Uptake of 2-NBDG as a method to monitor therapy response in breast cancer cell lines.

This study quantifies uptake of a fluorescent glucose analog, (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG), in a large panel of breast cancer cells and demonstrates potential to monitor changes in glycolysis caused by anticancer and endocrine therapies. Expressions of glucose transporter (GLUT 1) and hexokinase (HK I), which phosphorylates 2-NBDG, were measured via western blot in two normal mammary epithelial and eight breast cancer cell lines of varying biological subtype. Fluorescence intensity of each cell line labeled with 100 lM 2-NBDG for 20 min or unlabeled control was quantified. A subset of cancer cells was treated with anticancer and endocrine therapies, and 2-NBDG fluorescence changes were measured. Expression of GLUT 1 was necessary for uptake of 2-NBDG, as demonstrated by lack of 2-NBDG uptake in normal human mammary epithelial cells (HMECs). GLUT 1 expression and 2-NBDG uptake was ubiquitous among all breast cancer lines. Reduction and stimulation of 2-NBDG uptake was demonstrated by perturbation with anticancer agents, lonidamine (LND), and a-cyano-hydroxycinnamate (a-Cinn), respectively. LND directly inhibits HK and significantly reduced 2-NBDG fluorescence in a subset of two breast cancer cell lines. Conversely, when cells were treated with a-Cinn, a drug used to increase glycolysis, 2-NBDG uptake was increased. Furthermore, tamoxifen (tam), a common endocrine therapy, was administered to estrogen receptor positive and negative (ER?/-) breast cells and demonstrated a decreased 2-NBDG uptake in ER? cells, reflecting a decrease in glycolysis. Results indicate that 2-NBDG uptake can be used to measure changes in glycolysis and has potential for use in early drug development.

Optical redox ratio differentiates breast cancer cell lines based on estrogen receptor status.

Autofluorescence spectroscopy is a powerful imaging technique that exploits endogenous fluorophores. The endogenous fluorophores NADH and flavin adenine dinucleotide (FAD) are two of the principal electron donors and acceptors in cellular metabolism, respectively. The optical oxidation-reduction (redox) ratio is a measure of cellular metabolism and can be determined by the ratio of NADH/FAD. We hypothesized that there would be a significant difference in the optical redox ratio of normal mammary epithelial cells compared with breast tumor cell lines and that estrogen receptor (ER)-positive cells would have a higher redox ratio than ER-negative cells. To test our hypothesis, the optical redox ratio was determined by collecting the fluorescence emission for NADH and FAD via confocal microscopy. We observed a statistically significant increase in the optical redox ratio of cancer compared with normal cell lines (P < 0.05). Additionally, we observed a statistically significant increase in the optical redox ratio of ER(+) breast cancer cell lines. The level of ESR1 expression, determined by real-time PCR, directly correlated with the optical redox ratio (Pearson's correlation coefficient = 0.8122, P = 0.0024). Furthermore, treatment with tamoxifen and ICI 182,870 statistically decreased the optical redox ratio of only ER(+) breast cancer cell lines. The results of this study raise the important possibility that fluorescence spectroscopy can be used to identify subtypes of breast cancer based on receptor status, monitor response to therapy, or potentially predict response to therapy. This source of optical contrast could be a potentially useful tool for drug screening in preclinical models.

Preferential accumulation of 5-aminolevulinic acid-induced protoporphyrin IX in breast cancer: a comprehensive study on six breast cell lines with varying phenotypes.

We describe the potential of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence as a source of contrast for margin detection in commonly diagnosed breast cancer subtypes. Fluorescence intensity of PpIX in untreated and ALA-treated normal mammary epithelial and breast cancer cell lines of varying estrogen receptor expression were quantitatively imaged with confocal microscopy. Percentage change in fluorescence intensity integrated over 610-700 nm (attributed to PpIX) of posttreated compared to pretreated cells showed statistically significant differences between four breast cancer and two normal mammary epithelial cell lines. However, a direct comparison of post-treatment PpIX fluorescence intensities showed no differences between breast cancer and normal mammary epithelial cell lines due to confounding effects by endogenous fluorescence from flavin adenine dinucleotide (FAD). Clinically, it is impractical to obtain pre- and post-treatment images. Thus, spectral imaging was demonstrated as a means to remove the effects of endogenous FAD fluorescence allowing for discrimination between post-treatment PpIX fluorescence of four breast cancer and two normal mammary epithelial cell lines. Fluorescence spectral imaging of ALA-treated breast cancer cells showed preferential PpIX accumulation regardless of malignant phenotype and suggests a useful contrast mechanism for discrimination of residual cancer at the surface of breast tumor margins.

Effect of optical clearing agents on the in vivo optical properties of squamous epithelial tissue.

BACKGROUND AND OBJECTIVES: Optical clearing agents (OCAs) have previously been shown to increase depth penetration within turbid tissue ex vivo. This paper quantifies tissue optical properties of the hamster cheek pouch model in order to provide a means to assess the effect of OCAs quantitatively in vivo. STUDY DESIGN/MATERIALS AND METHODS: Diffuse reflectance spectra were obtained from both cheeks of 12 hamsters before and after immersion in dimethyl sulfoxide (DMSO), glycerol or a phosphate buffer saline (PBS) control for 20 minutes. A Monte Carlo model was then utilized to derive the wavelength dependent reduced scattering and absorption coefficients. RESULTS: DMSO caused a statistically significant decrease in the absorption and reduced scattering coefficients derived by the model. Glycerol caused a statistically significant increase in the wavelength dependent absorption coefficient, but no statistically significant changes in the reduced scattering coefficient. CONCLUSIONS: DMSO and glycerol act upon tissues differently as reflected by the tissue optical properties, implying that not all OCAs are equally effective in optically clearing tissues.