1 as highly expressed in glioblastoma vessel and investigated its role in tumor angiogenesis. Interestingly, deficiency of ELTD1 was associated with increased growth of orthotopic GL261 glioma and T241 fibrosarcoma, but did not affect vessel density in any model. Further investigation is warranted to evaluate whether ELTD1 serves a suitable vascular target for glioblastoma treatment. Anti-angiogenic drugs targeting VEGF signaling is widely used in the clinic for various types of cancer. However, the influences of anti-angiogenic treatment on tumor inflammation have not been thoroughly investigated. We demonstrate that VEGF inhibits TNF-α-induced endothelial activation by repressing NF-κB activation and expression of chemokines involved in T-cell recruitment. Sunitinib, a small molecule kinase inhibitor targeting VEGF/VEGFR2 signaling increased expression of chemokines CXCL10, CXCL11, and enhanced T-lymphocyte infiltration into tumors. Our study suggests that anti-angiogenic therapy may improve immunotherapy by enhancing endothelial activation and facilitating immune cell infiltration into tumors.

No description available.

Induction of an endogenous antibody response by therapeutic vaccination could provide an alternative to cost-intensive monoclonal antibody-based treatments for cancer. Since the target of a cancer vaccine will most likely be a self-antigen, self-tolerance of the immune system must be circumvented. Using fusion proteins consisting of the self-antigen to be targeted and a part derived from a foreign antigen, it is possible to break tolerance against the self-antigen. Furthermore, a potent adjuvant is required to support an immune response against a self-molecule. Currently no adjuvant suitable for this purpose is approved for use in humans. This thesis describes the development of a therapeutic vaccine targeting the vasculature of tumors. As tumor cells have developed strategies to escape immune surveillance, targeting of molecules associated with the tumor stroma is an interesting alternative. The alternatively spliced extra domain-A and B (ED-A and ED-B) of fibronectin and the glycan-binding protein galectin-1 are selectively expressed during events of tumor angiogenesis. We have designed recombinant proteins to target ED-B, ED-A and galectin-1, containing bacterial thioredoxin (TRX) as a non-self part, resulting in TRX-EDB, TRX-EDA and TRX-Gal-1. Vaccination against ED-B induced anti-ED-B antibodies and inhibited growth of subcutaneous fibrosarcoma. Immunization against ED-A decreased tumor burden and reduced the number of lung metastases in the MMTV-PyMT model for metastatic mammary carcinoma in a therapeutic setting. Analysis of the tumor tissue from ED-B and ED-A-immunized mice indicated an attack of the tumor vasculature by the immune system. Finally, we show that galectin-1 immunization reduced tumor burden and increased leukocyte numbers in the tumor tissue. Galectin-1 is pro-angiogenic and immunosuppressive, and therefore allows simultaneous targeting of fundamental characteristics of tumorigenesis. We furthermore show that the biodegradable squalene-based Montanide ISA 720 combined with CpG oligo 1826 (M720/CpG) is at least as potent as Freund’s adjuvant with respect to breaking self-tolerance, when comparing several immunological parameters. Freund’s is a potent but toxic adjuvant used in the majority of preclinical studies. The work presented in this thesis shows that therapeutic cancer vaccines targeting the tumor vasculature are a feasible and promising approach for cancer therapy.

The cell cycle is a highly complex process that is under the control of several pathways.  Failure to regulate and/or complete the cell cycle often leads to cell cycle arrest, which may be followed by programmed cell death (apoptosis). One cell type that has a variety of unique cell cycle properties is the horizontal cell of the chicken retina. In this thesis we aimed to characterize the final cell cycle of retinal horizontal cells. In addition, the regulation of the cell cycle and the resistance to apoptosis of retinal horizontal cells are investigated. Our results show that the final cell cycle of Lim1-expressing horizontal progenitor cells is heterogenic and three different cell cycle behaviors can be distinguished. The horizontal cells are generated by: (i) an interkinetic nuclear migration with an apical mitosis; (ii) a final cell cycle with an S-phase that is not followed by mitosis, such cells remain with a fully or partially replicated genome; or (iii) non-apical (basal) mitoses. Furthermore, we show that the DNA damage response pathway is not triggered during the heterogenic final cell cycle of horizontal progenitor cells. However, chemically induced DNA damage activated the DNA damage response pathway without leading to cell cycle arrest, and the horizontal progenitor cells entered mitosis in the presence of DNA damage. This was not followed by apoptosis, despite the horizontal cells being able to functionally activate p53, p21CIP1/waf1, and caspase-3. Finally, we show that FoxN4 is expressed in horizontal progenitor cells and is required for their generation. Over-expression of FoxN4 causes cell death in several neuronal retinal cell types, except horizontal cells, where it results in an overproduction. In conclusion, in this thesis, a novel cell cycle behavior, which includes endoreplication not caused by DNA damage and a basal mitosis that can proceed in the presence of DNA damage, is described. The cell cycle and cell survival processes are of particular interest since retinal horizontal cells are suggested to be the cell-of-origin for retinoblastoma.

