In-vivo singlet oxygen dosimetry of clinical 5-aminolevulinic acid photodynamic therapy.

Photodynamic therapy (PDT) is a viable treatment option for a wide range of applications, including oncology, dermatology, and ophthalmology. Singlet oxygen is believed to play a key role in the efficacy of PDT, and on-line monitoring of singlet oxygen during PDT could provide a methodology to establish and customize the treatment dose clinically. This work is the first report of monitoring singlet oxygen luminescence in vivo in human subjects during PDT, demonstrating the correlation of singlet oxygen levels during PDT with the post-PDT photobiological response.

In vivo optical molecular imaging of vascular endothelial growth factor for monitoring cancer treatment.

PURPOSE: Vascular endothelial growth factor (VEGF) expression is a critical component in tumor growth and metastasis. Capabilities to monitor VEGF expression in vivo can potentially serve as a useful tool for diagnosis, prognosis, treatment planning, monitoring, and research. Here, we present the first report of in vivo hyperspectral molecular imaging strategy capable of monitoring treatment-induced changes in VEGF expression. EXPERIMENTAL DESIGN: VEGF was targeted with an anti-VEGF antibody conjugated with a fluorescent dye and was imaged in vivo using a hyperspectral imaging system. The strategy was validated by quantitatively monitoring VEGF levels in three different tumors as well as following photodynamic treatment. Specificity of the molecular imaging strategy was tested using in vivo competition experiments and mathematically using a quantitative pharmacokinetic model. RESULTS: The molecular imaging strategy successfully imaged VEGF levels quantitatively in three different tumors and showed concordance with results from standard ELISA. Changes in tumoral VEGF concentration following photodynamic treatment and Avastin treatment were shown. Immunohistochemistry shows that (a) the VEGF-specific contrast agent labels both proteoglycan-bound and unbound VEGF in the extracellular space and (b) the bound VEGF is released from the extracellular matrix in response to photodynamic therapy. In vivo competition experiments and quantitative pharmacokinetic model-based analysis confirmed the high specificity of the imaging strategy. CONCLUSION: This first report of in vivo quantitative optical molecular imaging-based monitoring of a secreted cytokine in tumors may have implications in providing tools for mechanistic investigations as well as for improved treatment design and merits further investigation.

Real-time fluorescence monitoring of phenothiazinium photosensitizers and their anti-mycobacterial photodynamic activity against Mycobacterium bovis BCG in in vitro and in vivo models of localized infection.

An objective was to explore the photodynamic activity of two cationic photosensitizers (PS) (benzo[a]phenothiazinium chloride and benzo[a]phenoselenazinium chloride) against Mycobacterium bovis BCG both in vitro and in a murine model of BCG-granuloma. The hypothesis being tested in this study was that cationic molecules could best interact with the negatively charged membrane of BCG as a model for mycobacterial infection. Cells in culture were incubated with various concentrations of PS and subsequently illuminated using a 635 nm diode laser. Dark- and light-induced killing profiles were generated as a function of fluence and dye concentration. In vivo, local injection of the PS into subcutaneous Mycobacterium-induced granuloma sites in murine model was followed by red light illumination of the same area. A special microscope was fabricated for real-time in vivo fluorescent microscopy to monitor EtNBS delivery to subcutaneous murine granulomata. Both PS demonstrated good in vitro antimycobacterial photodynamic activity with varying degrees of toxicity under dark conditions. Real time in vivo monitoring of benzophenothiazine chloride in the mouse model indicated that this fluorescent photosensitizer was delivered rapidly to the subcutaneous granuloma site. In vivo, photosensitizer specific dark- and photo-toxicities depended on the structure, concentration of the photosensitizer and the light dose utilized. Cationic phenothiazine photosensitizers are promising candidates for use in anti-mycobacterial PDT for localized diseases such as cutaneous and pulmonary granulomata.

A mechanism-based combination therapy reduces local tumor growth and metastasis in an orthotopic model of prostate cancer.

