Changes in autofluorescence based organoid model of muscle invasive urinary bladder cancer (original) (raw)
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Biomedical Optics Express, 2015
The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.
Journal of Biomedical Optics, 1996
The aim of this study was to perform a preliminary evaluation of the diagnostic potential of laser-induced autofluorescence spectroscopy (LIAFS) for urothelial tumors using fluorescence intensity ratios at different wavelengths. After testing three laser excitation wavelengths (308, 337, and 480 nm) in normal and malignant bladder cell lines, 308 nm appeared to be the most promising wavelength since two fluorescence bands were observed at 360 and 440 nm; these were attributed to tryptophan (Trp) and reduced nicotinamide adenine dinucleotide (NADH) respectively. This study was then performed on freshly removed normal bladder and bladder tumor specimens exclusively using the 308-nm excitation wavelength. The tumor spectra, regardless of stage and grade, were very similar to the malignant cell spectra. However, a marked reduction of overall intensity was observed for carcinoma in situ (CIS). Normal bladder mucosa exhibited a shift of the first fluorescence band to 380 nm, indicating an overlap of Trp urothelial cell emission and collagen fluorescence derived from the lamina propria. The intensity of the NADH emission band was markedly reduced in tumor tissues compared with normal mucosa, which could indicate different redox conditions in urothelial tumors. A fluorescence intensity ratio at 360 and 440 nm can accurately discriminate normal or inflammatory mucosa from all bladder tumors, including CIS. These findings support the use of LIAFS as a new diagnostic technique for occult urothelial tumors.
Lund Reports in Atomic Physics, 1997
In this study three optical methods, autofluorescence, ALA-induced PpiX fluorescence and diffuse reflectance have been evaluated regarding their ability to detect malignant and dysplastic changes of human bladder tissue, in vivo. For the autofluorescence method, a single-fiber system based on a N2-laser was used. The 337 nm laser light excited the bladder tissue and the fluorescence was detected with an optical multichannel analyzer (OMA). The differences in the spectra from normal and malignant sites were investigated and the results showed a good demarcation between these tissue types. The sensitivity, specificity and positive and negative predictive values (non-malignant versus malignant) were found to be 73, 90, 73 and 90%, respectively. The second method utilized 5-aminolevulinic acid (ALA), which has a tendency to predominantly accumulate in malignant tissue. Through the heme cycle the ALA is converted to protoporphyrin IX (PpiX) which fluoresces red when excited with blue light. A filtered xenon-lamp was used to excite the PpiX and the red fluorescence was detected with a CCD camera mounted on a cystoscope. The images were stored on S-VHS tapes and the results were compared with the visual diagnosis made by the surgeon during the procedure as well as with the pathology report, if a biopsy of the site was taken. The easiness with which the PpiX is excited and the fluorescence detected, and the high tumor selectivity of ALA, make ALA-induced PpiX fluorescence a very helpful tool in finding tumors. The diffuse reflectance method utilized a white light source (xenon-lamp) and an OMA system to record the diffuse reflectance. By comparing the spectral differences between the malignant and non-malignant sites, a quantitative measure of the concentration of hemoglobin was obtained. In practice, this method showed only what was visible to the eye, that the tumor sites appeared red. Though, it provides a quantitative measure and avoids a subjective bias from the surgeon.
