The utility of photochemical internalization (PCI) for the treatment of malignant gliomas was investigated
in vitro using: (1) monolayers consisting of F98 rat glioma cells, and (2) human glioma spheroids
established from biopsy-derived glioma cells. In both cases, the cytotoxicity of AlPcS2a- based PCI of
bleomycin was compared to: (1) AlPcS2a-PDT, and (2) bleomycin. In all cases, monolayers and spheroids
were incubated in AlPcS2a (18 h), bleomycin (4 h), or AlPcS2a (18 h) + bleomycin (4 h) and were
subsequently exposed to 670 nm light. Toxicity was evaluated using colony formation assays or spheroid
growth kinetics.
Neither F98 rat glioma cells in monolayer nor human glioma spheroids were found to be particularly
sensitive to the effects of low irradiance (5 mW cm-2), low radiant exposure (1.5 J cm-2) AlPcS2a -PDT.
Bleomycin was found to be moderately toxic to F98 cells in monolayer at relatively low concentrations -
incubation of F98 cells in 0.1 μg ml-1 for 4 hours resulted in 80% survival. Under similar incubation
conditions, the effects of bleomycin on human glioma spheroids were negligible. In both in vitro systems
investigated, the PCI effect was found to be significant. For example, PCI consisting of a radiant exposure
of 1.5 J cm-2 together with 0.25 μg ml-1 bleomycin resulted in approximately 20 and 65 % survival of F98
rat glioma cells and human glioma spheroids respectively. These results show that AlPcS2a-mediated PCI
can be used to enhance the efficacy of chemotherapeutic agents such as bleomycin in malignant gliomas.
Introduction: Failure of treatment for high grade gliomas is usually due to local recurrence at the site of surgical resection
indicating that a more aggressive form of local therapy, such as PDT, could be of benefit. PDT causes damage to
both tumor cells as well as cerebral blood vessels leading to degradation of the blood brain barrier with subsequent increase
of brain edema. The increase in brain edema following ALA-PDT was evaluated in terms of animal survival,
histopatological changes in normal brain and tumor tissue and MRI scanning. The effect of steroid treatment, to reduce
post-treatment PDT induced edema, was also examined.
Methods:Tumors were established in the brains of inbred BD-IX and Fisher rats. At various times following tumor induction
the animals were injected with ALA ip. and four hours later light treatment at escalating fluences and fluence
rates were given. Nontumor bearing control animals were also exposed to ALA-PDT in a similar manner to evaluate
damage to normal brain and degree of blood brain barrier (BBB) disruption.
Results: Despite a very low level of PpIX production in normal brain, with a 200:1 tumor to normal tissue selectivity
ratio measured at a distance of 2 mm from the tumor border, many animals succumbed shortly after treatment. A total
radiant energy of 54 J to non-tumor bearing animals resulted in 50% mortality within 5 days of treatment. Treatment of
tumor bearing animals with moderate fluence levels produced similar brain edema compared to higher fluence levels.
ALA PDT in nontumor bearing animals produced edema that was light dose dependent. PDT appeared to open the BBB
for a period of 24-48 hrs after which it was restored. The addition of post operative steroid treatment reduced the incident
of post treatment morbidity and mortality.
Conclusions: T2 and contrast enhanced T1 MRI scanning proved to be a highly effective and non-evasive modality in
following the development of the edema reaction and the degree and time course of BBB dysfunction thus allowing the
use of fewer animals.
Light and heat distributions are measured in a rat glioma model used in photodynamic therapy. A fiber delivering 632-nm light is fixed in the brain of anesthetized BDIX rats. Fluence rates are measured using calibrated isotropic probes that are positioned stereotactically. Mathematical models are then used to derive tissue optical properties, enabling calculation of fluence rate distributions for general tumor and light application geometries. The fluence rates in tumor-free brains agree well with the models based on diffusion theory and Monte Carlo simulation. In both cases, the best fit is found for absorption and reduced scattering coefficients of 0.57 and 28 cm−1, respectively. In brains with implanted BT4C tumors, a discrepancy between diffusion and Monte Carlo–derived two-layer models is noted. Both models suggest that tumor tissue has higher absorption and less scattering than normal brain. Temperatures are measured by inserting thermocouples directly into tumor-free brains. A model based on diffusion theory and the bioheat equation is found to be in good agreement with the experimental data and predict a thermal penetration depth of 0.60 cm in normal rat brain. The predicted parameters can be used to estimate the fluences, fluence rates, and temperatures achieved during photodynamic therapy.
