HIF-1alpha role in oxygen-dependent radio- and chemosensitivity

Poor oxygenation (hypoxia) is a common characteristic of human solid tumours, and is associated with cell survival, metastasis and resistance to radio- and chemotherapies. Hypoxia-induced stabilisation of hypoxia-inducible factor-1α (HIF-1α) leads to changes in expression of various genes associated with growth, vascularisation and metabolism. However whether HIF-1α plays a causal role in promoting hypoxic resistance to antitumour therapies remains unclear. In this study we used pharmacological and genetic methods to investigate the HIF-1α contribution to radio- and chemoresistance in four cancer cell lines derived from cervical, breast, prostate and melanoma human tumours. Under normoxia or hypoxia (<0.2% or 0.5% oxygen) the cells were exposed to either a standard irradiation dose (6.2 Gy) or chemotherapeutic drug (cisplatin), and subsequent cell proliferation (after 7 days) was measured in terms of resazurin reduction. Oxygen-dependent radio- and chemosensitivity was evident in all wild type whereas it was reduced or abolished in HIF-1α (siRNA) knockdown cells. The effects of HIF-1α-modulating drugs (EDHB, CoCl2, deferoxamine to stabilise and R59949 to destabilise it) reflected both HIF-1α-dependent and independent mechanisms. Collectively the data show that HIF-1α played a causal role in our in vitro model of hypoxia-induced radioresistance whereas its contribution to oxygendependent sensitivity to cisplatin was less clear-cut. Although this behavior is likely to be conditioned by further biological and physical factors operating in vivo, it is consistent with the hypothesis that interventions directed at HIF-1α may improve the clinical effectiveness of tumour treatments.

Ex vivo culture of patient tissue and examination of gene delivery

Gene therapy has emerged as a realistic prospect for the treatment of cancer due to its potential for selective tumour cell targeting. The greatest challenge gene delivery vectors face is the ability to safely and efficiently deliver genes into target cells. The overall objectives of this thesis are to evaluate the efficacy of various gene delivery methods in a clinically relevant tumour model and to also investigate potential strategies for tumour selective delivery. We began with the development of a tumour slice model system using patient waste tissue. This model involves the use of fresh human tumour tissue, cut into thin slices and maintained ex vivo and is universally applicable to gene delivery methods, using a real-time luminescence detection method to assess gene delivery. The nature of the ex vivo culture system permitted examination of specific physiological variables, the influence of intratumoural factors and tissue specific effects on vector expression. Adenoviral vectors under the control of the human CXCR4 promoter demonstrated a 'tumour on' and 'normal off' expression profile when compared with the ubiquitously active CMV promoter when tested in patient tumour tissue. In addition, we developed an ex vivo system of changing oxygenation using the hypoxia inducer, cobalt, to mimic the transient hypoxic conditions found in solid tumours. We found that Adenoviral transgene expression was robust in the cycling hypoxic conditions relevant to solid tumours and re-oxygenation of chronically hypoxic tissue enhanced transgene expression. Finally, we demonstrated an AAV-based tumour targeting strategy using a tumour-selective promoter allowing for the efficient targeting of AAV vectors to cancer cells and the sparing of normal tissue in both murine metastatic liver tumours models and patient tissue. The thesis highlights the importance of indepth preclinical assessment of novel therapeutics and may serve as a platform for further testing of novel gene delivery approaches.

Multi-parametric metabolic assessment of cells under stress conditions

Glycolysis, glutaminolysis, the Krebs cycle and oxidative phosphorylation are the main metabolic pathways. Exposing cells to key metabolic substrates (glucose, glutamine and pyruvate); investigation of the contribution of substrates in stress conditions such as uncoupling and hypoxia was conducted. Glycolysis, O2 consumption, O2 and ATP levels and hypoxia inducible factor (HIF) signalling in PC12 cells were investigated. Upon uncoupling with FCCP mitochondria were depolarised similarly in all cases, but a strong increase in respiration was only seen in the cells fed on glutamine with either glucose or pyruvate. Inhibition of glutaminolysis reversed the glutamine dependant effect. Differential regulation of the respiratory response to FCCP by metabolic environment suggests mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function. At reduced O2 availability (4 % and 0 % O2), cell bioenergetics and local oxygenation varied depending on the substrate composition. Results indicate that both supply and utilisation of key metabolic substrates can affect the pattern of HIF-1/2α accumulation by differentially regulating iO2¬, ATP levels and Akt/Erk/AMPK pathways. Inhibition of key metabolic pathways can modulate HIF regulatory pathways, metabolic responses and survival of cancer cells in hypoxia. Hypoxia leads to transcriptional activation, by HIF, of pyruvate dehydrogenase (PDH) kinase which phosphorylates and inhibits PDH, a mitochondrial enzyme that converts pyruvate into acetyl-CoA. The levels of PDH (total and phosphorylated), PDH kinase and HIF-1α were analysed in HCT116 and HCT116 SCO2-/- (deficient in complex IV of the respiratory chain) grown under 20.9 % and 3 % O2. Data indicate that regulation of PDH can occur in a manner independent of the HIF-1/PDH kinase 1 axis, mitochondrial respiration and the demand for acetyl-CoA. Collectively these results can be applied to many diseases; reduced nutrient supply and O2 during ischemia/stroke, hypoglycaemia in diabetes mellitus and cancer associated changes in uncoupling protein expression levels.

New phosphorescence based probes and techniques for the analysis of cellular oxygen and respiration

Real time monitoring of oxygenation and respiration is on the cutting edge of bioanalysis, including studies of cell metabolism, bioenergetics, mitochondrial function and drug toxicity. This thesis presents the development and evaluation of new luminescent probes and techniques for intracellular O2 sensing and imaging. A new oxygen consumption rate (OCR) platform based on the commercial microfluidic perfusion channel μ-slides compatible with extra- and intracellular O2 sensitive probes, different cell lines and measurement conditions was developed. The design of semi-closed channels allowed cell treatments, multiplexing with other assays and two-fold higher sensitivity to compare with microtiter plate. We compared three common OCR platforms: hermetically sealed quartz cuvettes for absolute OCRs, partially sealed with mineral oil 96-WPs for relative OCRs, and open 96-WPs for local cell oxygenation. Both 96-WP platforms were calibrated against absolute OCR platform with MEF cell line, phosphorescent O2 probe MitoXpress-Intra and time-resolved fluorescence reader. Found correlations allow tracing of cell respiration over time in a high throughput format with the possibility of cell stimulation and of changing measurement conditions. A new multimodal intracellular O2 probe, based on the phosphorescent reporter dye PtTFPP, fluorescent FRET donor and two-photon antennae PFO and cationic nanoparticles RL-100 was described. This probe, called MM2, possesses high brightness, photo- and chemical stability, low toxicity, efficient cell staining and high-resolution intracellular O2 imaging with 2D and 3D cell cultures in intensity, ratiometric and lifetime-based modalities with luminescence readers and FLIM microscopes. Extended range of O2 sensitive probes was designed and studied in order to optimize their spectral characteristics and intracellular targeting, using different NPs materials, delivery vectors, ratiometric pairs and IR dyes. The presented improvements provide useful tool for high sensitive monitoring and imaging of intracellular O2 in different measurement formats with wide range of physiological applications.

Hypothermia protects brain mitochondrial function from hypoxemia in a murine model of sepsis

Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48 h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins, but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective.