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Exogenous administration of the photodynamic agent hexaminolevulinate induces Protoporphyrin IX (PpIX) accumulation in malignant tissue. This may enable differentiation from healthy tissues by emission of a distinctive red fluorescence. It provides the photo-specific detection when excited with blue light at 405 nm. This study determines the ex-vivo processing conditions (time, concentration, temperature and addition of a fluorescent dye) required for HAL-induced PpIX fluorescence to successfully discriminate between bladder cancer and benign fibroblast cells shed in urine at the single cell level. HAL-induced fluorescence was 4.5 times brighter in cancer cells than non-cancer cells when incubated in the optimum conditions, and could be used to correctly identified bladder cancer cells captured within a newly developed immunofunctionalized biosensor with 88% efficiency. This biosensor is designed to facilitate the immuno-capture of cancer cells by interaction with carcinoma specific anti Epithelial Cell Adhesion molecule (anti-EpCAM) antibodies. Anti-EpCAM antibodies were immobilized on polyoxazoline (POx) plasma polymers by covalent bonds in microfluidic channels. Combining photodynamic and immunoselective approach therefore constitute a promising approach for the non-invasive diagnosis of bladder cancer with two independent level of confidence., Objective: This study investigate the relationship between different regulatory factors (time, concentration, temperature and addition of a fluorescent dye) and Hexaminolevulinate (HAL)-mediated photodynamic diagnosis of bladder cancer (PDD) in vitro. We examine the natural photosensitizer Protoporphyrin IX (PpIX) fluorescence induced by HAL in several human bladder cancer cell lines and one non-cancer foreskin fibroblast cell line and identify the processing conditions that maximise the difference in fluorescence intensity between malign and benign cell types. The detection of HAL induced fluorescence at a single cell level by a selective cancer cell capture platform is also tested., Materials and Methods: Experiments were performed on cultured monolayer cells and cells in suspension. The cell lines examined included the transitional epithelium carcinoma cell lines HT1197, HT1376, EJ138 and RT4, and the non-cancer foreskin fibroblasts HFF. Cells were incubated with HAL in various doses, time and temperature settings. We also used the nuclear red as a tool to study the PpIX subcellular localization. PpIX fluorescence intensities were measured and analysed using fluorescence microscope software. Finally, we evaluated the possibility of using HAL to discriminate between cancer and non-cancer cells from a mixed cell population using a newly developed immunofunctionalized microfluidic platform., Results: The accumulation of PpIX in bladder cancer cells was significantly higher than in non-cancer cells, both cultured monolayer cells and cells in suspension. Effectively, the fluorescence intensity was 4.5 times brighter in bladder cancer cells than non-cancer foreskin fibroblast cells when incubated in the optimum condition, in which the nuclear stain adjuvant acted as a fluorescence enhancer. Cancer cells displayed PpIX accumulated mainly in mitochondria but none or very little PpIX was observed in non-cancer cells. HAL-induced fluorescence could be used to correctly identify bladder cancer cells within the EpCAM conjugated POx based microfluidic sensor with an 88% capture selectivity rate., Conclusions: These findings prove that the application of HAL-induced PpIX fluorescence can successfully distinguish between cancer and non-cancer cells in vitro. This test can provide advanced second level of confidence on the cancerous nature of cells captured by the immunofunctionalized bladder cancer diagnostic platform., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Photodynamic diagnosis and therapy have emerged as a promising tool in oncology. Using the visible fluorescence from photosensitisers excited by light, clinicians can both identify and treat tumour cells in situ. Protoporphyrin IX, produced in the penultimate step of the haem synthesis pathway, is a naturally occurring photosensitiser that visibly fluoresces when exposed to light. This fluorescence is enhanced considerably by the exogenous administration of the substrate 5-aminolaevulinic acid (5-ALA). Significantly, 5-ALA-induced protoporphyrin IX accumulates preferentially in cancer cells, and this enhanced fluorescence has been harnessed for the detection and photodynamic treatment of brain, skin and bladder tumours. However, surprisingly little is known about the mechanistic basis for this phenomenon. This review focuses on alterations in the haem pathway in cancer and considers the unique features of the cancer environment, such as altered glucose metabolism, oncogenic mutations and hypoxia, and their potential effects on the protoporphyrin IX phenomenon. A better understanding of why cancer cells fluoresce with 5-ALA would improve its use in cancer diagnostics and therapies.