This study aims to determine the effect of varying gadopentetate dimeglumine (Gd-DTPA) dose on
Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) tracking of brain tumor photodynamic therapy
(PDT) outcome. Methods: We injected 2.5 x 105 U87 cells (derived from human malignant glioma) into the brains of six
athymic nude rats. After 9, 12, and 13 days DCE-MRI images were acquired on a 9.4 T micro-MRI scanner before and
after administration of 100, 150, or 200 μL of Gd-DTPA. Results: Tumor region normalized DCE-MRI scan
enhancement at peak was: 1.217 over baseline (0.018 Standard Error [SE]) at the 100 μL dose, 1.339 (0.013 SE) at the
150 μL dose, and 1.287 (0.014 SE) at the 200 μL dose. DCE-MRI peak tumor enhancement at the 150 μL dose was
significantly greater than both the 100 μL dose (p < 3.323E-08) and 200 μL dose (p < 0.0007396). Discussion: In this
preliminary study, the 150 μL Gd-DTPA dose provided the greatest T1 weighted contrast enhancement, while
minimizing negative T2* effects, in DCE-MRI scans of U87-derived tumors. Maximizing Gd-DTPA enhancement in
DCE-MRI scans may assist development of a clinically robust (i.e., unambiguous) technique for PDT outcome assessment.
Post-operative verification of the specificity and sensitivity of photodynamic therapy (PDT) is most
pressing for deeply placed lesions such as brain tumors. We wish to determine whether Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) can provide a non-invasive and unambiguous quantitative measure of the
specificity and sensitivity of brain tumor PDT. Methods: 2.5 x 105 U87 cells were injected into the brains of six athymic
nude rats. After 5-6 days, the animals received 0.5 mg/kg b.w. of the phthalocyanine photosensitizer Pc 4 via tail-vein
injection. On day 7 peri-tumor DCE-MRI images were acquired on a 7T microMRI scanner before and after tail-vein
administration of 100 μL gadolinium and 400 μL saline. After this scan the animals received a 30 J/cm2 dose of 672-nm
light from a diode laser (i.e., PDT). The DCE-MRI scan protocol was repeated on day 13. Next, the animals were
euthanized and their brains were explanted for Hematoxylin and Eosin (H&E) histology. Results: No tumor was found
in one animal. The DCE-MRI images of the other five animals demonstrated significant tumor enhancement increase (p
< 0.053 two-sided t-test and p < 0.026 one-sided t-test) following PDT. H&E histology presented moderate to severe
tumor necrosis. Discussion: The change in signal detected by DCE-MRI appears to be due to PDT-induced tumor
necrosis. This DCE-MRI signal appears to provide a quantitative, non-invasive measure of the outcome of PDT in this
animal model and may be useful for determining the safety and effectiveness of PDT in deeply placed tumors (e.g.,
glioma).
Introduction: We have previously demonstrated the use of phthalocyanine Pc 4 for the photodynamic therapy (PDT) of
ectopic human glial tumors in the athymic nude rat brain. We wish to determine whether 18F-fluorodeoxy-glucose (18F-FDG)
Positron Emission Tomography (PET) imaging can detect the reduction in tumor metabolism that must occur after
Pc 4-PDT-induced necrosis. Methods: 2.5 x 105 U87 cells were injected into the brains of 12 athymic nude rats. After 7
days of tumor growth, all 12 animals were imaged functionally by 18F-FDG micro-PET (&mgr;PET) and structurally by
micro-CT and/or micro-MR. These animals received 0.5 mg/kg b.w. Pc 4 via tail-vein injection. One day later the scalp
was re-incised and the tumor illuminated with 30 J/cm2 of 672-nm light from a diode laser. The next day these animals
were again 18F-FDG &mgr;PET imaged. Next, the animals were euthanized and their brains were explanted for H&E
histology. Results: Histology showed that tumors in the 6 Pc 4-PDT-treated animals demonstrated necrosis ranging from
full to frank (severe). Preliminary analysis showed that 18F-FDG &mgr;PET activity in 3 of the 6 non-PDT group (i.e., no
tumor necrosis observed) animals was seen to increase 2.28 times following tumor photoirradiation, whereas 18F-FDG
&mgr;PET activity in 5 of the 6 PDT group (i.e., tumor necrosis observed) animals was seen to increase 1.15 times following
tumor photoirradiation. Discussion: The increased 18F-FDG &mgr;PET activity in the PDT group was unexpected. We had
expected this activity to decrease and are presently investigating the cause of this observation.
Introduction: Several workers have proposed the use of PET (Positron Emission Tomography) imaging for the outcome
assessment of photodynamic therapy (PDT), especially for deep-seated tumors. We report on our study of 18Ffluorodeoxy-
glucose (18F-FDG) PET imaging following brain tumor Pc4-PDT. Our working hypothesis was that the
tumor's metabolic activity would decline dramatically following Pc 4-PDT owing to tumor necrosis. Methods: Seven
days after intraparenchymal implantation of U87 cells, the brains of 12 athymic nude rats were imaged by micro-CT
and/or micro-MR. These animals were also 18F-FDG micro-PET (&mgr;PET) scanned before and after Pc 4-PDT. 18F-FDG
was used to trace metabolic activity that was monitored via &mgr;PET. Occurrence of PDT was confirmed on histology. The
analysis of 18F-FDG dose and animal weight normalized &mgr;PET activity was studied over the 90 minute µPET scan.