Cancer Gene Therapy, 2002
There has been no reliable orthotopic model available to visualize the growth of human superficial bladder cancer over time and to evaluate the efficacy of intravesical therapies. We have developed a novel approach to accomplish this task by generating human superficial bladder tumor cells to stably express high levels of green fluorescent protein ( GFP ) in vivo. Superficial bladder tumors were produced in athymic mice by intravesical instillation. In our initial studies tumors were quantitated by image analysis at a single time point, and the results compared to the estimation of the percentage of GFP cells present using flow cytometry after obtaining single cell suspensions of normal and tumor cells in the same bladder. A high correlation between the two methods was seen. Therefore, in subsequent studies, approximately 1 week after the intravesical instillation of the GFP expressing cancer cells a small incision was made to expose the bladder. The anterior, posterior, and lateral images of each bladder were captured to visualize GFPexpressing tumors. The ratio of green fluorescence pixel area, which represented the tumor burden, to the total area of the bladder was then calculated. A similar procedure was performed at 2, 3, and 4 weeks after instillation of the tumor cells. Using this procedure tumor progression over time could be measured in each mouse. By using this approach, it will now be possible to monitor the initial tumor sizes in the bladder of each mouse and then to evaluate the efficacy of various intravesical therapy protocols including intravesical gene therapy alone or in combination with other treatment modalities.
Ex Vivo Fluorescence Imaging of Normal and Malignant Urothelial Cells to Enhance Early Diagnosis
Photochemistry and Photobiology, 2007
Urinary cytology is a noninvasive and unconstraining technique for urothelial cancer diagnosis but lacks sensitivity for detecting low-grade lesions. In this study, the fluorescence properties of classical Papanicolaou-stained urothelial cytological slides from patients or from cell lines were monitored to investigate metabolic changes in normal and tumoral cells. Time- and spectrally-resolved fluorescence imaging was performed at the single cell level to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by urothelial cells. The results reveal quite different fluorescence distributions between tumoral urothelial cells, characterized by a perimembrane fluorescence localization, and the normal cells which exhibit an intracellular fluorescence. This is not caused by differences in the fluorescence emission of the endogenous fluorophores NAD(P)H, flavoproteins or porphyrins but by various localization of the EA 50 Papanicolaou stain as revealed by both the spectral and time-resolved parameters. The present results demonstrate that the use of single-cell endofluorescence emission of Papanicolaou-stained urothelial cytological slides can allow an early ex vivo diagnosis of low-grade bladder cancers.
Fluorescence detection of bladder cancer using urine cytology
International Journal of Oncology, 2007
Bladder cancer is the fourth most common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate in bladder cancer can be reduced by early treatment following pre-cancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimens, which is both invasive and time-consuming. In this study, ex vivo urine fluorescence cytology was investigated to offer an alternative timely and biopsy-free means for detecting bladder cancers. Sediments in patient urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cancer cells from the other cells based on the cellular size. Intensity histograms of each targeted cell and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cancer cells were observed. A diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this report. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder cancer diagnosis.
Photodiagnosis and Photodynamic Therapy, 2015
Background: Non-muscle invasive bladder cancer can be missed during white light endoscopy in up to 50% of cases. We aimed to test whether or not we could find a difference between benign and cancerous tissue wavelengths using laser induced autofluorescence spectroscopy can increase cancer detection. Materials and methods: We analysed 67 tissue samples using spectral analysis. The WavSTAT (Spectra Science) optical biopsy device was used to record fluorescence spectra from biopsied tissue enabling calculation of an AUC for each spectrum, a measure of the mean spectral wavelength ((nm)) and a dimensionless fluorescence ratio. Mann-Whitney test was used to compare the two groups. Results: We found that 49.3% (33/67) of the tissue was benign, 44.8% (30/67) was CIS/cancerous tissue, and the remaining 4/67 samples were atypia (2) and dysplasia (2). The median AUC for the benign tissue was 19.53 (interquartile range [IQR]: 5.35-30.39) and that for CIS/cancerous tissue was 7.05 (IQR: 2.89-14.24) (P = 0.002). The median wavelengths for the benign tissue and malignant tissue were 502.4 nm (IQR: 500.3-504.3 nm) and 505.2 nm (IQR: 502.1-513.2 nm), respectively (P = 0.003). The median fluorescence ratio was 0.080 (IQR: 0.070-0.088) for benign tissue and 0.096 (IQR: 0.079-0.221) for CIS/cancerous tissue (P = 0.002).