Photodynamic therapy (PDT) is ideally suited for the treatment of head and neck cancer (HNC) in humans. Developing useful PDT protocols for HNC is challenging due to the expense of Phase I and II clinical trials. Moreover, the often-poor predictive value of murine models means that photosensitizers may proceed far into development before problems are noted. Dogs and cats with spontaneous oral squamous cell carcinoma (SCC) share striking similarities with humans affected with oral SCC. These similarities include viral and environmental tobacco smoke as risk factors, location-dependent prognoses, and relative resistance to chemotherapy. The relatively large oral cancers encountered in veterinary patients allow for light and drug dosimetry that are directly applicable to humans. The irregular shape of oral SCC allows a rigorous evaluation of novel photodynamic therapy protocols under field conditions. Because spontaneous tumors in dogs and cats arise in an outbred animal population it is possible to observe an intact host response to PDT. The shorter lifespan of dogs and cats allows rapid accrual of endpoint data. External beam radiation therapy and chemotherapy are commonplace in veterinary medicine, making dogs and cats with spontaneous SCC a useful resource to study the interactions with PDT and other cancer treatment modalities. Our preliminary results demonstrate that PDT is well-tolerated by dogs with oral cancer, and a Phase II clinical trial of zinc-phthalocyanine-based photodynamic therapy is underway in dogs with oral SCC. The usefulness of 5-aminolevulinic acid methyl ester-based PDT is being investigated in cats with oral SCC.
Approximately 12,000 people are diagnosed with invasive transitional cell carcinoma of the urinary bladder (InvTCC) each year in the United States. Surgical removal of the bladder (cystectomy) and regional lymph node dissection are considered frontline therapy. Cystectomy causes extensive acute morbidity, and 50% of patients with InvTCC have occult metastases at the time of diagnosis. Better staging procedures for InvTCC are greatly needed. This study was performed to evaluate an intra-operative near infrared fluorescence imaging (NIRF) system (Frangioni laboratory) for identifying sentinel lymph nodes draining InvTCC. NIRF imaging was used to map lymph node drainage from specific quadrants of the urinary bladder in normal dogs and pigs, and to map lymph node drainage from naturally-occurring InvTCC in pet dogs where the disease closely mimics the human condition. Briefly, during surgery NIR fluorophores (human serum albumen-fluorophore complex, or quantum dots) were injected directly into the bladder wall, and fluorescence observed in lymphatics and regional nodes. Conditions studied to optimize the procedure including: type of fluorophore, depth of injection, volume of fluorophore injected, and degree of bladder distention at the time of injection. Optimal imaging occurred with very superficial injection of the fluorophore in the serosal surface of the moderately distended bladder. Considerable variability was noted from dog to dog in the pattern of lymph node drainage. NIR fluorescence was noted in lymph nodes with metastases in dogs with InvTCC. In conclusion, intra-operative NIRF imaging is a promising approach to improve sentinel lymph node mapping in invasive urinary bladder cancer.
Photodynamic therapy (PDT) involves the light activation of a drug within a tumor causing selective tumor cell death. Unfortunately, some photosensitizing drugs have been associated with adverse reactions in veterinary patients. Zinc phthalocyanine tetrasulfonate (ZnPcS4) is a promising second-generation photosensitizer for use in veterinary medicine, however, it cannot be applied clinically until safety and efficacy data are available. ZnPcS4 was given to Swiss Webster mice to assess acute toxicity. Doses >100 mg/kg were associated with acute toxicity and mortality, and doses >100 mg/kg resulted in renal tubular nephrosis, suggesting that the minimum toxic dose is approximately 100 mg/kg. Based on these data, a Phase I clinical trial of ZnPcS4-based PDT in tumor-bearing dogs is underway, with ZnPcS4 doses up to 2 mg/kg producing no apparent toxicity. Tumor response has been observed after ZnPcS4-based PDT using doses as low as 0.25 mg/kg, suggesting that conventional phase I clinical trials may not be appropriate for PDT protocols.
