This PDF file contains the front matter associated with SPIE Proceedings Volume 8568, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Using two agents that preferentially target lysosomes for photodamage but produce different reactive oxygen species (ROS) upon irradiation, we examined effects of the degree of oxygenation on photokilling. Irradiation of the chlorin NPe6 results in a high yield of singlet oxygen but the bacteriopheophorbide WST11 forms only oxygen radicals. We found that PDT efficacy of WST11 was impaired when the ambient oxygen concentration was reduced to 1%, while that of NPe6 was essentially unaffected. These result were correlated with photobleaching effects. Use of fluorescent probes for singlet oxygen vs. OH radical revealed that photobleaching was correlated with ¹O₂ but not ^.OH formation during irradiation of solutions containing NPe6. Photoproduct formation could be followed by changes in absorbance spectra. In the presence of mercaptoethanol, different photoproducts were formed, showing that environmental factors could influence photochemical reactions.

A complete understanding of the biological mechanisms regulating devastating disease such as cancer remains elusive. Pancreatic and brain cancers are primary among the cancer types with poor prognosis. Molecular biomarkers have emerged as group of proteins that are preferentially overexpressed in cancers and with a key role in driving disease progression and resistance to chemotherapy. The epidermal growth factor receptor (EGFR), a cell proliferative biomarker is particularly highly expressed in most cancers including brain and pancreatic cancers. The ability of EGFR to sustain prolong cell proliferation is augmented by biomarkers such as Bax, Bcl-XL and Bcl-2, proteins regulating the apoptotic process. To better understand the role and effect of the microenvironment on these biomarkers in pancreatic cancer (PaCa); we analysed two pancreatic tumor lines (AsPc-1 and MiaPaCa-2) in 2D, 3D in-vitro cultures and in orthotopic tumors at different growth stages. We also investigated in patient derived glioblastoma (GBM) tumor cultures, the ability to utilize the EGFR expression to specifically deliver photosensitizer to the cells for photodynamic therapy. Overall, our results suggest that (1) microenvironment changes affect biomarker expression; thereby it is critical to understand these effects prior to designing combination therapies and (2) EGFR expression in tumor cells indeed could serve as a reliable and a robust biomarker that could be used to design targeted and image-guided photodynamic therapy.

Mathematic models were developed to simulate the complex dynamic process of photodynamic therapy (PDT). Macroscopic or microscopic modeling of singlet oxygen (¹O₂) is particularly of interest because it is the major cytotoxic agent causing biological effects during PDT. Our previously introduced macroscopic PDT model incorporates the diffusion equation for the light propagation in tissue and the macroscopic kinetic equations for the production of the ¹O₂. The distance-dependent distribution of ³O₂ and reacted ¹O₂ can be numerically calculated using finite-element method (FEM). We recently improved the model to include microscopic kinetic equations of oxygen diffusion from uniformly distributed blood vessels and within tissue. In the model, the cylindrical blood capillary has radius in the range of 2-5 μm and a mean length of 300 μm, and supplies oxygen into tissue. The blood vessel network is assumed to form a 2-D square grid perpendicular to a linear light source. The spacing of the grid is 60 μm. Oxygen can also diffuse along the radius and the longitudinal axial of the cylinder within tissue. The oxygen depletion during Photofrin-PDT PDT can be simulated using both macroscopic and microscopic approaches. The comparison of the simulation results have reasonable agreements when velocity of blood flow is reduced during PDT.

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases. EGFR activation upon binding of ligands (such as EGF and TGF-α) results in cell signaling cascades that promote cell proliferation, survival and apoptosis inhibition. As reported for many solid tumors, EGFR overactivation is associated with tumor development and progression, resistance to cancer therapies and poor prognosis. Therefore, inhibition of EGFR function is a rational cancer therapy approach. We have shown previously that 280 nm UV illumination of two cancer cell lines overexpressing EGFR could prevent phosphorylation of EGFR and of its downstream signalling molecules despite the presence of EGF. Our earlier studies demonstrated that UV illumination of aromatic residues in proteins leads to the disruption of nearby disulphide bridges. Since human EGFR is rich in disulphide bridges and aromatic residues, it is likely that structural changes can be induced upon UV excitation of its pool of aromatic residues (Trp, Tyr and Phe). Such changes may impair the correct binding of ligands to EGFR which will halt the process of tumor growth. In this paper we report structural changes induced by UV light on the extracellular domain of human EGFR. Steady state fluorescence spectroscopy and binding immunoassays were carried out. Our goal is to gain insight at the protein structure level that explains the way the new photonic cancer therapy works. This technology can be applicable to the treatment of various forms of cancer, alone or in combination with other therapies to improve treatment outcome.