Therapy-induced stimulation of angiogenic molecules can promote tumor angiogenesis leading to enhanced tumor growth and cancer metastasis. Several standard and emerging therapies, such as radiation and photodynamic therapy (PDT), can induce angiogenic molecules, thus limiting their effectiveness. PDT is approved for the treatment of several cancers; however, its induction of vascular endothelial growth factor (VEGF) creates conditions favorable to enhanced tumor growth and metastasis, therefore mitigating its cytotoxic and antivascular effects. This is the first report showing that subcurative PDT in an orthotopic model of prostate cancer (LNCaP) increases not only VEGF secretion (2.1-fold) but also the fraction of animals with lymph node metastases. PDT followed by administration of an antiangiogenic agent, TNP-470, abolished this increase and reduced local tumor growth. On the other hand, administration of TNP-470 before PDT was less effective at local tumor control. In addition, animals in all groups, except in the PDT + TNP-470 group, had a weight loss of >3 g at the time of sacrifice; the weight of the animals in the PDT + TNP-470 group did not change. The significant reduction (P < 0.05) in tumor weight and volume observed between the PDT + TNP-470 group and the control group suggests that the combination of PDT and antiangiogenic treatment administered in the appropriate sequence was not only more effective at controlling local tumor growth and metastases but also reduced disease-related toxicities. Such molecular response-based combinations merit further investigations as they enhance both monotherapies and lead to improved treatment outcomes.

Mechanistic investigation and implications of photodynamic therapy induction of vascular endothelial growth factor in prostate cancer.

Photodynamic therapy (PDT) is now an approved therapeutic modality, and induction of vascular endothelial growth factor (VEGF) following subcurative PDT is of concern as VEGF may provide a survival stimulus to tumors. The processes that limit the efficacy of PDT warrant investigation so that mechanism-based interventions may be developed. This study investigates VEGF increase following subcurative PDT using the photosensitizer benzoporphyrin derivative (BPD) both in an in vitro and in an orthotopic model of prostate cancer using the human prostate cancer cell line LNCaP. The two subcurative doses used, 0.25 and 0.5 J/cm(2), mimicked subcurative PDT and elicited a 1.6- and 2.1-fold increase, respectively, in secreted VEGF 24 hours following PDT. Intracellular VEGF protein measurement and VEGF mRNA showed a 1.4- and 1.6-fold increase only at 0.5 J/cm(2). In vivo subcurative PDT showed an increase in VEGF by both immunohistochemistry and ELISA. In vitro analysis showed no activation of hypoxia-inducible factor-1alpha (HIF-1alpha) or cyclooxygenase-2 (COX-2) following subcurative PDT; furthermore, small interfering RNA inhibition of HIF-1alpha and COX-2 inhibitor treatment had no effect on PDT induction of VEGF. PDT in the presence of phosphatidylinositol 3-kinase/AKT inhibitor or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor still induced VEGF. However, subcurative PDT increased phosphorylated p38 and stress-activated protein kinase/c-Jun NH(2)-terminal kinase. The p38 MAPK inhibitor abolished PDT induction of VEGF. The results establish the importance of VEGF in subcurative BPD-PDT of prostate cancer and suggest possible molecular pathways for its induction. These findings should provide the basis for the development of molecular-based interventions for enhancing PDT and merit further studies.

Model-based analysis of clinical fluorescence spectroscopy for in vivo detection of cervical intraepithelial dysplasia.

We present a mathematical model to calculate the relative concentration of light scatterers, light absorbers, and fluorophores in the epithelium and stroma. This mathematical description is iteratively fit to the fluorescence spectra measured in vivo, yielding relative concentrations of each molecule. The mathematical model is applied to a total of 493 fluorescence measurements of normal and dysplastic cervical tissue acquired in vivo from 292 patients. The estimated parameters are compared with histopathologic diagnosis to evaluate their diagnostic potential. The mathematical model is validated using fluorescence spectra simulated with known sets of optical parameters. Subsequent application of the mathematical model to in vivo fluorescence measurements from cervical tissue yields fits that accurately describe measured data. The optical parameters estimated from 493 fluorescence measurements show an increase in epithelial flavin adenine dinucleotide (FAD) fluorescence, a decrease in epithelial keratin fluorescence, an increase in epithelial light scattering, a decrease in stromal collagen fluorescence, and an increase in stromal hemoglobin light absorption in dysplastic tissue compared to normal tissue. These changes likely reflect an increase in the metabolic activity and loss of differentiation of epithelial dysplastic cells, and stromal angiogenesis associated with dysplasia. The model presented here provides a tool to analyze clinical fluorescence spectra yielding quantitative information about molecular changes related to dysplastic transformation.

Calibration standards for multicenter clinical trials of fluorescence spectroscopy for in vivo diagnosis.

In the context of clinical trials, calibration protocols for optical instruments that ensure measurement accuracy and the ability to carry out meaningful comparisons of data acquired from multiple instruments are required. A series of calibration standards and procedures are presented to assess technical feasibility of optical devices for cervical precancer detection. Measurements of positive and negative standards, and tissue are made with two generations of research grade spectrometers. Calibration accuracy, ability of standards to correct and account for changes in experimental conditions, and device components are analyzed. The relative frequency of measured calibration standards is investigated retrospectively using statistical analysis of trends in instrument performance. Fluorescence measurements of standards and tissue made with completely different spectrometers show good agreement in intensity and lineshape. Frequency of wavelength calibration standards is increased to every 2 h to compensate for thermal drifts in grating mount. Variations in illumination energy detected between standards and patient measurements require probe redesign to allow for simultaneous acquisition of illumination power with every patient measurement. The use of frequent and well-characterized standards enables meaningful comparison of data from multiple devices and unambiguous interpretation of experiments among the biomedical optics community.

