Prompted by a study of traumatic brain injury (TBI) in a model system of cultured astrocytes, we
discovered that low level laser illumination (LLL) at 660nm elevates the level of intracellular Ca2+. The coherence
of the illumination was not essential since incoherent red light also worked. For cells bathed in low Ca2+ saline so
that influx was suppressed, the Ca2+ level rose with no significant latency following illumination and consistent
with a slow leak of Ca2+ from storage such as from the endoplasmic reticulum and/or mitochondria. When the cells
were bathed in normal Ca2+ saline, the internal Ca2+ rose, but with a latency of about 17 seconds from the beginning
of illumination. Pharmacologic studies with ryanodine inhibited the light effect. Testing the cells with fluid shear
stress as used in the TBI model showed that mechanically induced elevation of cell Ca2+ was unaffected by
illumination.
Limited therapeutic options exist when chest wall recurrence form breast cancer progresses despite standard salvage treatment. As photodynamic therapy offers excellent response for cutaneous lesions this may be a possible indication for PDT. A total of 102 treatment fields were illuminated on 9 women with biopsy proven chest wall recurrence of breast cancer which was progressing despite salvage surgery, radiation, and chemi-hormonal therapy. PDT consisted of outpatient IV infusion of Photofrin at 0.8 mg/kg followed 48 hours laser by illumination at 140-170 J/cm2 via a KTP Yag laser coupled to a dye unit. No patient was lost to follow up. At 6 months post PDT; complete response, defined as total lesion elimination was 89 percent, partial response 8 percent, and no response 3 percent. No photosensitivity was seen and no patient developed scarring, fibrosis, or healing difficulties. Low dose Photofrin induced PDT is very active against chest wall lesions. Despite fragile and heavily pre-treated tissues, excellent clinical and cosmetic outcome was obtained. PDT is an underutilized modality for this indication.
A system for in vivo, fluorescence image-guided, non-contact point fluorescence spectroscopy is presented. A 442 nm HeCd laser is used as the fluorescence excitation source. An intensified CCD serves as the detector for both imaging and spectroscopy, on which two regions of 300 X 300 pixels were used for green (500 +/- 18 nm) and red (630 +/- 18 nm) imaging channels, and a strip of 600 X 120 pixels are used for emission spectroscopy (450 - 750 nm). At a working distance of 40 mm, the system has a spatial resolution of 0.16 mm and a spectral resolution of 5 nm. System performance is demonstrated in a carcinogenesis model in hamsters, where tumors were induced by painting DMBA in the cheek pouch. Autofluorescence and Photofrin-induced fluorescence measurements were performed every 2 weeks during the 18 weeks of tumor induction. Punch biopsies on selected animals were taken for histological staging. The results show that autofluorescence fluorescence can distinguish dysplasia from normal mucosal tissue model, utilizing the peak red intensity (or the red-to-green intensity ratio). Photofrin-induced fluorescence was superior to autofluorescence for differentiating high grade dysplasia from invasive cancer.
We investigated the in vivo fluorescence detection of premalignant and malignant lesions in carcinogen-induced tumors of the hamster buccal cheek pouch and micrometastases in rat lymph nodes using hexylpyropheophorbide-a (HPPH) at 0.5 mg/kg and He-Ne laser-induced fluorescence photometry. Time course studies of HPPH in hamster tumor model showed maximal relative fluorescence readings at 48 hours after i.p. injection in each stage (dysplasia: 0.35, papilloma: 0.58, squamous cell carcinoma: 1.04). Squamous cell carcinoma showed significantly greater fluorescence than papillomas and dysplasias at all time points (p < 0.01). Metastatic lymph nodes were significantly greater than normal lymph nodes at all time points (p < 0.01). Maximal fluorescence levels of normal lymph nodes were observed at 6 hours after i.v. injection. Metastatic lymph nodes still showed high fluorescence levels at 72 hours. Micrometastases showed fluorescence levels between the levels of metastatic and normal lymph nodes (normal: 0.40 < micrometastasis: 0.62 < metastasis: 0.75 at 48 hours after i.v. injection). The results demonstrated good correlation between fluorescence levels and histopathological developments at all time points. Therefore, HPPH may be a promising fluorophore for the detection of developing malignancies and occult disease.
Recently, a new photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-1 (HPPH) was developed for PDT which possesses more rapid clearance from skin and greater cytotoxicity per drug dose than Photofrin. The spectral characteristics of HPPH shows an absorption band at 665 nm (50,000 M-1cm(superscript -1 and peak emission at 680 nm. The aid of this study was to examine HPPH as a fluorescent diagnostic compound for the detection of transformed tissues using the in vivo fluorescence photodetector. The model of tissue transformation was the carcinogen (DMBA [9, 10-dimethyl-1, 2- benzanthracene])-induced premalignant and malignant lesions of the hamster buccal cheek pouch. The results demonstrated significant correlations between fluorescence levels and histological developments at all time points after injection. Time course studies of HPPH showed highest fluorescence readings at 48 hours after injection of 0.5 mg/kg HPPH (mild-moderate dysplasia, 0.35 +/- 0.17 volts; papillary disease with severe dysplasia, 0.58 +/- 0.33 volts; and squamous cell carcinoma, 1.04 $OM 0.32 volts). Therefore, it appears that HPPH may be a promising fluorophore for the detection of transformed tissues.
