Ng, J; (2017) Bioluminescence-Mediated Photodynamic Therapy: A Novel Treatment for Grade 4 Astrocytoma. Doctoral thesis , UCL (University College London).

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Abstract

BACKGROUND AND AIMS: Despite advances in surgery, radiation and chemotherapy, grade 4 astrocytoma, the commonest primary brain tumour, remains incurable with a dire prognosis related to its diffusely infiltrative nature. With photodynamic therapy, pre-treatment with a photosensitising drug and subsequent exposure to light of a specific wavelength can mediate selective tumour destruction. However, it is limited by light penetration. Bioluminescence results from conversion of chemical energy into light. This thesis explores whether bioluminescence generated inside astrocytoma cells in vitro and in in vivo can mediate PDT: light generation by target cells requires no knowledge of the exact location of every cell, thereby potentially overcoming the diffuse, infiltrative nature of astrocytomas. METHODS: in vitro: genetically modified U87 glioma cells expressing firefly luciferase (U87-luc) were generated, pre-treated with the photosensitisers hypericin or mTHPC, then incubated with d-luciferin to induce bioluminescence. Cell viability was assessed by MTT assay, haemocytometry, and a growth assay. Inhibition by lycopene, an antioxidant that suppresses PDT, was assessed. Control studies used untransfected U87 cells. METHODS: in vivo: U87-luc cells were xenografted subcutaneously and intracranially into CD1 nu/nu mice. Mice were pre-treated with intraperitoneal mTHPC then given a 7 day infusion of d-luciferin (subcutaneously implanted pump). Tumour response was followed by bioluminescence imaging, volume measurements, and survival. Tumours were harvested for pathological examination and BrdU immunohistochemistry. RESULTS: in vitro: bioluminescence-mediated PDT produced significant cell death with both photosensitisers in U87-luc cells, which was suppressed by lycopene. There was no effect in untransfected cells that could not generate bioluminescence. RESULTS: in vivo: in 3 out of 4 trials, including one in an intracranial model, a treatment effect was demonstrated by a significant reduction of proliferation, as assessed by the proportion of BrdU positive cells in the ‘treatment’ group compared to controls. CONCLUSION: Bioluminescence-mediated PDT kills tumour cells in vitro. Preliminary evidence of an effect in vivo supports the concept being explored further.

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