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Bioluminescence-activated photodynamic therapy for luciferase transfected, grade 4 astrocytoma cells in vitro
Jane Ng1, Nico Henriquez2, Alexander MacRobert3
1UCL Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom; National Medical Laser Centre (now Division of Surgery and Interventional Science), University College London, Charles Bell House 43-45 Foley Street, London W1W 7TS, United Kingdom.
Background:
. Grade 4 astrocytoma is incurable due to the diffusely infiltrative nature of the disease. Photodynamic therapy (PDT) is a promising therapeutic option, but external light delivery is not feasible when cancer cells infiltrate unknown areas of normal brain. Hence the search for endogenous sources such as bioluminescence that can generate light at cancer cells. This requires a substrate (a luciferin) and an enabling enzyme (a luciferase), neither seen in mammalian cells.
Methods:
. Preliminary studies confirmed that U87 cells (derived from a human grade 4 astrocytoma) could be killed by conventional PDT using the photosensitizers hypericin or mTHPC. U87 cells were then transfected with firefly and other luciferases and light generating cell lines (U87-luc, U87-hRluc, U87-CBG68luc) identified using the appropriate substrate. Reagent doses and conditions were optimized and U87-luc cells incubated with hypericin or mTHPC with d-luciferin added to initiate bioluminescence activated PDT (bPDT). Cell survival was assessed by MTT assay, haemocytometry and growth assay. Control groups included U87-luc cells with no added active reagents, substrate only, photosensitizer only and non-transfected U87 cells. Results were expressed as a percentage of surviving cells compared with untreated U87-luc controls.
Results:
. There was no bPDT effect on non-transfected cells. The mean survival of treated transfected cells was 36%, (P<0.001) using hypericin and 35% (P<0.001) using mTHPC, compared with untreated U87-luc cells. bPDT effects were suppressed by the anti-oxidant, lycopene.
Conclusions:
. bPDT can kill Grade 4 astrocytoma cells transfected with luciferase in vitro. This justifies progression to in vivo studies.
Insights
Bioluminescence-activated photodynamic therapy (bPDT) effectively killed Grade 4 astrocytoma cells in vitro. This novel approach uses luciferase-transfected cells and endogenous light for targeted cancer therapy.
Area of Science:
- Oncology
- Biotechnology
- Photochemistry
Background:
- Grade 4 astrocytoma is an incurable brain cancer due to its infiltrative nature.
- Photodynamic therapy (PDT) is a potential treatment, but light delivery is challenging for deep-seated or infiltrating tumors.
- Bioluminescence offers an endogenous light source for PDT, requiring a luciferin substrate and luciferase enzyme, which are not naturally present in mammalian cells.
Purpose of the Study:
- To investigate the efficacy of bioluminescence-activated photodynamic therapy (bPDT) against Grade 4 astrocytoma cells in vitro.
- To determine if genetically engineered astrocytoma cells expressing luciferase can be killed by bPDT using common photosensitizers.
Main Methods:
- Human Grade 4 astrocytoma U87 cells were transfected to express firefly luciferase, creating light-generating cell lines.
- Optimized conditions involved incubating transfected U87-luc cells with photosensitizers (hypericin or mTHPC) and d-luciferin to activate bPDT.
- Cell survival was quantified using MTT assays, haemocytometry, and growth assays, with comparisons to control groups.
Main Results:
- bPDT significantly reduced the survival of transfected U87-luc astrocytoma cells by approximately 64-65% (P<0.001) compared to untreated controls.
- No significant bPDT effect was observed in non-transfected U87 cells.
- The bPDT-induced cell death was partially inhibited by the antioxidant lycopene, suggesting a role for reactive oxygen species.
Conclusions:
- bPDT is a viable strategy for eliminating Grade 4 astrocytoma cells expressing luciferase in an in vitro setting.
- These findings support the further investigation of bPDT in preclinical in vivo models for brain tumor treatment.
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