Photodynamic therapy (PDT) with aminolevulinate (ALA) is widely accepted as an effective treatment for superficial carcinomas and pre-cancers. However, PDT is still suboptimal for deeper tumors, mainly due to inadequate ALA penetration and subsequent conversion to PpIX. We are interested in improving the effectiveness of photodynamic therapy (PDT) for deep tumors, using a combination approach (cPDT) in which target protoporphyrin (PpIX) levels are significantly enhanced by differentiation caused by giving Vitamin D or methotrexate (MTX) for 3 days prior to ALAPDT. In LNCaP and MEL cells, a strong correlation between inducible differentiation and expression of C/EBP transcription factors, as well as between differentiation and mRNA levels of CPO (a key heme-synthetic enzyme), indicates the possibility of CPO transcriptional regulation by the C/EBPs. Sequence analysis of the first 1300 base pairs of the murine CPO upstream region revealed 15 consensus C/EBP binding sites. Electrophoretic Mobility Shift Assays (EMSA) proved that these sites form specific complexes that have strong, moderate or weak affinities for C/EBPs. However, in the context of the full-length CPO promoter, inactivation of any type of site (strong or weak) reduced CPO promoter activity (luciferase assay) to nearly the same extent, suggesting cooperative interactions. A comparative analysis of murine and human CPO promoters revealed possible protein-protein interactions between C/EBPs and several neighboring transcription factors such as NFkB, Sp1, AP-1, CBP/p300 and CREB (an enhanceosome complex). Overall, these results confirm that C/EBP’s are important for CPO expression via complex mechanisms which upregulate PpIX and enhance the outcome of cPDT.

Aminolevulinic acid (ALA)-induced Protoporphyrin IX (PpIX)-based photodynamic therapy (PDT) is an effective treatment for skin cancers including basal cell carcinoma (BCC). Topically applied ALA promotes PpIX production preferentially in tumors, and many strategies have been developed to increase PpIX distribution and PDT treatment efficacy at depths > 1mm is not fully understood. While surface imaging techniques provide useful diagnosis, dosimetry, and efficacy information for superficial tumors, these methods cannot interrogate deeper tumors to provide in situ insight into spatial PpIX distributions. We have developed an ultrasound-guided, white-light-informed, tomographics spectroscopy system for the spatial measurement of subsurface PpIX. Detailed imaging system specifications, methodology, and optical-phantom-based characterization will be presented separately. Here we evaluate preliminary in vivo results using both full tomographic reconstruction and by plotting individual tomographic source-detector pair data against US images.

Photosensitizer fluorescence emitted during photodynamic therapy (PDT) is of interest for monitoring the local concentration of the photosensitizer and its photobleaching. In this study, we use Monte Carlo (MC) simulations to evaluate the relationship between treatment light and fluorescence, both collected by an isotropic detector placed on the surface of the tissue. In treatment of the thoracic and peritoneal cavities, the light source position changes continually. The MC program is designed to simulate an infinitely broad photon beam incident on the tissue at various angles to determine the effect of angle. For each of the absorbed photons, a fixed number of fluorescence photons are generated and traced. The theoretical results from the MC simulation show that the angle theta has little effect on both the measured fluorescence and the ratio of fluorescence to diffuse reflectance. However, changes in the absorption and scattering coefficients, μa and μ'_s do cause the fluorescence and ratio to change, indicating that a correction for optical properties will be needed for absolute fluorescence quantification. Experiments in tissue-simulating phantoms confirm that an empirical correction can accurately recover the sensitizer concentration over a physiologically relevant range of optical properties.