Cultured 9L cells were incubated with graded doses of pheophorbide-a-hexyl ether (HPPH) and exposed to 665 nm red light from either a noncoherent light source or a KTP-pumped dye laser. Cell death was observed after irradiation using either light source, with the noncoherent light being most effective at the highest HPPH concentrations. To determing the practicality of using the noncoherent light source for clinical PDT, dogs and cats with spontaneous tumors were injected intravenously with 0.15 mg/kg HPPH one hour before their tumors were irradiated with 665 nm noncoherent light (50 mW cm-2, 100 J cm-2). Of the 9 tumors treated, 8 complete responses were observed, all of which occurred in animals with squamous cell carcinoma. After 68 weeks of follow up, the median initial disease free interval had not been reached. These data support the use of noncoherent light sources for PDT of spontaneous tumors in animals, representing a cost-effective alternative to medical lasers in both veterinary and human dermatology and oncology.
Laser immunotherapy is a novel method developed to treat metastatic tumors. The selective photothermal interaction using a near-infrared laser and indocyanine green destroys living tumor cells while preserving antigenic tissue derived protein. The treated tumor remained in the host as a source of exposed tumor antigens. Working in tandem with the applications of immunoadjuvant, laser immunotherapy has shown to induce an anti-tumor immunity in a metastatic mammary tumor model in rats. To further study the effect of laser immunotherapy, the immunogenicity of the rat model, DMBA-4, was investigated through surgical removal or primary tumors and by tumor immunization using freeze-thaw tumor cell lysates. The surgical removal of the primary tumors did not have any effect on the recurrence of the tumors at the primary sites. The metastases at remote sites also developed after the surgery. In the tumor challenges following the immunization, the immunized rats only had delayed emergence of the primary tumors and the metastases. All the immunized rats died with multiple tumors. The laser immunotherapy cured rats, on the other hand, showed strong tumor resistance in repeated tumor rechallenges. Our results showed that the laser immunotherapy could induce a strong anti-tumor immunity in a poorly immunogenic tumor model. Furthermore, the effect of laser immunotherapy on the metastases at remote sites indicated that this novel method could become an effective method in treating the metastatic tumors.
Induced anti-tumor immunity can be the most effective and long-term cure for cancers, particularly for metastatic tumors. Laser immunotherapy has been developed to induce such immunological responses in rats bearing DMBA-4 metastatic mammary tumors. It involves an intratumoral administration of a laserabsorbing dye (indocyanine green) and a specially formulated immunoadjuvant (glycated chitosan), followed by an irradiation of a near-infrared laser (805-nm diode laser). To understand the immunity induced in this tumor model, immunization using freeze-thaw cell lysates against the DMBA-4 tumors was performed, followed by the tumor challenge twenty-one days later. Also performed is the surgical removal of the primary tumors of the rats before the observation of metastatic tumors. The immunization only delayed the emergence of the primary and metastases in the rats but did not provide immunity against the tumor challenge. After surgical removal of the primary tumors, the tumors re-emerged at the primary sites and the metastases developed at multiple remote sites. In contrast, laser immunotherapy cured rats experienced tumor regression and eradication. Our research has provided strong support for the working mechanism of laser immunotherapy. The experimental results showed that selective photothermal laser-tissue interaction with a complementary use of immunoadjuvant could be a potential therapy for treatment of metastatic tumors by inducing a tumor-specific, long-lasting immunity.