The use of the photosensitizer Photofrin' (dihematoporphyrin ether, DHE)' in the photodynarnic treatment of human disease is becoming well known and widely used. While a number of photosensitizers may be used for photodynamic therapy (PDT), the porphyrins, in particular Photofrin', have received the most attention. This is specifically true for human trials.25 Clinical studies in photodynamic therapy (PDT) have utilized lasers to take advantage of coupling efficiencies to optical fibers allowing light to be delivered to many areas of the body. This is particularly true in endoscopic PDT. Both interstitial and superficial delivery techniques can be applied using one of a variety of delivery fibers available. A fiber with an optically flat end with a lens to produce a spot with a homogeneous intensity is used for superficial applications. Diffusers of various lengths, at the tip of a fiber, produce a cylindrical isotropic pattern and are suited for either intraluminal or interstitial illuminations, Previous studies have measured space irradiance produced by cylindrical fibers to determine the extent to which conditions of use would affect light distribution.6 Further, those studies attempted to assess the impact of variations in light distribution on the therapeutic effect. The therapeutic effect, however, was based upon the use of the cylindrical fiber as an interstitial implant and conclusions, therefore, were made in that context. The manner in which energy is distributed in air by fiberoptics can be described in terms of those surfaces through which the energy flux (energy per unit area per unit time) is everywhere constant. Since the therapeutic light dose has units of energy per unit area, these surfaces are properly called 'isodose-rate' surfaces. A field of treatment that conforms to an isodose-rate surface receives an incidental uniform light dose in a time interval (sec) given by the numerical ratio of the light dose (Joules/cni2) to the dose rate (watts/cm2). The isodose-rate surfaces in the air of the microlens and the sphere diffuser are simple surfaces, namely planes perpendicular to the light beam and spheres centered on the light source, respectively. The isodose-rate surfaces of the cylinder diffuser do not have a simple cycometry. In the following, these surfaces are presented for a line source which is a good approximation of the cylinder diffuser.
KEYWORDS: Tumors, Photodynamic therapy, Tissues, In vitro testing, Pancreatic cancer, Pancreas, Tumor growth modeling, Resistance, Data modeling, Control systems
Photodynamic therapy (PDT) is a promising alternative in the treatment of pancreatic cancer in man, due to the low sensitivity of the normal pancreas to PDT as shown in preclinical studies. Investigations on four human pancreatic cancer lines (MIA PaCa-2, PaCa 1, PaCa 3, and CAPAN 2) in vitro demonstrated a considerable variety in PDT-sensitivity proportional to the degree of differentiation, which was related to photosensitizer-uptake (PhotofrinTM). The well differentiated pancreatic tumor line Capan 2 showed a close relationship between high cell density and increased PDT-resistance. The Photofrin uptake of Capan 2 at high cell densities could be increased by short trypsinization prior to photosensitizer exposure. The data supports the hypothesis that a complex intercellular organization reduces the cell surface available for photosensitizer uptake and may cause the relative PDT resistance of normal pancreatic tissues and highly differentiated tumors.
Neoplastic tissue can be detected by its increased fluorescence compared to surrounding normal tissue after the injection of the tumor-localizing compound Photofrin®. In vivo fluorescence photometry is a non-imaging photodetector which detects the 690 nm fluorescence of the porphyrin. The sensitivity of the instrumentation has allowed the detection of micrometastases in both pre-clinical and clinical studies using low, non-photosensitizing levels of the drug. The technique is now being applied to the 9,10 dimethyl-1,2-.benzanthracene (DMBA)induced hamster buccal cheek pouch carcinoma model to obtain data on the correlation between Photofrmn® uptake and tumor development. This model shows consistent time patterns of tumor development as well precancerous leukoplakia lesions and has been well documented as an animal model of oral epidermoid carcinogenesis. The buccal cheek pouches of Syrian Golden hamsters were exposed to a 0.5% DMBA in acetone thrice weekly for specified time durations. Hamsters were subsequently injected with 1.0 mgfkg of Photofrmn® within the various stages of tumor development. Twenty-four hours post-injection, fluorescence due to drug uptake was measured by in vivo fluorescence photometry. Mucosal tissues were subsequently biopsied and used for extraction assays. Results demonstrate that Photoflin® is retained in DMBA treated tissue with a linear relationship between length of application and Photofrin® uptake and fluorescence. This relationship establishes that premalignant lesions can be distinguished from normal tissue by Photofrmn® uptake and fluorescence and suggest that Photofrmn® uptake and fluorescence can be used in a predictive manner to diagnose and determine the progression of individual lesions.