Photodynamic Therapy (PDT) has proven to be an effective treatment option for nonmelanoma skin cancers. The ability to quantify the concentration of drug in the treated area is crucial for effective treatment planning as well as predicting outcomes. We utilized spatial frequency domain imaging for quantifying the accurate concentration of protoporphyrin IX (PpIX) in phantoms and in vivo. We correct fluorescence against the effects of native tissue absorption and scattering parameters. First we quantified the absorption and scattering of the tissue non-invasively. Then, we corrected raw fluorescence signal by compensating for optical properties to get the absolute drug concentration. After phantom experiments, we used basal cell carcinoma (BCC) model in Gli mice to determine optical properties and drug concentration in vivo at pre-PDT.

Infra-red lasers (1268 nm) were reported to induce irreversible oxidative stress in cancer cells through direct triplet→single oxygen transition designating a novel cancer treatment equally with photodynamic therapy. We using in vitro and in silico approaches revealed that main impact on the cell oxidative state makes cascade of secondary reactive oxygen species triggered by primary laser-pulse-induced singlet oxygen and irreversible depletion of cellular antioxidative thioredoxin system in tumour. Based on these cancer cell features we can propose laser impulse strategy of killing cancer cells where initial impulse(s) may deplete antioxidant system making cancer cells deadly vulnerable to the next cascade of ROS by following impulse(s) at non-thermal doses.

Advanced stage ovarian carcinoma is characterized by poor prognosis and peritoneal micronodules that exhibit treatment resistance. This is partially due to interactions between multifocal disease and the tumor microenvironment, which includes tumor endothelial cells (TECs) and extracellular matrix components (ECM). Here we describe the development of a three-dimensional model of ovarian cancer that incorporates TECs and ECM. A comparison of several methodologies to generate endothelialized ovarian micronodules along with a preliminary physical characterization is described. This model will allow for detailed investigation of tumor-stroma interactions and how they impact disease progression and treatment response.

Photosensitizer-conjugated polyacrylamide nanoparticles were prepared for in vivo characterization of the minimally invasive and localized treatment of photodynamic therapy (PDT) on brain tumors. By incorporating a variety of nanoparticle matrixes, choosing methylene blue as a photosensitizer, and targeting the nanoparticle by the use of F3 peptide we have made nanoparticle-based PDT improvements to current PDT efficiency. Quantitative growth patterns were determined through visual observation of the tumorigenic response to various treatments by the use of an animal cranial window model. PDT treatments with methylene blue-polyacrylamide (MB-PAA) nanoparticles produced significant adjournment of tumor growth over control groups, clearly demonstrating the advantages of nanoparticle-based PDT agents for the eradication of local tumors, leading to the potential palliation of the advancing disease.

We undertook a phase I dose-escalation study of verteporfin photodynamic therapy (PDT) in 15 patients with locally advanced pancreatic cancer. Needle placement and laser delivery were technically successful in all patients. Thirteen patients were treated with a single laser fibre. Three treatments were carried out each at 5, 10 and 20 J/cm²; and 5 treatments (4 patients) at 40 J/cm². A further 2 patients were treated with 2 or 3 laser fibres at 40 J/cm². Tumour necrosis was measured on CT (computed tomography) by two radiologists 5 days after treatment. There was a clear dosedependent increase in necrosis with a median area of 20 x 16 mm (range 18 x 16 to 35 x 30 mm) at 40 J/cm². In the 2 patients treated with multiple fibres, necrosis was 40 x 36 mm and 30 x 28 mm, respectively. There were no early complications in patients treated with a single fibre. Both patients treated with multiple fibres had evidence on CT of inflammatory change occurring anterior to the pancreas but without clinical deterioration. These results suggest that single fibre verteporfin PDT is safe in a clinical setting up to 40J/cm² and produces a dose-dependent area of pancreatic necrosis.