Reflectance spectroscopy for diagnosis of epithelial precancer: model-based analysis of fiber-optic probe designs to resolve spectral information from epithelium and stroma.

Reflectance spectroscopy is a promising technology for detection of epithelial precancer. Fiber-optic probes that selectively collect scattered light from both the epithelium and the underlying stroma are likely to improve diagnostic performance of in vivo reflectance spectroscopy by revealing diagnostic features unique to each layer. We present Monte Carlo models with which to evaluate fiber-optic probe geometries with respect to sampling depth and depth resolution. We propose a probe design that utilizes half-ball lens coupled source and detector fibers to isolate epithelial scattering from stromal scattering and hence to resolve spectral information from the two layers. The probe is extremely compact and can provide easy access to different organ sites.

Ball lens coupled fiber-optic probe for depth-resolved spectroscopy of epithelial tissue.

A ball lens coupled fiber-optic probe design is described for depth-resolved measurements of the fluorescence and reflectance properties of epithelial tissue. A reflectance target, fluorescence targets, and a two-layer tissue phantom consisting of fluorescent microspheres suspended in collagen are used to characterize the performance of the probe. Localization of the signal to within 300 microm of the probe tip is observed by use of reflectance and fluorescence targets in air. Differential enhancement of the fluorescence signal from the top layer of the two-layer tissue phantom is observed.

Combined reflectance and fluorescence spectroscopy for in vivo detection of cervical pre-cancer.

Optical technologies, such as reflectance and fluorescence spectroscopy, have shown the potential to provide improved point-of-care detection methods for cervical neoplasia that are sensitive, specific, and cost-effective. Our specific goals are to analyze the diagnostic potential of reflectance and fluorescence spectra, alone and in combination, to discriminate normal and precancerous cervical tissue in vivo and to identify which classification features contain significant diagnostic information. Reflectance spectra are measured at four source-detector separations and fluorescence emission spectra are measured at 16 excitation wavelengths, from 324 sites in 161 patients. These 20 spectral features are permuted in all possible combinations of one, two, and three; and classification algorithms are developed to evaluate the diagnostic performance of each combination. Algorithms based on fluorescence spectra alone yield better diagnostic performance than those based on reflectance spectra alone. The combination of fluorescence and reflectance do not significantly improve diagnostic performance compared to fluorescence alone, except in the case of discriminating high-grade precancers from columnar normal tissue. In general, fluorescence emission spectra at 330- to 360-nm and 460- to 470-nm excitation provide the best diagnostic performance for separating all pairs of tissue categories.

Analytical model to describe fluorescence spectra of normal and preneoplastic epithelial tissue: comparison with Monte Carlo simulations and clinical measurements.

Fluorescence spectroscopy has shown promise for the detection of precancerous changes in vivo. The epithelial and stromal layers of tissue have very different optical properties; the albedo is relatively low in the epithelium and approaches one in the stroma. As precancer develops, the optical properties of the epithelium and stroma are altered in markedly different ways: epithelial scattering and fluorescence increase, and stromal scattering and fluorescence decrease. We present an analytical model of the fluorescence spectrum of a two-layer medium such as epithelial tissue. Our hypothesis is that accounting for the two different tissue layers will provide increased diagnostic information when used to analyze tissue fluorescence spectra measured in vivo. The Beer-Lambert law is used to describe light propagation in the epithelial layer, while light propagation in the highly scattering stromal layer is described with diffusion theory. Predictions of the analytical model are compared to results from Monte Carlo simulations of light propagation under a range of optical properties reported for normal and precancerous epithelial tissue. In all cases, the mean square error between the Monte Carlo simulations and the analytical model are within 15%. Finally, model predictions are compared to fluorescence spectra of normal and precancerous cervical tissue measured in vivo; the lineshape of fluorescence agrees well in both cases, and the decrease in fluorescence intensity from normal to precancerous tissue is correctly predicted to within 5%. Future work will explore the use of this model to extract information about changes in epithelial and stromal optical properties from clinical measurements and the diagnostic value of these parameters.

Detecting the signal of the menstrual cycle in fluorescence spectroscopy of the cervix.