Laser immunotherapy is a special cancer treatment modality using an intratumor injection of a special formulation consisting of a novel immunoadjuvant and a laser-absorbing dye, followed by a non-invasive near-IR laser irradiation. Our early experiments using a metastatic mammary rat tumor model showed that laser immunotherapy could cause acute selective photothermal tumor destruction and induce a systemic, long-term specific anti-tumor immunity. In the current study, laser immunotherapy was used to treat metastatic prostate tumors in Copenhagen male rats. The transplantable tumors metastasize mainly to the lung and the lung cancer is usually the cause of death. Two experimental were performed in our study. The first was to study the effect of laser immunotherapy on the tumor burdens, both the primary and the metastasis in the lung. The second was to study the effect of laser immunotherapy on the long-term survival of the tumor-bearing rats. For comparison, some rat tumors were also treated by the laser-dye combination to study the photothermal effect. Tour results showed that both the photothermal effect and the laser immunotherapy could slow the growth of primary tumors and the metastatic tumors. The laser-dye-immunoadjuvant treatment resulted in more than 20 percent long-term survival rate in tumor-bearing rats. Our experimental results indicate that the laser immunotherapy has a great potential in treating metastatic tumors.
Photothermal interaction of an 805-nm diode laser with an absorptive dye, indocyanine green (ICG), has been shown to be an efficacious therapy for metastatic breast tumors in a rat model when combined with immunoadjuvant. When ICG solution was injected into the target tissue, selective tissue destruction can be achieved. To study the selective photothermal interaction, temperature change in irradiated tissue was achieved. To study the selective photothermal interaction, temperature change in irradiated tissue was studied using chicken breast tissue and phantom composed of gelatin and intralipid. ICG solution was mixed with gel phantom to simulate dye-enhanced target tissue. The target gel was then embedded in chicken breast tissue. The temperature change of irradiated chicken and gel phantom was measured by needle temperature probes at different tissue depths and radii from the center of the laser beam to construct a two-dimensional array of temperature change in a cylindrical coordinate system. It was shown that it is possible to selectively raise the temperature of deep target tissue while not substantially heating nontargeted tissue. A Nd:YAG laser was also used to irradiate the tissue-gel system and the photothermal results were compared with that using the 805-nm laser.
Tumor cell destruction usually induces host immune responses, such as local inflammation and increased activities of macrophages and neutrophils. Use of immunoadjuvant can usually enhance such immune activities. Laser immunotherapy is designed to use the combination of laser photothermal and immunological interactions to induce long-term antitumor immunity with the help of immunoadjuvant. It uses a selective hyperthermia for acute tumor destruction through an intratumor administration of indocyanine green and a noninvasive irradiation by an 805-nm laser. The concurrent in situ administration of immunoadjuvant helped achieve the desired effect: tumor eradication and antitumor immunity. The current study further explores the function of immunoadjuvants in laser immunotherapy by testing four different adjuvants: glycated chitosan, complete Freund's adjuvant, incomplete Freund's adjuvant, and c-parvum. Each adjuvant provided long-term tumor cure in the treatment of a metastatic mammary tumor model in rats. However, glycated chitosan and complete Freund's adjuvant were most effective with 25% and 18% long- term cure rates, respectively. Different concentrations of glycated chitosan were also used in treatment of rats bearing metastatic breast tumors.
The Ho:YAG laser, used successfully in humans, may be effective for treating canine urolithiasis. The purpose of this study was to determine the in vitro effects of various Ho:YAG laser operating parameters on canine uroliths and a urethra model. Canine uroliths of known composition and cadaveric porcine urethral tissue were irradiated with the Ho:YAG laser. Fragmentation time was analyzed as a function of urolith composition, water weight, Hertz, pulse mode, and energy. All uroliths were completely fragmented with 20 seconds or less of laser irradiation. Calcium oxalate, cystine, and silica uroliths had significantly longer fragmentation times than struvite and urate uroliths. As Hertz rate increased, fragmentation time significantly decreased. No significant differences were noted in fragmentation time or total energy required for fragmentation between single and double pulse mode. Grossly visible urethral damage increased as total energy increased. Urethral damage scores were significantly higher in tissues treated with a 90° incident beam compared to those tissues treated with a 30° incident beam. Likewise, urethral damage scores were significantly higher in contact mode treated tissues compared to non-contact mode treated tissues. Based on these findings, the Ho:YAG laser may be useful for treating clinical cases of canine urolithiasis.