Photodynamic therapy (PDT) using aminolevulinic acid or its methyl ester, methyl-aminolevulinate (MAL), is an increasingly recognized approach for treating squamous neoplasia of the skin. Advantages of MAL-PDT include its ability to cover broad diseased areas (field treatment), and to do multiple sessions with little-to-no risk of scarring or mutagenesis. MAL-PDT is especially valuable in certain populations at high risk for skin cancer, including Caucasian patients with extensive solar damage, and organ transplant recipients (OTR) who take immunosuppressive drugs to prevent graft rejection. The latter group has a 65-200 fold increased risk of developing squamous cell carcinoma (SCC), a major cause of mortality. Therapeutic options for those patients, other than frequent surgeries, are very limited. Topical 5-Fluorouracil (5-FU), frequently prescribed in normal patients for pre-SCC of the skin, is only minimally effective in the OTR group. MAL-PDT, however, has ~40% efficacy for pre-SCC in OTR patients. Based upon our preclinical studies in mouse tumor models, which showed that preconditioning with 5-FU can drive higher accumulation of target protoporphyins (PpIX), we proposed a rational combination regimen of 5-FU and MAL-PDT in humans. A clinical trial was designed to test the hypothesis that a combination of 5-FU followed by MAL-PDT will elevate PpIX levels and achieve better clinical outcomes in high-risk OTR patients. Primary endpoints include PpIX levels and biochemical markers (p53) measured noninvasively and in skin biopsies. Lesion clearance and recurrence (via photographs and clinical exam) are secondary endpoints. Ongoing results of this clinical trial are presented.

Pleural photodynamic therapy (PDT) has been used as an adjuvant treatment with lung-sparing surgical treatment for mesothelioma with remarkable results. In the current intrapleural PDT protocol, a moving fiber-based point source is used to deliver the light and the light dose are monitored by 7 detectors placed in the pleural cavity. To improve the delivery of light dose uniformity, an infrared (IR) camera system is used to track the motion of the light sources. A treatment planning system uses feedback from the detectors as well as the IR camera to update light fluence distribution in real-time, which is used to guide the light source motion for uniform light dose distribution. We have improved the GUI of the light dose calculation engine to provide real-time light fluence distribution suitable for guiding the surgery to delivery light more uniformly. A dual-correction method is used in the feedback system, so that fluence calculation can match detector readings using both direct and scatter light models. An improved measurement device is developed to automatically acquire laser position for the point source. Comparison of the effects of the guidance is presented in phantom study.

In fluorescence molecular tomography, optical measurements at the surface are used with diffusion theory modeling to reconstruct the maps of the fluorophore distribution in the tissue using an iterative error minimization algorithm. While normalizing the fluorescence signal with the excitation signal has been shown to correct for source and detector inconsistencies somewhat, this approach does not always correct for tissue heterogeneities and inaccuracies that are not matched by the forward diffusion model. Using computer simulations and an ultrasound-guided fluorescence tomography (FT) system designed for spatial mapping of Protoporphyrin IX (PpIX), the errors in fluorophore concentration recovery by assignment of incorrect optical properties are analyzed. Using simulations and experiments, white light spectroscopy was used to obtain more accurate tissue properties for forward diffusion model, prior to FT. Using white light spectroscopy the accuracy in FT values improved by 97% on average and the minimal detectable concentration of PpIX with the system was 0.025μg/ml.

A custom-made robotic multichannel platform for interstitial photodynamic therapy (PDT) and diffuse optical tomography (DOT) was developed and tested in a phantom experiment. The system, which was compatible with the operating room (OR) environment, had 16 channels for independent positioning of light sources and/or isotropic detectors in separate catheters. Each channel’s motor had an optical encoder for position feedback, with resolution of 0.05 mm, and a maximum speed of 5 cm/s. Automatic calibration of detector positions was implemented using an optical diode beam that defined the starting position of each motor, and by means of feedback algorithms controlling individual channels. As a result, the accuracy of zero position of 0.1 mm for all channels was achieved. We have also employed scanning procedures where detectors automatically covered the appropriate range around source positions. Thus, total scan time for a typical optical properties (OP) measurement throughout the phantom was about 1.5 minutes with point sources. The OP were determined based on the measured light fluence rates. These enhancements allow a tremendous improvement of treatment quality for a bulk tumor compared to the systems employed in previous clinical trials.

