Many common types of solid cancerous tumours exhibit overexpression of certain biomarkers that can be used to distinguish tumour from adjacent normal tissue. Molecular probes conjugated to ligands (peptides, antibodies) that bind to overexpressed biomarkers can then enable cancer-targeted imaging for early diagnosis, tumour staging and ultimately image-guided surgery. For example, prostate specific membrane antigen(PSMA) is overexpressed in several solid tumours (prostate and renal cell carcinoma, glioblastoma) and PET probe targeting by the PSMA glutamate-based ligand has been clinically validated in prostate (Uijen et al Eur. J. Nuc. Med. Mol. Imaging, 2021). Fluorescence imaging is very sensitive and capable of high sub-mm resolution,and is well suited to real-time intraoperative and endoscopic/laparoscopic imaging unlike PET.However, the heterogenous nature of the tumour microenvironment and overlapping tissue autofluorescence have complicated the realisation of this approach. In addition, the fluorescence properties of the probe molecule, including the fluorescence lifetime, may change following internalisation of the probe after binding to the surface receptor. This has led to increasing interest in the use of fluorescence lifetime imaging (FLIM) which is capable of in situ mapping of the fluorescence lifetime of the targeted probe in cells and tissue. The fluorescence lifetime is a key photophysical property of any fluorescent agent, and FLIM has recently been investigated clinically for imaging of glioblastomas (Alfonso-Garcia et al, J. Biomed. Optics, 2022). Following ultrashort pulsed laser excitation, FLIM is capable of distinguishing between the fluorescence decay lifetimes between the fluorescence probe, typically 5-15 ns, and the tissue autofluorescence, which is generally shorter <5 ns) and may differ between the normal and cancerous tissue. The application of FLIM using time-gated imaging therefore has the potential to improve discrimination between the targeted cancer cells and the normal tissue and enhance sensitivity and specificity.The hypothesis to be investigated is that fluorescence lifetime imaging can be used to enhance cancer imaging selectivity.
Aims and Experimental Schedule
- To investigate the photophysical properties cancer-selective fluorescence probes and map their fluorescence lifetimes using FLIM in prostate cancer cells, 3D cancer constructs and tissue.
- To analyse the spectral and time-resolved fluorescence lifetime properties and assess the potential of FLIM for improving discrimination between targeted cancer cells and normal tissue and translation to preclinical studies.
A combination of techniques will be used combining optical spectroscopy with chemical biology and tissue culture, for which the necessary equipment is already in place. The cell lines are already available and animal tissues will be provided from parallel unrelated projects in our department. The contrast agents are already available through existing projects, 5-aminolevulinic acid and ester derivatives, and a porphyrin PSMA conjugate. The 3D construct will use the model developed at UCL (Nyga et al Acta Biomat. 2013) which incorporates an extracellular matrix component.
Yr 1-2: The in vitro binding efficiency and imaging selectivity of the targeted contrast agents will be validated as a function of dose and time using in vitro 2D and 3D models with well characterised human prostate tumour cell lines vs. control cell lines. Techniques: confocal microscopy, spectrophotometry and FACs. Milestone: validation of targeting
Yr 1-2: Time-correlated single photon counting detection (TCSPC) will be employed to resolve the fluorescence lifetimes of the agents with sub-nanosecond resolution in appropriate solvents in a cuvette. In situ studies of the fluorescence lifetime and emission spectrumusing a fibre-optic sensing probe placed on the tissue surface from sensitised rodent tissue will be carried out. Milestone: characterisation of photophysical properties of the agents
Yr 2-4: Fluorescence lifetime imaging (FLIM) on cells and tissue cryosections will be carried out using a fluorescence microscope equipped with an array of sensitive SPAD detectors and laser excitation with a multi-beam beam array for rapid scanning. These microscopic studies will provide sub-cellular resolution mapping of fluorescence lifetime dynamics and efficiencies in different structures within the tissue(tumour cells, connective tissue, as identified using immunostaining) and other tissues. In parallel data analysis and correlation with tumour cell selectivity will be carried out. Milestone: identification of optimised FLIM parameters (eg optimum time-gating)for preclinical assessment.
Supervision & Risk management: The principles of this technology have been shown to be feasible at UCL therefore we are confident that this project can be implemented. Risk mitigation will be constantly reviewed throughout. The project will be conducted jointly between the Dept. of Surgical Biotechnologyin the DSIS and the Cancer Institute,and provide multidisciplinary training in chemical biology and photophysics (both EPSRC themes).The student will be supervised day-to-day by SM and EY, and the supervisory team will meet monthly. SM has a 100% record of successful PhD completion to date. SM, EY and PB have an established track record in biophotonics including FLIM and SMalso has expertise in laser-based cancer theranostics. The development of new techniques for image-guided surgery is a key research theme of the Division and SM and EY collaborate with the Depts. of Medical Physics and Bioengineering and Computer Science through the Wellcome/EPSRC Centre for Interventional Surgical Sciences(WEISS).
Studentships are expected to start on 26th September 2022 unless exceptional circumstances require an alternate start date.
Eligibility
Successful applicants must fulfil the academic entry requirements for the programme they are applying for. Further eligibility criteria are based on nationality and residence, see EPSRC regulations on Student eligibility.
These studentships are open to those with Home and International fee status (including EU); however, the number of students with International fee status which can be recruited is capped according to the EPSRC terms and conditions.
We particularly encourage applications from Black, Asian, and Minority Ethnic candidates, who are currently under-represented within UCL at this level.
How to Apply
Please submit applications in the following format:
- A CV, including full details of all University course grades to date.
- Contact details for two academic or professional referees.
- A personal statement (750 words maximum) outlining (i) your suitability for the project with reference to the criteria above, (ii) what you hope to achieve from the PhD and (iii) your research experience to-date.
Only shortlisted candidates will be contacted.
Please send your applications to [Email Address Removed]
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