High-dose Photodynamic Therapy Research Articles

Interstitial fluid pressure: A novel biomarker to monitor photo‐induced drug uptake in tumor and normal tissues

Low-dose photodynamic therapy PDT (photoinduction) can modulate tumor vessels and enhance the uptake of liposomal cisplatin (Lipoplatin®) in pleural malignancies. However, the photo-induction conditions must be tightly controlled as overtreatment shuts down tumor vessels and enhances normal tissue drug uptake. In a pleural sarcoma and adenocarcinoma rat model (n = 12/group), we applied photoinduction (0.0625 mg/kg Visudyne®, 10 J/cm2 ) followed by intravenous Lipoplatin® (5 mg/kg) administration. Tumor and normal tissue IFP were assessed before and up to 1 hour following photoinduction. Lipoplatin® uptake was determined 60 minutes following photoinduction. We then treated the pleura of tumor-free minipigs with high dose photodynamic therapy (PDT) (0.0625 mg/kg Visudyne®, 30 J/cm2 , n = 5) followed by Lipoplatin (5 mg/kg) administration. In rodents, photoinduction resulted in a significant decrease of IFP (P < 0.05) in both tumor types but not in the surrounding normal lung, equally exposed to light. Also, photoinduction resulted in a significant increase of Lipoplatin® uptake in both tumor types (P < 0.05) but not in normal lung. Tumor IFP variation and Lipoplatin® uptake fitted an inverted parabola. In minipigs, high dose photodynamic treatment resulted in pleural IFP increase of some animals which predicted higher Lipoplatin® uptake levels. Normal and tumor vasculatures react differently to PDT. Continuous IFP monitoring in normal and tumor tissues is a promising biomarker of vessel photoinduction. Moderate drop in tumor with no change in normal tissue IFP are predictive of specific Lipoplatin® uptake by cancer following PDT. Lasers Surg. Med. 49:773-780, 2017. © 2017 Wiley Periodicals, Inc.

Evaluation of the effect of photodynamic therapy with hematoporphyrin monomethyl ether on VX2 tumors implanted in the rectal submucosa of rabbits

We evaluated the influence of the photodynamic therapy (PDT) dosage with hematoporphyrin monomethyl ether (HMME) on its therapeutic efficacy in a rabbit model of in situ rectal cancer. VX2 cells were injected into the rectal submucosa of a rabbit to establish a carcinoma model in situ. After 10–14days, the tumors were treated with PDT at three dosages: a low dose (5mg/kg HMME; 60J/cm2 laser power), intermediate dose (5mg/kg; 240J/cm2), and high dose (10mg/kg; 360J/cm2). Tumor growth, animal survival, histopathological changes, general conditions, and adverse reactions were analyzed. PDT showed inhibitive effects on rectal cancer in all PDT groups, and the efficacy was correlated with the PDT dosage. The high dose PDT group had the best efficacy with a remarkable response rate of 20% and slight response rate of 80%, but it also had the highest death rate of 80% at day 7. The intermediate dose PDT group had a total response rate of 80% (60% remarkable plus 20% slight) and a 40% death rate. Comparably, the low dose PDT group had a 40% slight response rate and 60% death rate. Therefore, the intermediate dose of PDT was considered to be optimal among the three groups. Based on endoscopy findings, we also found that high dose PDT presented more side effects including inflammation, intestinal obstruction, rectal dysfunction, and death when PDT was performed on tumors inside the rectal tract. Our results indicate that a moderate PDT dose is more appropriate to treat tumors located in tract or cavity tissues.

Tetra-sulfonate phthalocyanine zinc-bovine serum albumin conjugate-mediated photodynamic therapy of human glioma