Fluorescence spectroscopy of the cervix has been shown to be an effective noninvasive diagnostic tool for cervical intraepithelial neoplasia (precancer). To assess the effect of the menstrual cycle on fluorescence spectroscopy, daily measurements were made on ten subjects for the length of their cycle. These measurements were analyzed to determine if there was a statistically significant signal associated with the menstrual cycle. A signal was found for emission wavelengths between 425 and 445 nm inclusive--near the main hemoglobin absorption band, the Soret band, at 420 nm. We suspect that the slight displacement of the Soret band is due to the nearby dominant NAD(P)H peak, which increases the signal-to-noise ratio and affects statistical significance. The signal consists of a reduction in fluorescence intensity for the first few days of the cycle. This analysis indicates that hemoglobin absorption is the main menstrual-cycle effect on the use of fluorescence spectroscopy on the cervix. The effect is confined to a small set of excitation/emission wavelengths and to approximately the first 8 days of the cycle. This suggests that any problems from the menstrual cycle can be avoided with a simple requirement that the device not be used during the period of menstrual bleeding.

Reflectance spectroscopy for in vivo detection of cervical precancer.

Optical technologies, in particular fluorescence spectroscopy, have shown the potential to provide improved detection methods for cervical neoplasia that are sensitive and cost effective through accurate, objective, instantaneous point-of-care diagnostic tools. The specific goals of this study were to analyze reflectance spectra of normal and neoplastic cervical tissue in vivo and to evaluate the data for use in diagnostic algorithm development. Spectroscopic measurements were obtained at four distinct source-detector separations from 324 sites in 161 patients. As the source-detector separation increases, greater tissue depth is probed. The average spectra of each diagnostic class differed at all source-detector separations, with the greatest differences occurring at the smallest source-detector separations. Algorithms, based on principal-component analysis and Mahalanobis distance classification, were developed and evaluated for all combinations of source-detector separations relative to the gold standard of colposcopically directed biopsy. The diagnostic combination of squamous normal versus high-grade squamous intraepithelial lesions gave good discrimination with a sensitivity of 72% and a specificity of 81%; discrimination of columnar normal versus high-grade squamous intraepithelial lesions also was good, with sensitivity of 72% and specificity of 83%. Thus, reflectance spectroscopy appears promising for in vivo detection of cervical precancer. Strategies that combine fluorescence and reflectance spectroscopy may enhance the discrimination capabilities.

Fluorescence spectroscopy for cervical precancer detection: Is there variance across the menstrual cycle?

This study assesses one possible cause of inter-patient variation in fluorescence spectroscopy of the cervix: the menstrual cycle. Ten patients with no history of an abnormal Pap smear were seen daily throughout 30 consecutive days of their cycle. Fluorescence excitation-emission matrices were measured from three cervical sites on each patient. Principal component analysis was used to determine which spectral regions varied with the day of the cycle. Classification was performed to assess the influence of menstrual cycle on precancer diagnosis. Variations in the principal component scores and the redox ratio values show that the fluorescence emission spectra at 340-380 nm excitation appear to correlate with the cell metabolism of the cervical epithelium throughout the menstrual cycle; these changes do not affect diagnostic classification. The menstrual cycle affects intra-patient variation but does not appear to cause a significant level of inter-patient variation. It does not need to be controlled for in optical detection strategies based on fluorescence spectroscopy.

Optimal excitation wavelengths for discrimination of cervical neoplasia.

Fluorescence spectroscopy has shown promise for the in vivo, real-time detection of cervical neoplasia. However, selection of excitation wavelength has in the past been based on in vitro studies and the availability of light sources. The goal of this study was to determine optimal excitation wavelengths for in vivo detection of cervical neoplasia. Fluorescence excitation-emission matrices (EEMs) were measured in vivo from 351 sites in 146 patients. Data were analyzed in pairs of diagnostic classes to determine which combination of excitation wavelengths yields classification algorithms with the greatest sensitivity and specificity. We find that 330-340-, 350-380-, and 400-450-nm excitation yield the best performance. The sensitivity and specificity for discrimination of squamous normal tissue and high-grade squamous intraepithelial lesion (HGSIL) were 71% and 77% on cross validation using three excitation wavelengths. These results are comparable with those found in earlier in vivo studies; however, in this study we find that the proportion of samples which are HGSIL influences performance. Furthermore stratification of samples within low-grade squamous intraepithelial lesion and HGSIL also appears to influence diagnostic performance. Future diagnostic studies should be carried out at these excitation wavelengths in larger groups so that data can be stratified by diagnostic subcategory, age and menopausal status. Similarly, large studies should be done in screening populations.