Investigating the effects of chemotherapy and radiation therapy in a prostate cancer model system using SERS nanosensors
Camus, Victoria Louise
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Intracellular redox potential (IRP) is a measure of how oxidising or reducing the environment is within a cell. It is a function of numerous factors including redox couples, antioxidant enzymes and reactive oxygen species. Disruption of the tightly regulated redox status has been linked to the initiation and progression of cancer. However, there is very limited knowledge about the quantitative nature of the redox potential and pH gradients that exist in cancer tumour models. Multicellular tumour spheroids (MTS) are three-dimensional cell cultures that possess their own microenvironments, similar to those found in tumours. From the necrotic core to the outer proliferating layer there exist gradients of oxygen, lactate, pH and drug penetration. Tumours also have inadequate vasculature resulting in a state of hypoxia. Hypoxia is a key player in metabolic dysregulation but can also provide cells with resistance against cancer treatments, particularly chemotherapy and radiation therapy. The primary hypoxia regulators are HIFs (Hypoxia Inducible Factors) which under low O2 conditions bind a hypoxia response element, inhibiting oxidative phosphorylation and upregulating glycolysis which has two significant implications: the first is an increase in levels of NADPH/NADH, the main electron donors found in cells which impacts the redox state, whilst the second is a decrease in intracellular pH (pHi) because of increased lactate production. Thus, redox state and intracellular pHi can be used as indicators of metabolic changes within 3D cultures and provide insight into cellular response to therapy. Surface-Enhanced Raman Spectroscopy (SERS) provides a real-time, high resolution method of measuring pHi and IRP in cell culture. It allows for quick and potentially portable analysis of MTS, providing a new platform for monitoring response to drugs and therapy in an unobtrusive manner. Redox and pH-active probes functionalised to Au nanoshells were readily taken up by prostate cancer cell lines and predominantly found to localise in the cytosol. These probes were characterised by density functional theory and spectroelectrochemistry, and their in vitro behaviour modelled by the chemical induction of oxidative and reductive stress. Next, targeting nanosensors to different zones of the MTS allowed for spatial quantification of redox state and pHi throughout the structure and the ability to map the effects of drug treatments on MTS redox biology. The magnitude of the potential gradient can be quantified as free energy (ΔG) and used as a measurement of MTS viability. Treatment of PC3 MTS with staurosporine, an apoptosis inducer, was accompanied by a decrease in free energy gradients over time, whereas treatment of MTS with cisplatin, a drug to which they are resistant, showed an increase in viability indicating a compensatory mechanism and hence resistance. Finally, using this technique the effects of ionising radiation on IRP and pHi in the tumour model was explored. Following exposure to a range of doses of x-ray radiation, as well as single and multi-fractionated regimes, IRP and pHi were measured and MTS viability assessed. Increased radiation dosage diminished the potential gradient across the MTS and decreased viability. Similarly, fractionation of a single large dose was found to enhance MTS death. This novel SERS approach therefore has the potential to not only be used as a mode of drug screening and tool for drug development, but also for pre-clinical characterisation of tumours enabling clinicians to optimise radiation regimes in a patient-specific manner.