The dismal survival statistics for pancreatic cancer are due in large part to the notoriously poor response of these tumors to conventional therapies. Here we examine the ability of photodynamic therapy (PDT), using the photosensitizer verteporfin to enhance of the efficacy of traditional chemotherapy agents and/or eradicate populations that are nonresponsive to these agents. Using an in vitro 3D tumor model of pancreatic cancer combined with an imaging-based methodology for quantifying therapeutic response, we specifically examine PDT combination treatments with gemcitabine and oxaliplatin. We show that our 3D cell culture model recapitulates a more clinically-relevant dose response to gemcitabine, with minimal cytotoxic response even at high doses. The same cultures exhibit modest response to PDT treatments, but are also less responsive to this modality relative to our previous reports of monolayer dose response in the same cells. In combination we found no evidence of any enhancement in efficacy of either PDT or gemcitabine treatment regardless of dose or sequence (PDT before gemcitabine, or gemcitabine before PDT). However, when oxaliplatin chemotherapy was administered immediately after treatment with 2.5J/cm² verteporfin PDT, there was an observable enhancement in response that appears to exceed the additive combination of either treatment alone and suggesting there may be a synergistic interaction. This observation is consistent with previous reports of enhanced efficacy in combinations of PDT with platinum-based chemotherapy. The contrast in results between the combinations examined here underscores the need for rational design of mechanism-based PDT combinations.

Photodynamic therapy (PDT) dosimetry is an active area of study that is motivated by the need to reliably predict treatment outcomes. Implicit dosimetric parameters, such as photosensitizer (PS) photobleaching, may indicate PDT efficacy and could establish a framework to provide patient-customized PDT. Here, tumor destruction and benzoporphryin-derivative (BPD) photobleaching are characterized by systematically varying BPD-light combinations to achieve fixed PDT doses (M * J * cm^-2) in a three-dimensional (3D) model of micrometastatic ovarian cancer (OvCa). It is observed that the BPD-light parameters used to construct a given PDT dose significantly impact nodule viability and BPD photobleaching. As a result, PDT dose, when measured by the product of BPD concentration and fluence, does not reliably predict overall efficacy. A PDT dose metric that incorporates a term for BPD photobleaching more robustly predicts PDT efficacy at low concentrations of BPD. These results suggest that PDT dose metrics that are informed by implicit approaches to dosimetry could improve the reliability of PDT-based regimens and provide opportunities for patient-specific treatment planning.

A thorough understanding of light distribution in the desired tissue is necessary for accurate light dosimetry in PDT. Solving the problem of light dose depends, in part, on the geometry of the tissue to be treated. When considering PDT in the thoracic cavity for treatment of malignant, localized tumors such as those observed in malignant pleural mesothelioma (MPM), changes in light dose caused by the cavity geometry should be accounted for in order to improve treatment efficacy. Cavity-like geometries demonstrate what is known as the “integrating sphere effect” where multiple light scattering off the cavity walls induces an overall increase in light dose in the cavity. We present a Monte Carlo simulation of light fluence based on a spherical and an elliptical cavity geometry with various dimensions. The tissue optical properties as well as the non-scattering medium (air and water) varies. We have also introduced small absorption inside the cavity to simulate the effect of blood absorption. We expand the MC simulation to track photons both within the cavity and in the surrounding cavity walls. Simulations are run for a variety of cavity optical properties determined using spectroscopic methods. We concluded from the MC simulation that the light fluence inside the cavity is inversely proportional to the surface area.

In-vivo light dosimetry for patients undergoing photodynamic therapy (PDT) is critical for predicting PDT outcome. Patients in this study are enrolled in a Phase I clinical trial of HPPH-mediated PDT for the treatment of non-small cell lung cancer with pleural effusion. They are administered 4mg per kg body weight HPPH 48 hours before the surgery and receive light therapy with a fluence of 15-45 J/cm² at 661 and 665nm. Fluence rate (mW/cm²) and cumulative fluence (J/cm²) are monitored at 7 sites during the light treatment delivery using isotropic detectors. Light fluence (rate) delivered to patients is examined as a function of treatment time, volume and surface area. In a previous study, a correlation between the treatment time and the treatment volume and surface area was established. However, we did not include the direct light and the effect of the shape of the pleural surface on the scattered light. A real-time infrared (IR) navigation system was used to separate the contribution from the direct light. An improved expression that accurately calculates the total fluence at the cavity wall as a function of light source location, cavity geometry and optical properties is determined based on theoretical and phantom studies. The theoretical study includes an expression for light fluence rate in an elliptical geometry instead of the spheroid geometry used previously. The calculated light fluence is compared to the measured fluence in patients of different cavity geometries and optical properties. The result can be used as a clinical guideline for future pleural PDT treatment.