Glioma is the most common brain malignancy with poor prognosis. The current treatments for gliomas are mainly based on surgery, chemotherapy, and radiotherapy, which exhibit limited efficacy. Photodynamic therapy (PDT) using photosensitizers has been applied to glioma therapy. However, different photosensitizers usually lead to different therapeutic effects and adverse reactions. This study investigates the anti-tumor effect of photosensitizer ZnPcS4-BSA in xenograft glioma tumors. The xenograft glioma tumor model was established by inoculating nude mice with U251 cells. Tumor growth was evaluated by tumor volume, weight, and inhibition rate. Cell apoptosis was evaluated using TUNEL staining. Vascular endothelial growth factor (VEGF) expression and microvessel density were measured by immunohistochemistry. Significant decreases in tumor volume and weight as well as significant increases in tumor inhibition rate, cell apoptosis, VEGF expression, and microvessel density were observed in mice in the low- and high-dose PDT groups compared to the control, irradiation alone, and photosensitizer alone groups. No significant difference in cytotoxicity was observed between control group and photosensitizer alone group. Photosensitizer ZnPcS4-BSA significantly inhibited xenograft glioma tumor growth through induction of apoptosis. PDT using ZnPcS4-BSA may be effective for the therapy of gliomas.

Treatment of posterior uveal melanoma with multi-dose photodynamic therapy

AimsTo report on the use of multi-dose photodynamic therapy (PDT) in the treatment of posterior uveal melanoma.MethodsProspective case series. 18 patients with posterior uveal melanoma were treated with a minimum...

Apoptotic cell death induced by 5-aminolaevulinic acid-mediated photodynamic therapy of hypertrophic scar-derived fibroblasts

Objective: To investigate the effects of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on dermal fibroblasts from hypertrophic scars (HSs). Methods: HS samples were obtained from five patients who underwent surgery, and normal skin from healthy donors was used as a control. Dermal fibroblasts were isolated and cultured with various concentrations of ALA for 6 h. Intracellular protoporphyrin IX (PpIX) was measured by confocal microscopy. After 5 h of ALA treatment, cells were irradiated by a red laser (635 nm wavelength) at a power density of 10 mW/cm2 with an energy density from 0.5 to 4 J/cm2. Cell survival was measured by a CCK-8 Kit after 24 h. Cell death was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and flow cytometric analysis of annexin V. Results: Intracellular PpIX accumulation was observed in fibroblasts from HS patients and healthy donors after ALA treatment. After laser irradiation, viable cells were decreased among both cell types in a dose-dependent manner with energy density. In addition, apoptotic cell death was observed with low dose PDT, whereas necrotic cells were increased by high dose PDT. Conclusion: HS-derived fibroblasts efficiently accumulate PpIX after ALA treatment and can be eliminated via apoptosis by controlled laser irradiation.

Nitric oxide-mediated activity in anti-cancer photodynamic therapy

Cell recurrence in cancer photodynamic therapy (PDT) is an important issue that is poorly understood. It is becoming clear that nitric oxide (NO) is a modulator of PDT. By acting on the NF-κB/Snail/RKIP survival/anti-apoptotic loop, NO can either stimulate or inhibit apoptosis. We found that pheophorbide a/PDT (Pba/PDT) induces the release of NO in B78-H1 murine amelanotic melanoma cells in a concentration-dependent manner. Low-dose PDT induces low NO levels by stimulating the anti-apoptotic nature of the above loop, whereas high-dose PDT stimulates high NO levels inhibiting the loop and activating apoptosis. When B78-H1 cells are treated with low-dose Pba/PDT and DETA/NO, an NO-donor, intracellular NO increases and cell growth is inhibited according to scratch-wound and clonogenic assays. Western blot analyses showed that the combined treatment reduces the expression of the anti-apoptotic NF-κB and Snail gene products and increases the expression of the pro-apoptotic RKIP gene product. The combined effect of Pba and DETA/NO was also tested in C57BL/6 mice bearing a syngeneic B78-H1 melanoma. We used pegylated Pba (mPEG-Pba) due to its better pharmacokinetics compared to free Pba. mPEG-Pba (30mg/Kg) and DETA/NO (0.4mg/Kg) were i.p. injected either as a single molecule or in combination. After photoactivation at 660nM (fluence of 193J/cm2), the combined treatment delays tumor growth more efficiently than each individual treatment (p<0.05). Taken together, our results showed that the efficacy of PDT is strengthened when the photosensitizer is used in combination with an NO donor.