Results: Currently, &mgr;PET data have been studied for: (1) three of the animals that did not indicate tumor necrosis on
histology and were assigned to a "Non-PDT" group, and (2) six animals that exhibited tumor necrosis on histology and
were assigned to a "PDT" group. The &mgr;PET-detected 18F-FDG uptake activity in the tumor region before and after
photoirradiation increased in the Non-PDT group an average of 2.28 times, and in the PDT group it increased an average
of 1.15 times. Discussion: We are investigating the cause of the increase in 18F-FDG &mgr;PET activity that we observed in
the PDT group. The methodology used in this study should be useful in determining whether this or other PET, SPECT,
or MR functional imaging protocols will detect both the specificity and sensitivity of brain tumor necrosis following Pc
4-PDT.
Introduction: Given the length of procedures and the brightness of operating room lights, there is concern that
photosensitizers used to locate brain tumors and treat them with photodynamic therapy (PDT) may photobleach before
they can be fully utilized. The phthalocyanine photosensitizer Pc 4 is resistant to photobleaching. In this study, we tested
the hypothesis that exposure of Pc 4-loaded glioma cells to photoactivating light will result in continuing fluorescence of
Pc 4. Methods: U87 human glioma cells were cultured in MEM with 5% penicillin/streptomycin, 5% sodium pyruvate,
10% fetal bovine serum, and 25 mM HEPES. These cultures were given 0 or 125 nM Pc 4, followed 2 hours later by
three separate exposures of 200 J/cm2 of red light (&lgr;max = 675 nm). Confocal fluorescence images were collected before
and after each exposure. Results: Pc 4 fluorescence was localized to cytoplasmic membranes of the U87 glioma cells, as
previously seen in other types of cells. After exposure to PDT, Pc 4 fluorescence was not reduced and even increased.
Discussion: Pc 4 may be useful for the intra-operative detection of glioma by fluorescence and for PDT, since neither Pc
4 level nor its fluorescence is likely to decrease during exposure to operating room lights.
Small animal imaging devices are now commonly used to study gene activation and model the effects of
potential therapies. We are attempting to develop a protocol that non-invasively tracks the affect of Pc 4-mediated
photodynamic therapy (PDT) in a human glioma model using structural image data from micro-CT and/or micro-MR scanning and functional data from 18F-fluorodeoxy-glucose (18F-FDG) micro-PET imaging.
Methods: Athymic nude rat U87-derived glioma was imaged by micro-PET and either micro-CT or micro-MR prior to Pc 4-PDT.
Difficulty insuring animal anesthesia and anatomic position during the micro-PET, micro-CT, and micro-MR scans required adaptation
of the scanning bed hardware. Following Pc 4-PDT the animals were again 18F-FDG micro-PET scanned, euthanized one day
later, and their brains were explanted and prepared for H&E histology. Histology provided the gold standard for tumor
location and necrosis. The tumor and surrounding brain functional and structural image data were then isolated and coregistered.
Results: Surprisingly, both the non-PDT and PDT groups showed an increase in tumor functional activity
when we expected this signal to disappear in the group receiving PDT. Co-registration of the functional and structural
image data was done manually. Discussion: As expected, micro-MR imaging provided better structural discrimination of the
brain tumor than micro-CT. Contrary to expectations, in our preliminary analysis 18F-FDG micro-PET imaging does not readily
discriminate the U87 tumors that received Pc 4-PDT. We continue to investigate the utility of micro-PET and other methods
of functional imaging to remotely detect the specificity and sensitivity of Pc 4-PDT in deeply placed tumors.
Introduction: Photodynamic therapy (PDT) for Barrett’s esophagus, advanced esophageal cancer, and both early and late inoperable lung carcinoma is now FDA-approved using the first generation photosensitizer PhotofrinTM (Axcan Pharma, Birmingham, AL). Photofrin-mediated PDT of glioma is now in Phase III clinical trials. A variety of second generation photosensitizers have been developed to provide improved: (1) specificity for the target tissue, (2) tumoricidal capability, and (3) rapid clearance the vascular compartment, skin, and eyes. The phthalocyanine Pc 4 is a second generation photosensitizer that is in early phase I clinical trials for skin cancer. We have undertaken a preclinical study that seeks to determine if Pc 4-mediated PDT can be of benefit for the intra-operative localization and treatment of glioma. Methods: Using a stereotactic frame, 250,000 U87 cells were injected via Hamilton syringe through a craniotomy, and the dura, 1-2 mm below the cortical surface of nude (athymic) rat brains (N=91). The craniotomy was filled with a piece of surgical PVC and the scalp closed. After two weeks of tumor growth, the animals received 0.5 mg/kg Pc 4 via tail vein injection. One day later the scalp was re-incised, and the PVC removed. The tumor was then illuminated with either 5 or 30 Joule/cm2 of 672-nm light from a diode laser at 50 mW/cm2. The animals were sacrificed one day later and the brain was cold-perfused with formaldehyde. Two thirds of the explanted brains are now being histologically surveyed for necrosis after staining with hematoxylin and eosin and for apoptosis via immunohistochemistry (i.e., TUNEL assay). The other third were analyzed by HPLC-mass spectrometry for the presence of drug in tumor, normal brain, and plasma at sacrifice. Initial histological results show PDT-induced apoptosis and necrosis confined to the growing (live) portion of the tumor. Preliminary analysis shows an average selectivity of Pc 4 uptake in the bulk tumor to be 3.8 times greater than in normal brain tissue. Discussion: The observed specificity and tumoricidal activity of Pc 4 warrants further preclinical studies to determine the preferred Pc 4 drug and light dose for future glioma patient clinical trials.
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