The standard method of performing investigations in cell biology consists of studying the behavior of average populations to extract information of the events occurring at a cellular and molecular level. Despite the unquestionable amount of knowledge accumulated with this approach, a closer look at the behavior in a single-cell and even subcellular scale is becoming more desirable as a way to study rare event populations. This becomes possible with the advent of an increasing number of fluorescent probes, which are compatible with cell viability and permit the monitoring of the morphological and physiological changes of a single cell following a particular challenge. The number of observed events can be kept large to ensure adequate population statistics. Changes associated with morphological and physiological responses are encoded with fluorescence parameters such as intensity, wavelength and spatial localization variations and fluorescence lifetime. In addition to this, following a particular challenge, confocal microscopy is a powerful tool to track these changes over time. It also provides a method of following the behavior of a large number of cells even at high magnifications, due to its versatility and automatization capabilities.
Interstitial Photodynamic therapy (IPDT) has been under intense investigation in recent years, with multiple clinical trials underway. This effort has demanded the development of optimization strategies that determine the best locations and output powers for light sources (cylindrical or point diffusers) to achieve an optimal light delivery. Furthermore, we have recently introduced cylindrical diffusers with customizable emission profiles, placing additional requirements on the optimization algorithms, particularly in terms of the stability of the inverse problem.
Here, we present a general class of linear feasibility algorithms and their properties. Moreover, we compare two particular instances of these algorithms, which are been used in the context of IPDT: the Cimmino algorithm and a weighted gradient descent (WGD) algorithm. The algorithms were compared in terms of their convergence properties, the cost function they minimize in the infeasible case, their ability to regularize the inverse problem, and the resulting optimal light dose distributions.
Our results show that the WGD algorithm overall performs slightly better than the Cimmino algorithm and that it converges to a minimizer of a clinically relevant cost function in the infeasible case. Interestingly however, treatment plans resulting from either algorithms were very similar in terms of the resulting fluence maps and dose volume histograms, once the diffuser powers adjusted to achieve equal prostate coverage.
Tailored light diffusers offer the flexibility of shaping the delivered light dose (fluence rate) distribution, potentially
leading to conformal light delivery. Because of scattering and absorption, tissue acts as a spatial low pass
filter of the diffuser's emission profile, and therefore some dose distributions with high spatial frequencies cannot
be delivered. We characterize the set of attainable light dose distributions in terms of the spatial frequency of
the emission profile and identify regimes where such distributions are less sensitive to changes in optical properties.
Furthermore, we contrast two different algorithms to solve the inverse problem: Simulated Annealing
(SA) and Non-negative Least Squares (NNLS). SA is plagued by superimposed high frequency components that
do not contribute significantly to the cost. We present an iterative low pass filter that smooths the emission
profile without considerably increasing the cost. A non-negative least square (NNLS) algorithm is also tested.
We conclude that non-negative least squares (NNLS) is superior to simulated annealing (SA) in terms of time
performance and cost minimization.
Enhanced green fluorescent protein (EGFP)-expressing cells are customarily used in a variety of in vitro and in vivo studies and assays to ease visualization and localization. Nonetheless, the effects of EGFP expression on cellular responsivity to Photodynamic therapy (PDT), a combination therapy combining a photoactive drug and light, have yet to be characterized. To address this effect, rat astrocytoma cells (CNS-1), a lentivirus-transduced EGFP variant (CNS-1 GFP), human glioblastoma (U-87), and the transfected EGFP variant (U-87 GFP) are analyzed in terms of cell survival following PDT mediated by two different photoactive drugs. Cell survival is quantified via colony forming assays and Alamar blue assays, as a function of light dose, using the photosensitizers Photofrin (1ug ml-1 for 24h) and ALA (200ug ml-1 for 5h). Furthermore, effect of GFP expression on the responsivity to Cisplatin, a DNA-binding chemotherapeutic agent is determined for these cell lines. Our results show that EGFP expression does not affect the responsivity of Photofrin-PDT in comparison to parental cell lines (non GFP expressing cells), but does alter that of ALA-PDT. No change in responsivity is observed for Cisplatin treatment for either cell line. These results can be explained by oxidative stress induced by EGFP expression. This work will establish under which circumstances it is appropriate to use EGFP-expressing cell lines in the context of PDT preclinical research in vivo and in vitro.