LEDs have more and more influence on daily life as well as on scientific equipment. In this paper we want to report results gained with the first LED powered setup for time resolved detection of the singlet oxygen luminescence in solution as well as in cell suspension. The results show, that this setup can compete with the best laser powered setups worldwide. The high sensitivity comes along with a superior long term stability and wavelength versatility. Setups based on LED excitation can simplify the technical part of such measurement very much and reduce the costs, making this technology available for a wider scientific community.

Photodynamic therapy with aminolevulinic acid can be modified by pretreatment regimens with drugs such as 5- Fluorouracil (5-FU) or Vitamin D (calcitriol) that enhance accumulation of protoporphyrin IX (PpIX) within tumor tissue which presumably will enhance the therapeutic response to light. However, histological approaches for monitoring therapeutic responses are poorly suited for studying long term survival because large numbers of mice need to be sacrificed. To address this limitation, a non-invasive model to monitor tumor regression and regrowth has been established. Breast cancer cells, stably transfected with firefly luciferase (MDA-Luc cell line), are implanted orthotopically in nude mice (0.25 - 1 x 10⁶ cells/site), and monitored 0-60 min after s.c. injection of luciferin, with Xenogen in-vivo imaging system. Luminescence is detectable at day 1 post-implantation. Tumors are suitable for experimentation on day 6, when daily injections of pretreatment agents (5-FU, 300 mg/kg; calcitriol, 1 μg/kg) begin. On day 9, ALA (75 mg/kg i.p.) is given for 4 hr, followed by illumination (633 nm, 100 J/cm²). Tumor luminescence post- PDT is monitored daily and compared with caliper measurements. Pretreatments (5-FU, calcitriol) by themselves do not inhibit luciferase expression, and all tumors grow at a similar rate during the pretreatment period. Results from in vivo survival experiments can be correlated to survival responses of MDA-Luc cells grown in monolayer cultures ± PDT and ± pretreatments, and additional mechanistic information (e.g. Ki67 and E-cadherin expression) obtained. In summary, this noninvasive model will permit testing of the therapeutic survival advantages of various pretreatments during cPDT.

The goal of this work was to determine the light dose required to induce necrosis in verteporfin-based photodynamic therapy, in the VERTPAC-1 trial. Patient CT scans were obtained of the abdomen, including the entire treatment zone of pancreas and surrounding tissues, before and after treatment, as well as fast scans during needle placement. These scans were used to estimate arterial and venous blood content, and provide structural information of the pancreas and nearby blood vessels. Using NIRFAST, a finite-element based package for modeling diffuse near-infrared light transport in tissue, simulations were run to create maps of light fluence within the pancreas. These maps provided visualizations of light dose overlaid on the original CT scans, and were used to estimate light dose at the boundary of the zone of necrosis, as observed in follow up treatment outcome CT scans. The aim of these simulation studies was to assist pre-treatment planning by informing the light treatment parameters. This paper presents a case study of the process used on a single patient.

Photodynamic therapy (PDT) offers a cancer treatment modality capable of providing minimally invasive localized tumor necrosis. To accurately predict PDT treatment outcome based on pre-treatment patient specific parameters, an explicit dosimetry model is used to calculate apparent reacted ¹O₂ concentration ([¹O₂]rx) at varied radial distances from the activating light source inserted into tumor tissue and apparent singlet oxygen threshold concentration for necrosis ([¹O2]_rx, sd) for type-II PDT photosensitizers. Inputs into the model include a number of photosensitizer independent parameters as well as photosensitizer specific photochemical parameters ξ σ, and β. To determine the specific photochemical parameters of benzoporphyrin derivative monoacid A (BPD), mice were treated with BPDPDT with varied light source strengths and treatment times. All photosensitizer independent inputs were assessed pre-treatment and average necrotic radius in treated tissue was determined post-treatment. Using the explicit dosimetry model, BPD specific ξ σ, and β photochemical parameters were determined which estimated necrotic radii similar to those observed in initial BPD-PDT treated mice using an optimization algorithm that minimizes the difference between the model and that of the measurements. Photochemical parameters for BPD are compared with those of other known photosensitizers, such as Photofrin. The determination of these BPD specific photochemical parameters provides necessary data for predictive treatment outcome in clinical BPD-PDT using the explicit dosimetry model.