KEYWORDS: Luminescence, Tumors, Lymphatic system, Tissue optics, Fluorescence spectroscopy, Tumor growth modeling, In vivo imaging, Animal model studies, Photometry, Cancer
The growth of microscopic tumor lesions at or beyond treatment field lesions poses major problems in the diagnosis and curative treatment of numerous cancers. Early detection techniques which clearly define the extent of condemned or field spread of disease may improve the primary treatment of the disease. In vivo fluorescence photometry is a non-imaging technique which digitally displays relative fluorescence values in volts. The sensitivity of the instrument has allowed the detection of micrometastases in both pre-clinical and clinical studies using drug doses that are 80-90 lower than those used therapeutically. This technique is now being applied in preliminary experiments to the hamster cheek pouch models to (1) discern varying grades of dysplasia; (2) levels of uptake of the drug in normal growing and quiescent tumors. Results will be shown in two models in which this technique has shown to be efficacious preclinically in the Pollard rat adenocarcinoma model in which micrometastases in the lymph node have been detected, and preliminary studies involving the hamster cheek pouch model in which the pouch is painted with 9, 10 dimethyl-1, 2-benzanthracene (DMBA) for initiation and promotion of tumors. Clinically results will be shown in which fluorescence detection, confirmed by biopsy and histopathological examination, was capable of detecting the existence of micrometastatic involvement of less than 100 cells.
Extraction procedures to quantitate porfimer sodium concentration in tissues were correlated with fluorescence measurements made in vivo, on hamster and rat normal pancreas and intra-pancreatic tumors. The uptake of photosensitizer has been shown to be high in both normal and malignant pancreatic tissues, in both animal models studied. Photobleaching of the drug, as evidenced by both techniques within the pancreatic tumor, occurs in a typical manner during PDT, with resultant tissue destruction. In contrast, when the normal pancreas is exposed to PDT, a negligible photobleaching effect, as well as a lack of tissue response, is observed. The lack of observable response is corroborated by a lack of measurable physiological response. Both serum amylase and serum glucose show acute changes up to 12 hours post treatment but quickly return to normal. HPLC analysis shows that the drug extracted from both the normal pancreas and intrapancreatic tumor is essentially the same as that extracted from other tissues and similar to that which has been injected into the animal. Fluorescence microscopy has shown that at time points between 12-120 hours the drug is associated with lymphatic channels. This would not, however, necessarily preclude normal tissue destruction. Similar results have been found with other photosensitizers. Understanding the lack of response in the pancreas may lead to a deeper understanding of the diseased state which is normally refractory to all therapy as well as understanding the fundamental concepts of the mechanisms of PDT.
A significant clinical problem in the local treatment of cutaneous
metastases of breast cancer (by any modality--surgery, radiation
therapy or photodynainic therapy) is the fact that the disease almost
always extends beyond the boundary of visible lesions in the form of
microscopic deposits. These deposits may be distant from the site of
visible disease but are often in close proximity to it and are
manifested sooner or later by the development of recurrent lesions at
the border of the treated area, thus the "marginal miss" in radiation
therapy, the "rim recurrence" in photodynamic therapy, and the
"incisional recurrence" following surgical excision. More intelligent
use of these treatment modalities demands the ability to detect
microscopic deposits of tumor cells using non-invasive methodology.
In vivo fluorescence measurements have been made possible by the
development of an extremely sensitive fiber optic in vivo fluorescence
photometer. The instrument has been used to verify that fluorescence
correlated with injected porphyrin levels in various tissues. The
delivery of light to excite and detect background fluorescence as well
as photosensitizer fluorescence in tissues has been accomplished using
two HeNe lasers emitting at 632.8 nm and 612 nm delivered through a
single quartz fiber optic. Chopping at different frequencies,
contributions of fluorescence may be separated. Fluorescence is
picked up via a 400 micron quartz fiber optic positioned appropriately
near the target tissue. Validation of these levels was made by
extraction of the drug from the tissues with resultant quantitation.
Recently, an extensive study was undertaken to determine if
fluorescence could be used for the detection of occult, clinically
non-palpable metastases in the lymph node of rats. This unique model
allowed for the detection of micrometastases in lymph nodes using very
low injected doses of the photosensitizer Photofrin II. Data obtained
revealed the ability to detect on the order of 50-100 cells using 0.25
mg/kg of sensitizer, a level 20 times lower than normally used for
treatment of animal tumors. These results indicate that Photofrin II
could be used for fluorescence detection of small metastatic tumors,
while substantially reducing the major side effect of PDT; namely,
prolonged photosensitivity. Results to be presented demonstrate the
ability of this technique to detect microscopic deposits of malignant
tumor cells in patients with metastatic breast cancer. These deposits
were found in clinically negative areas of the chest wall.
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