The increased interest in the use of light ion therapy is due to the high dose conformity to the target and the dense energy deposition along the tracks resulting in increased relative biological effectiveness compared to conventional radiation therapy. In spite of the good clinical experience, fundamental research on the characteristics of the ion beams is still needed in order to be able to fully explore their use. Therefore, a Monte Carlo track structure code, KITrack, simulating the transport of electrons in liquid water, has been developed and used for calculation of parameters of interest for beam characterization. The influence of the choice of the cross sections for the physical processes on the electron tracks has also been explored. As an alternative to Monte Carlo calculations a semi-analytical approach to calculate the radial dose distribution from ions, has been derived and validated. In advanced radiation therapy, accurate characterization of the beams has to be complemented by comprehensive radiobiological models, which relate the dose deposition into the cells to the outcome of the treatment. The second part of the study has therefore explored the influence of heterogeneity in the dose deposition into the cells as well as the heterogeneity in the cells sensitivity to radiation on the probability of controlling the tumor. Analytical expressions for tumor control probability including heterogeneous dose depositions or variation of radiation sensitivity of cells and tumors have been derived and validated with numerical simulations. The more realistic case of a combination of these effects has also been explored through numerical simulations. The MC code KITrack has evolved into an extremely useful tool for beam characterization. The tumor control probability, given by the analytical derived expression, can help improve radiation therapy. A novel anisotropy index has been proposed. It is a measure of the absence of isotropy and provides deeper understanding of the relationship between beam quality and biological effects. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Antigen induced activation of Th1 cells in the peripheral blood leads to elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that have been directly linked to disease induction and neuropathic pain. It was hypothesized that following antigenic induction, cytokines gain access to the spinal cord and participate in direct cellular interaction with dorsal horn neurons. Using an animal model of MS, we show that TNF-α gene and protein expression in the dorsal root ganglia (DRG) and spinal cord tissue is increased in the active group. In addition, our findings show TNF-α mRNA expression in the dorsal root entry point. Therefore, our results support the hypothesis that antigen induced DRG derived TNF-α can transport to the spinal cord via the dorsal roots and is involved in the underlying pathogenesis of MS induced neuropathic pain.

Background: Multiple sclerosis (MS) is a chronic, neurological disease characterized by targeted destruction on central nervous system (CNS) myelin. The autoimmune theory is the most widely accepted explanation of disease pathology. Circulating Th-1 cells become activated by exposure to CNS-specific antigens such as myelin basic protein. The activated Th-1 cells secrete inflammatory cytokines, which are pivotal for inflammatory responses. We hypothesize that enhanced production of inflammatory cytokines triggers cellular events within the dorsal root ganglia (DRG) and/or spinal cord, facilitating the development of neuropathic pain (NPP) in MS. NPP, the second worst disease-induced symptom suffered by patients with MS, is normally regulated by DRG and/or spinal cord. Objective: To determine gene and protein expression levels of tumor necrosis factor-alpha (TNF ) within DRG and/or spinal cord in an animal model of MS. Methods: Experimental autoimmune encephalomyelitis (EAE) was induced in adolescent female Lewis rats. Animals were sacrificed every 3 days post-disease induction. DRG and spinal cords were harvested for protein and gene expression analysis. Results: We show significant increases in TNF expression in the DRG and of EAE animals at peak disease stage, as assessed by clinical symptoms. Conclusion: Antigen-induced production of inflammatory cytokines such as TNF within the DRG identifies a potential noel mechanism for MS-induced NPP.