During HPPH-mediated pleural photodynamic therapy (PDT), it is critical to determine the anatomic geometry of the pleural surface quickly as there may be movement during treatment resulting in changes with the cavity. We have developed a laser scanning device for this purpose, which has the potential to obtain the surface geometry in real-time. A red diode laser with a holographic template to create a pattern and a camera with auto-focusing abilities are used to scan the cavity. In conjunction with a calibration with a known surface, we can use methods of triangulation to reconstruct the surface. Using a chest phantom, we are able to obtain a 360 degree scan of the interior in under 1 minute. The chest phantom scan was compared to an existing CT scan to determine its accuracy. The laser-camera separation can be determined through the calibration with 2mm accuracy. The device is best suited for environments that are on the scale of a chest cavity (between 10cm and 40cm). This technique has the potential to produce cavity geometry in real-time during treatment. This would enable PDT treatment dosage to be determined with greater accuracy. Works are ongoing to build a miniaturized device that moves the light source and camera via a fiber-optics bundle commonly used for endoscopy with increased accuracy.

Photodynamic therapy (PDT) has recently emerged as a potential treatment alternative for head and neck cancer. There is strong evidence that imprecise PDT dosimetry results in variations in clinical responses. Quantitative tools are likely to play an essential role in bringing PDT to a full realization of its potential benefits. They can provide standardization of site-specific individualized protocols that are used to monitor both light and photosensitizer (HPPH) dose, as well as the tissue response for individual patients. To accomplish this, we used a custom instrument and a hand-held probe that allowed quantification of blood flow, blood volume, blood oxygen saturation and drug concentration.

Diffuse reflectance spectroscopy (DRS) is a common technique for assessing tissue optical parameters (absorption, scattering) non-invasively. However, choosing the correct model for light-tissue interaction is needed for accurate quantification. The diffusion approximation is only valid for certain ranges of tissue optical properties and specific probe geometries. For improved quantification with DRS over a wide range of optical properties and for a short source detector separation, a probe-specific light transport model can provide more accurate analysis of optical parameters. We developed and tested a probe-specific empirical model by using tissue simulating phantoms with promising results. We will apply it for the analysis of patients from a clinical trial for head and neck cancer.

PDT has become a treatment of choice especially for the cases with multiple sites and large areas. However, the efficacy of PDT is limited for thicker and deeper tumors. Depth and size information as well as vascularity can provide useful information to clinicians for planning and evaluating PDT. High-resolution ultrasound and photoacoustic imaging can provide information regarding skin structure and vascularity. We utilized combined ultrasound-photoacoustic microscopy for imaging a basal cell carcinoma (BCC) tumor pre-PDT and the results indicate that combined ultrasound-photoacoustic imaging can be useful tool for PDT planning by providing both structural and functional contrasts.

PDT dose is the product of the photosensitizer concentration and the light fluence in target tissue. Although existing systems are capable of measuring the light fluence in vivo, the concurrent measurement of photosensitizer in the treated tissue so far has been lacking. We have developed and tested a new method to simultaneously acquire light dosimetry and photosensitizer fluorescence data via the same isotropic detector, employing treatment light as the excitation source. A dichroic beamsplitter is used to split light from the isotropic detector into two fibers, one for light dosimetry, the other, after the 665 nm treatment light is removed by a band-stop filter, to a spectrometer for fluorescence detection. The light fluence varies significantly during treatment because of the source movement. The fluorescence signal is normalized by the light fluence measured at treatment wavelength. We have shown that the absolute photosensitizer concentration can be obtained by an optical properties correction factor and linear spectral fitting. Tissue optical properties are determined using an absorption spectroscopy probe immediately before PDT at the same sites. This novel method allows accurate real-time determination of delivered PDT dose using existing isotropic detectors, and may lead to a considerable improvement of PDT treatment quality compared to the currently employed systems. Preliminary data in patient studies is presented.