Photodynamic therapy (PDT) is a potential improvement over conventional therapy for urinary bladder cancer, especially using the pro-photosensitizer 5-aminolevulinic acid (ALA), which is metabolized to its active form, protoporphyrin IX (PpIX), within both normal and neoplastic epithelial tissue. The purpose of this study was to evaluate ALA-based PDT in healthy dogs as a prelude to treating dogs with transitional cell carcinoma of the urinary bladder. Methods: Twelve dogs were given oral ALA at 0, 30, 60, or 90 mg/kg. Four hours later, the dogs were sacrificed and bladder tissue was collected and frozen for low-intensity fluorescence microscopy. Fifteen dogs were given oral ALA at 0, 30, 60, or 90 mg/kg and anesthetized four hours later. The trigone was irradiated with 635 nm laser light. All dogs were necropsied after PDT (48 h or 21 d). Results: PpIX fluorescence was confined to the urinary bladder mucosa with no detectable fluorescence in the muscularis or serosa. Histopathological lesions after PDT were limited to mild inflammation. Conclusions: Oral ALA appears to be well tolerated by healthy dogs at the dosages used. Because PpIX fluorescence was confined to the mucosa, ALA-based PDT may be a useful treatment for canine lower urinary tract neoplasia.
A novel immunoadjuvant, glycated chitosan, has been used in combinations with a near-infrared laser and a laser- absorbing dye to treat metastatic tumors in rats. The laser-dye combination provides selective photothermal tumor destruction. The addition of the in situ immunoadjuvant works in tandem with the photothermal interaction to induce a host antitumor immunity. Our previous experiments have shown the efficacy of this novel modality against a metastatic breast cancer in rat model, using the three components. The current study is to investigate the roles of different components, namely, the laser, the dye and the immunoadjuvant. Firs, the selective photothermal laser- tissue interactions are studied in vivo using rat leg muscles and rat tumors. Our results showed that with appropriate combination of laser parameter and dye does, an optimal selective photothermal tissue interaction could be achieved. The immune response is crucial in control of tumor metastasis and the immunoadjuvant has played pivotal role in the induction of the immunity in our experiment. Therefore, the role of immunoadjuvants in the laser cancer treatment is also investigated in the current study. Specifically, three different concentrations of glycated chitosan solutions - 0.5%, 1% and 2% - were used. In comparison, the 1% solution provided the best treatment outcome. Two additional immunoadjuvants, incomplete Freund's adjuvant and complete Freund's adjuvant were also used in the same laser-dye-adjuvant treatment protocol. The functions of different adjuvants are compared.
The effects of heat on malignant tumors are well known. Laser-induced hyperthermia is an alternative to microwave or radio wave tissue heating for cancer treatment. In situ injection of a chromophore that converts light into heat allows selective tissue heating. Our attempts to reproduce published results, using the chromophore ICG to enhance tissue heating, produced unacceptable skin damage. This was likely a function of light delivery methods or ICG diffusion into the skin from the tumor. We therefore set about to investigate the effects of ICG on normal and tumor tissue heating, and to develop methods of light delivery and skin cooling to minimize damage to normal tissues. ICG significantly increased tissue heating during 810 nm laser irradiation. Both argon gas and argon gas with water mist cooled the skin; only the addition of water mist maintained normal skin temperatures during laser treatments. In situ injection of ICG, combined with active cooling of the skin produced selective heating of the tumor. Laser light delivery through a microlens produced lower energy densities in the skin while sufficiently heating ICG-containing tumors. Selective laser-induced hyperthermia may become a useful cancer therapy with refinements in laser energy delivery, chromophore localization, and active cooling methods.