The effect of non-ideal cylindrical diffusers on the fluence rate distribution is studied in intraluminal and interstitial light delivery settings. Two types of diffuser non-uniformities are modeled: a forward-directed polar emission profile, and a longitudinal emission profile with a peak at the distal tip of the diffuser. These effects were compared with an ideal diffuser constructed via a superposition of isotropic point sources placed along the length of the diffuser. Monte Carlo simulations were run for a wide range of optical properties and the resulting fluence rate distribution were analyzed. Parameters describing the shape of these distributions were defined. The longitudinal profile most strongly influenced the shape of the fluence rate distribution displaying high local fluence rates, high degrees of asymmetry, and deeper penetration into tissue. These characteristics are particularly evident for interstitial illumination. In intraluminal illumination, both non-ideal profiles produced a shift of the fluence rate, but, while remaining largely insensitive to optical properties for the longitudinal diffuser, the position of the peak fluence rate varied to a considerable extent for the polar anisotropic diffuser, particularly as a function of albedo. Measurement of the polar emission profile and its inclusion in treatment planning, based on the tissue optical properties, is recommended for the intraluminal geometry. The longitudinal emission profile should be determined regardless of the application, together with knowledge of the tissue optical properties.
The potential for the use of photodynamic therapy (PDT) after resection of brain tumors is currently limited by the ensuing side effects. These include direct non-specific tissue damage due to the photodynamic action and elevated intracranial pressure as a result of edema and subsequent indirect tissue damage. Erythropoietin (EPO) has been recognized to confer resistance to apoptosis of neurons and endothelial cells in the brain. Here we present preliminary results of the combination of Photofrin – PDT and EPO in the treatment of rat brain astrocytomas in vivo and its ability to increase the therapeutic ratio versus stand alone PDT. The effects of the combination treatment are characterized in normal rat brain based on tissue damage (using TTC as a live cell stain) and monitoring intracranial pressure (ICP) for 24 hours following surgery, using a piezoelectric transducer. To access tumor cell kill, EGFP transduced astrocytoma cell line, CNS-1_gfp, is implanted in the cortex of Lewis rats through a craniotomy, allowed to grow to a diameter of 3mm. Immediately after PDT tumors are excised with the aid of a fluorescence microscope, desaggregated, counted under fluorescence and plated for colony forming assays. Tumor cell kill due to PDT is compared in the presence and absence of EPO.
Glioblastoma Multiforme is the most common form of malignant brain tumors and accounts for approximately 25% of all primary brain tumors. Only 5% of these patients survive longer than 2 years. The standard form of treatment is radiation therapy and surgery if the site is accessible. Different forms of adjuvant chemotherapy have been largely proven unsuccessful. Another form of adjuvant therapy, Photodynamic Therapy (PDT), has undergone preliminary trials
showing some promising results but at the cost of increased side effects like rise in intracranial blood pressure and neurological deficiency. Apoptotic cell kill used as a biological treatment endpoint can possibly ameliorate these side effects. This study evaluates the significance of apoptotic cell death in the 9L rat gliosarcoma using the aminolevulinic acid (ALA) induced endogenous photosensitizer Protophorphyrin IX (PpIX). A strong influence of drug incubation time with cell kill was observed. The percentage of apoptotic cell death was less than 10% for 2 and 4 hours incubation times and irradiation times ensuring up to 70 and 80% cell kill respectively. Accumulation of PpIX in the mitochondria and cytoplasm was quantified by confocal fluorescence microscopy showing a linear relationship of PpIX fluorescence with concentration. The possibility of an in vitro threshold in the PDT dose is discussed, above which cell repair mechanisms may become exhausted. In conclusion for the range of parameters investigated, apoptotic cell kill may be hard to exploit therapeutically in this tumor model.
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