Probing Hexaminolevulinate Mediated PpIX Fluorescence in Cancer Cell Suspensions in the Presence of Chemical Adjuvants

Abstract

Exogenous administration of hexaminolevulinate (HAL) induces fluorescent protoporphyrin IX (PpIX) accumulation preferentially in cancer cells. However, the PpIX fluorescence intensities between noncancer and cancer cells are highly variable. The contrast between cancer and noncancer cells may be insufficient to reliably discriminate, especially at the single cell level in cancer diagnostics. This study examines the use of the chemical adjuvants dimethylsulphoxide (DMSO) or deferoxamine (DFO) to enhance the HAL induced PpIX accumulation in cancer cells. Our results showed that in some of the incubation conditions tested, the addition of DFO with HAL significantly increased PpIX 21 fluorescence of adherent monolayer cancer cells, but this was never the case for cells in suspension. Permeabilisation with DMSO did not increase PpIX fluorescence. Cell-to-cell interaction may well play an important role in the PpIX accumulation when suspended cells are treated in HAL and adjuvant chemicals.presence of intracellular PpIX. 5-ALA based PDD is used in the detection of a wide range of cancers, such as brain [5], ovarian [6], colorectal [7], breast [8] and bladder [9]. In urology, blue light cystoscopy PPD is now recommended by several guidelines around the world. Yet the underlying mechanisms responsible for the specificity of PpIX accumulation in tumour cells remains poorly understood [10]. To date, it has been attributed to the distinctive nature of cancer cells, primarily, to reduced FECH activity [11] and to the limited availability of iron in cancer cells [12,13] or to the reduced availability of nicotinamide adenine dinucleotide phosphate (NADPH) provision. However, the difference in PpIX fluorescence intensity between normal and cancer cells is highly variable from one cell type to another [14]. In fact, the contrast is, in some cases, insufficient to reliably discriminate between the two, especially when applying PDD principles ex vivo, at the single cell level [15,16]. To enable the development of noninvasive diagnostic methods using PDD on body fluid sediments, such as urine [17][18][19][20], practical ways to enhance the PpIX fluorescence contrast ex vivo are required [21]. Strategies to do so may well be different from those that are successful in vivo, simply because the cell microenvironments are drastically different in those two scenarios.

Effects of Supplemental Drugs on Hexaminolevulinate (HAL)-Induced PpIX Fluorescence in Bladder Cancer Cell Suspensions

“…Various inhibitors of these chelating and efflux steps of the metabolism have been shown to be effective in increasing PpIX accumulation in some cancer types [15], although results for bladder cancer have not been reported. Results from our previous works indicated that the cell microenvironment plays an important role in the regulation of PpIX accumulation when suspended bladder cells are treated with HAL [16]. In this study, we hypothesize that reducing the PpIX efflux and FECH activity with inhibitors and iron chelators could enhance HAL-induced PpIX fluorescence within bladder cancer cells (Figure 1).…”

“…The cells were then treated with the inhibitors under investigation (EDTA, selumetinib, U0126, genistein, SA, deferiprone and trametinib), for a different amount of time and at different temperatures, as summarized in Table 2 . The effect of the seven PpIX conversion and/or membrane transport inhibitors on the overall HAL-induced fluorescence of the cells was then quantified following established fluorescence microscopy procedures [ 16 , 23 , 24 ].…”

“…Results from our previous works indicated that the cell microenvironment plays an important role in the regulation of PpIX accumulation when suspended bladder cells are treated with HAL [ 16 ]. In this study, we hypothesize that reducing the PpIX efflux and FECH activity with inhibitors and iron chelators could enhance HAL-induced PpIX fluorescence within bladder cancer cells ( Figure 1 ).…”

Peptide-targeted dendrimeric prodrugs of 5-aminolevulinic acid: A novel approach towards enhanced accumulation of protoporphyrin IX for photodynamic therapy

“…To address this, many prodrugs of ALA have been studied to offset the hydrophilic nature of ALA itself, enhance stability at physiological pH, and to improve targeting of cancerous cells [17,18]. The formation of lipophilic esters as in the clinically approved methyl ester (Metvix) and hexyl ester (Hexvix), has been shown to be highly effective for the enhancement of ALA uptake, but the use of simple ALA-esters presents a challenge with respect to specificity, causing a nonspecific distribution of ALA in all cell types post administration of the prodrugs [19].…”

Fluid Flow Dependency in Immunoselective Cell Capture via Liquid Biopsy

“…The device couples EpCAM-based immunoaffinity with photodynamic principles established in clinical urology , to specifically identify malignant cells of epithelial origin shed in urine samples. Our approach has achieved promising results with model cell lines from both bladder and prostate cancer origin. , However, results in patients samples were challenged by a number of false-positive results . As the cells lining the urinary tract (squamous, renal tubular, and transitional cells) are both epithelial and adherent in nature, a careful examination of the conditions in which the specificity of the immune-capture toward urogenital cancer cells can be enhanced is critical for future clinical use.…”