Our group has constructed a new type of viral nanoparticles (VNPs) from genome-depleted plant infecting brome mosaic virus (BMV) that encapsulates the FDA-approved near infrared (NIR) indocyanine green (ICG)[1]. We refer to these VNPs as optical viral ghosts (OVGs) since the constructs lack the genomic content of wild-type BMV. One of our areas of interest is the application of OVGs for real-time intraoperative NIR fluorescence imaging of small peritoneal ovarian tumor nodules. We target human epidermal growth factor receptor-2 (HER-2) expression in ovarian cancer as a biomarker associated with ovarian cancer, since its over-expression is linked to the disease’s progression to death. We functionalize the OVGs with anti-HER-2 monoclonal antibodies using reductive amination methods. We used fluorescence imaging to visualize the SKOV-3 cells (high HER-2 expression) after incubation with free ICG, OVGs, and functionalized OVGs. Our results suggest the possibility of using anti-HER2 conjugated OVGs in conjunction with cytoreductive surgery to detect small tumor nodules (<5cm) which currently are not excised during surgery.

An ultrasound-guided fluorescence molecular tomography system is under development for in vivo quantification of Protoporphyrin IX (PpIX) during Aminolevulinic Acid - Photodynamic Therapy (ALA-PDT) of Basal Cell Carcinoma. The system is designed to combine fiber-based spectral sampling of PPIX fluorescence emission with co-registered ultrasound images to quantify local fluorophore concentration. A single white light source is used to provide an estimate of the bulk optical properties of tissue. Optical data is obtained by sequential illumination of a 633nm laser source at 4 linear locations with parallel detection at 5 locations interspersed between the sources. Tissue regions from segmented ultrasound images, optical boundary data, white light-informed optical properties and diffusion theory are used to estimate the fluorophore concentration in these regions. Our system and methods allow interrogation of both superficial and deep tissue locations up to PpIX concentrations of 0.025ug/ml.

Glioblastoma (GBM) is an aggressive cancer with dismal survival rates and few new treatment options. Fluorescence guided resection of GBM followed by photodynamic therapy (PDT) has shown promise in several chemo- or radiotherapy non-responsive GBM treatments clinically. PDT is an emerging light and photosensitizer (PS) mediated cytotoxic method. However, as with other therapeutic modalities, the outcomes are variable largely due to the nonpersonalization of dose parameters. The variability can be attributed to the differences in heterogeneous photosensitizer accumulation in tumors. Building upon our previous findings on utilizing PS fluorescence for designing tumor-specific PDT dose, we explore the use of photoacoustic imaging, a technique that provides contrast based on the tissue optical absorption properties, to obtain 3D information on the tumoral photosensitizer accumulation. The findings of this study will form the basis for customized photodynamic therapy for glioblastoma and have the potential to serve as a platform for treatment of other cancers.

Advances in biophotonic medicine require new information on photodynamic mechanisms. In photodynamic therapy (PDT), a photosensitizer (PS) is injected into the body and accumulates at higher concentrations in diseased tissue compared to normal tissue. The PS absorbs light from a light source and generates excited-state triplet states of the PS. The excited triplet states of the PS can then react with ground state molecular oxygen to form excited singlet - state oxygen or form other highly reactive species. The reactive species react with living cells, resulting in cel l death. This treatment is used in many forms of cancer including those in the prostrate, head and neck, lungs, bladder, esophagus and certain skin cancers. We developed a novel numerical method to model the photophysical and photochemical processes in the PS and the subsequent energy transfer to O₂, improving the understanding of these processes at a molecular level. Our numerical method simulates light propagation and photo-physics in PS using methods that build on techniques previously developed for optical communications and nonlinear optics applications.

The goal of this work was to develop and validate a pancreas tumor animal model to investigate the relationship between photodynamic therapy (PDT) effectiveness and photosensitizer drug delivery. More specifically, this work lays the foundation for investigating the utility of dynamic contrast enhanced blood perfusion imaging to be used to inform subsequent PDT. A VX2 carcinoma rabbit cell line was grown in the tail of the pancreas of three New Zealand White rabbits and approximately 3-4 weeks after implantation the rabbits were imaged on a CT scanner using a contrast enhanced perfusion protocol, providing parametric maps of blood flow, blood volume, mean transit time, and vascular permeability surface area product.