A significant contributory factor to the poor prognosis of patients with glioblastoma multiforme is the inability of conventional treatments to eradicate infiltrating glioma cells. A syngeneic rat brain tumor model is used to investigate the effects of aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on small clusters of tumor cells sequestered in normal brain. The intrinsic sensitivity of rat glioma cells to PDT was investigated by exposing ALA-incubated cells to a range of radiant exposures and irradiances using 635 nm light. Biodistribution studies were undertaken on tumor-bearing animals in order to determine the tumor selectivity of the photosensitizer following systemic administration (i.p.) of ALA. Effects of ALA-PDT on normal brain and gross tumor were evaluated using histopathology. Effects of PDT on isolated glioma cells in normal brain were investigated by treating animals 48 h after tumor cell implantation: a time when the micro-clusters of cells are protected by an intact blood-brain-barrier (BBB). Rat glioma cells in monolayer are susceptible to ALA-PDT - lower irradiances are more effective than higher ones. Fluorescence microscopy of frozen tissue sections showed that photosensitizer is produced with better than 200:1 tumor-to-normal tissue selectivity following i.p. ALA administration. ALA-PDT resulted in significant damage to both gross tumor and normal brain, however, the treatment failed to prolong survival of animals with newly implanted glioma cells compared to non-treated controls if the drug was delivered either i.p. or directly into the brain. In contrast, animals inoculated with tumor cells pre-incubated in vitro with ALA showed a significant survival advantage in response to PDT.
Introduction: Failure of treatment for high grade gliomas is usually due to local recurrence at the site of surgical resec-tion indicating that a more aggressive form of local therapy could be of benefit. Photodynamic therapy (PDT) is a local form of treatment involving the administration of a tumor-localizing photosensitizing drug that is activated by light of a specific wavelength The results of in vitro experiments indicated that PDT, given at low fluence rates was substantially more effective at inhibiting glioma spheroid growth than short term high fluence rate regimes. This prompted the initia-tion of in vivo studies of low fluence rate 5-aminolevulinic acid (ALA) PDT in a rat glioma model.
Methods:BT4C cell line tumors were established in the brains of inbred BD- IX rats. Eighteen days following tumor induction the animals were injected with 125 mg/kg ALA ip. and four hours later light treatment at various fluences and fluence rates were given after the introduction of an optical fiber. Tumor histology and animal survival were examined.
Results: In vitro experiments verified that the cell line was sensitive to ALA PDT. Microfluorometry of frozen tissue sections showed that PpIX is produced with a greater than 20:1 tumor to normal tissue selectivity ratio four hours after ALA injection. Histological examination demonstrated neutrophil infiltration and tumor central necrosis in low fluence rate treated tumors.
Conclusions: Low fluence rate long term ALA mediated PDT had a more pronounced effect on tumor histology than single shot short duration treatments at similar total fluence levels.
The usefulness of repetitive ALA mediated PDT is being evaluated in a rat brain tumor model. In preliminary experiments an optical fiber was repeatedly introduced directly into the brain to apply light, requiring multiple surgical procedures. In an attempt to refine the animal model, an indwelling light applicator, using a modified microdialysis guide cannula has been developed. The cannula is fixed to the scull and can remain in the rat brain for several weeks without complications. In studies of repetitive PDT the need for repeated mounting in a stereotactic frame and surgical procedures can be eliminated. The applicator also makes it feasible to employ low fluence rates with long treatment times, which correspond to future clinically relevant protocols. The fluence rate distribution was measured in vivo and compared to diffusion theory and Monte Carlo simulations to estimate the tissue optical properties of the rat brain for light at 632 nm. The modeling suggested a reduced scattering coefficient 38 cm-1, an absorption coefficient 0.46 cm-1 and a penetration depth 0.14 cm.
The probability of achieving local control with current single-shot, intraoperative PDT treatments of intracerebral gliomas seems improbable due to the length of time required to deliver adequate light fluences to depths of 1 - 2 cm in the resection margin. The results of in vitro experiments indicated that PDT, repeated at weekly intervals, was substantially more effective at inhibiting glioma spheriod growth than single treatment regimes. This prompted the initiation of in vivo studies of repetitive PDT in a rat glioma model. BT4C cell line tumors were established in the brains of inbred BD-IX rats. Three days following tumor induction, the animals were injected with 250 mg/kg ALA i.p. and four hours later, after the introduction of an optical fiber, light treatment at various radiant energies was given over a 10- to 30-minute interval. Two additional treatments were given at weekly intervals. In vitro experiments verified that spheroids derived from the cell line were sensitive to ALA PDT. Microfluorometry of frozen tissue sections showed that PpIX is produced with a 10 - 20:1 tumor to normal tissue selectivity ratio four hours after 250 mg/kg ALA i.p. Toxic radiant energy levels for ALA PDT have been determined.
MDCK II cells in colonies of eight cells and in confluent monolayers were treated with two different sensitizers and blue light. In both cases cooperation between the treated cells were detected. Cells photosensitized with Photofrin died by necrosis after irradiation, while cells photosensitized with tetra(3-hydroxyphenyl)porphyrin died by apoptosis. The cooperative interactions between the cells are contrary to one of the fundamental paradigms of radiation biology, namely that the targets of radiation are independent of each other.
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