Photodynamic therapy (PDT) is a treatment option for several forms of human cancer, and like traditional chemotherapy and ionizing radiation therapy, PDT alone is not curative for some cases. Recent efforts have aimed at developing strategies for adjuvant therapy for PDT. Given the nature of PDT-mediated cell damage, immunotherapy is a promising adjuvant for long-term control of solid tumors. A candidate immune stimulant for use with PDT is monophosphoryl lipid A (MLA), a non-toxic fraction of the endotoxin molecule. The hypothesis is that adjuvant MLA immunotherapy with PDT will improve local tumor control and prevent growth of subsequently implanted tumor cells when compared to PDT alone. To date, no significant differences in circulating leukocyte populations or tumor infiltrating lymphocyte populations have been identified in 9L tumor-bearing F344 rats after systemic administrations of MLA. Likewise, no significant difference has been identified in local tumor control following PDT of 9L tumors with or without adjuvant MLA. Further results are pending.
Given exogenously, ALA defeats intrinsic regulatory feedback mechanisms allowing intracellular accumulation of protoporphyrin IX (PpIX), a highly efficient photosensitizer. In vivo, PpIX synthesis in neoplastic mammary tissues averages 20-fold higher than in normal mammary tissues. PpIX is retained intracellularly, unlike perivascular localization of other photosensitizers, and it is then cleared quickly from the body. In vitro, ALA induced PpIX production in our laboratory in 6 cell lines tested, including an established feline kidney cell line and dermal fibroblasts from primary skin biopsy explant, resulting in photosensitization. Fluorescent microscopy confirmed PpIX production in skin adnexae following ALA administration in a normal cat. To evaluate toxicity, three cats were treated with a single i.v. dose of ALA (either 100, 200, of 400 mg/kg) and followed for 7 days. Cats receiving 100 or 200 mg/kg ALA i.v. had elevated liver enzymes and bilirubin within 24 hours. Histopathology revealed hydropic changes in the liver and renal fibrosis. The cat receiving 400 mg/kg ALA intravenously had cutaneous flush, bradycardia and apnea associated with ALA administration; within 24 hours the cat was lethargic, anorectic and icteric. ALT, AST and bilirubin concentrations had increased significantly. At necropsy the liver had a prominent lobular pattern; histopathology revealed severe periportal hepatitis and splenic necrosis. Systemically administered ALA induces PpIX production, but toxicity may preclude its clinical application in the cat. PpIX levels seem to be more time dependent than those dependent at these three ALA doses and they are well beyond the saturation point for adequate PpIX conversion. The literature is scant regarding toxicity associated with parenteral administration of ALA.
For soft tissue sarcomas with high local recurrence rates following seemingly adequate surgical excision, intraoperative photodynamic therapy (IOPDT) provides a treatment strategy to eradicate microscopic, residual disease. Six Fischer 344 rats were given bilateral subcutaneous syngeneic 9L gliosarcoma tumors. When tumors reached 1.0 cm3, the rats were given 200 mg/kg ALA by intraperitoneal injection. Four hours later the rats were anesthetized, both tumors were incompletely excised and one surgical field was treated with 635 nm laser light (75 mW/cm2, 100 J/cm2). The surgical sites were then closed and the rats were monitored for local recurrence of the tumor. Low intensity fluorescent microscopic images captured with a cooled CCD camera confirmed that the tumors converted ALA into PpIX. Post-IOPDT swelling of the treated leg lasted 48 hours. An 80% local recurrence rate was observed in both groups 14 days following treatment. There was no difference in wound healing between IOPDT-treated sites and sites treated with surgery alone. Recurrent tumors were smaller in IOPDT sites than those treated with surgery alone. Additionally, the IOPDT recurrences were at the margin of the treatment field, suggesting a photoirradiation 'geographical miss.' These preliminary results, although disappointing, suggest that with further refinement IOPDT is a promising treatment modality for traditionally difficult to treat tumors.
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