This thesis investigated the cooperation of the Kras2 oncogene with the tumor suppressor gene Dlc1 in lung tumor development. Dlc1 is a negative regulator of RhoGTPase proteins, which are mainly involved in the regulation of the actin cytoskeleton and cell migration. We hypothesized that loss of Dlc1 expression leads to more aggressive tumors, which should also result in increased incidence of metastasis. All experiments were performed in mice containing a heterozygous oncogenic Kras allele and a heterozygous gene trapped Dlc1 allele (KD) and in mice only carrying the oncogenic Kras allele (K+). Throughout all experiments we have consistently found no significant differences between the two groups in terms of tumor burden (tumor numbers, sizes and areas), metastases or methylation patterns. These results suggest that heterozygous downregulation of Dlc1 is not enough to increase tumor formation and metastasis development in the Kras lung tumors.

This work describes the development and application of a novel combination of single-cell Raman spectroscopy (RS), automated data processing, and principal component analysis (PCA) for investigating radiation induced biochemical responses in human tumour cells. The developed techniques are first validated for the analysis of large data sets (~200 spectra) obtained from single cells. The effectiveness and robustness of the automated data processing methods is demonstrated, and potential pitfalls that may arise during the implementation of such methods are identified. The techniques are first applied to investigate the inherent sources of spectral variability between single cells of a human prostate tumour cell line (DU145) cultured {\it in vitro}. PCA is used to identify spectral differences that correlate with cell cycle progression and the changing confluency of a cell culture during the first 3-4 days after sub-culturing. Spectral variability arising from cell cycle progression is (i) expressed as varying intensities of protein and nucleic acid features relative to lipid features, (ii) well correlated with known biochemical changes in cells as they progress through the cell cycle, and (iii) shown to be the most significant source of inherent spectral variability between cells. This characterization provides a foundation for interpreting spectral variability in subsequent studies. The techniques are then applied to study the effects of ionizing radiation on human tumour cells. DU145 cells are cultured in vitro and irradiated to doses between 15 and 50 Gy with single fractions of 6 MV photons from a medical linear accelerator. Raman spectra are acquired from irradiated and unirradiated cells, up to 5 days post-irradiation. PCA is used to distinguish radiation induced spectral changes from inherent sources of spectral variability, such as those arising from cell cycle. Radiation induced spectral changes are found to correlate with both the irradiated dose and the incubation time post-irradiation, and to arise from biochemical differences in lipids, nucleic acids, amino acids, and conformational protein structures between irradiated and unirradiated cells. This study is the first use of RS to observe radiation induced biochemical effects in single cells, and is the first use of vibrational spectroscopy to observe such effects independent from cell cycle or cell death related processes. The same methods are then applied to a panel of human tumour cell lines, derived from prostate (DU145, PC3, LNCaP and PacMet), breast (MDA-MB-231 and MCF7) and lung (H460), which vary by p53 gene status and intrinsic radiosensitivity. One radiation induced PCA component is detected for each cell line by statistically significant changes in the PCA score distributions for irradiated samples, as compared to unirradiated samples, in the first 24 to 72 hours post-irradiation. These RS response signatures arise from radiation induced changes in cellular concentrations of aromatic amino acids, conformational protein structures, and certain nucleic acid and lipid functional groups. Correlation analysis between the radiation induced PCA components separates the cell lines into three unique RS response categories: R1 (H460, MCF7 and PacMet), R2 (MDA-MB-231 and PC3), and R3 (DU145 and LNCaP). These RS categories partially segregate according to radiosensitivity; the R1 and R2 cell lines are radioresistant and the R3 cell lines are radiosensitive (PacMet radiosensitivity (R1) unknown). The R1 and R2 cell lines further segregate according to p53 gene status, corroborated by cell cycle analysis post-irradiation. Preliminary results obtained from a mouse prostate tumour cell line (TRAMP-C2), irradiated both in vitro and in vivo, indicate that RS signatures of radiation response may also be detectable from tumour cells obtained from an in vivo system during radiation therapy treatment. These results indicate the potential for future RS studies designed to investigate, monitor, or predict radiation response. / Graduate

No description available.