In Vitro Studies on Erythrosine-Based Photodynamic Therapy of Malignant and Pre-Malignant Oral Epithelial Cells

Photodynamic Therapy (PDT) involves the administration of a tumor localizing photosensitizing agent, which upon activation with light of an appropriate wavelength leads to the destruction of the tumor cells. The aim of the present study was to determine the efficacy of erythrosine as a photosensitizer for the PDT of oral malignancies. The drug uptake kinetics of erythrosine in malignant (H357) and pre-malignant (DOK) oral epithelial cells and their susceptibility to erythrosine-based PDT was studied along with the determination of the subcellular localization of erythrosine. This was followed by initial investigations into the mechanism of cell killing induced following PDT involving both high and low concentrations of erythrosine. The results showed that at 37°C the uptake of erythrosine by both DOK and H357 cells increased in an erythrosine dose dependent manner. However, the percentage of cell killing observed following PDT differed between the 2 cell lines; a maximum of ∼80% of DOK cell killing was achieved as compared to ∼60% killing for H357 cells. Both the DOK and H357 cell types exhibited predominantly mitochondrial accumulation of erythrosine, but the mitochondrial trans-membrane potential (ΔΨm) studies showed that the H357 cells were far more resistant to the changes in ΔΨm when compared to the DOK cells and this might be a factor in the apparent relative resistance of the H357 cells to PDT. Finally, cell death morphology and caspase activity analysis studies demonstrated the occurrence of extensive necrosis with high dose PDT in DOK cells, whereas apoptosis was observed at lower doses of PDT for both cell lines. For H357 cells, high dose PDT produced both apoptotic as well as necrotic responses. This is the first instance of erythrosine-based PDT's usage for cancer cell killing.

Photodynamic Therapy Enhances Liposomal Doxorubicin Distribution in Tumors during Isolated Perfusion of Rodent Lungs

Background: Photodynamic therapy (PDT) at low drug-light conditions can enhance the transport of intravenously injected macromolecular therapeutics through the tumor vasculature. Here we determined the impact of PDT on the distribution of liposomal doxorubicin (Liporubicin™) administered by isolated lung perfusion (ILP) in sarcomas grown on rodent lungs. Methods: A syngeneic methylcholanthrene-induced sarcoma cell line was implanted subpleurally in the left lung of Fischer rats. Treatment schemes consisted in ILP alone (400 µg of Liporubicin), low-dose (0.0625 mg/kg Visudyne®, 10 J/cm² and 35 mW/cm²) and high-dose left lung PDT (0.125 mg/kg Visudyne, 10 J/cm² and 35 mW/cm²) followed by ILP (400 µg of Liporubicin). The uptake and distribution of Liporubicin in tumor and lung tissues were determined by high-performance liquid chromatography and fluorescence microscopy in each group. Results: Low-dose PDT significantly improved the distribution of Liporubicin in tumors compared to high-dose PDT (p < 0.05) and ILP alone (p < 0.05). However, both PDT pretreatments did not result in a higher overall drug uptake in tumors or a higher tumor-to-lung drug ratio compared to ILP alone. Conclusions: Intraoperative low-dose Visudyne-mediated PDT enhances liposomal doxorubicin distribution administered by ILP in sarcomas grown on rodent lungs which is predicted to improve tumor control by ILP.

Differential cytotoxic responses to low- and high-dose photodynamic therapy in human gastric and bladder cancer cells

Here, we present differential cytotoxic responses to two different doses of photodynamic therapies (PDTs; low-dose PDT [LDP] and high-dose PDT [HDP]) using a chlorin-based photosensitizer, DH-II-24, in human gastric and bladder cancer cells. Fluorescence-activated cell sorting analysis using Annexin V and propidium iodide (PI) showed that LDP induced apoptotic cell death, whereas HDP predominantly caused necrotic cell death. The differential cytotoxic responses to the two PDTs were further confirmed by a DiOC(6) and PI double-staining assay via confocal microscopy. LDP, but not HDP, activated caspase-3, which was inhibited by Z-VAD, Trolox, and BAPTA-AM. LDP and HDP demonstrated opposite effects on intracellular reactive oxygen species (ROS)/Ca(2+) signals; LDP stimulated intracellular ROS production, contributing to a transient increase of intracellular Ca(2+) , whereas HDP induced a massive and prolonged elevation of intracellular Ca(2+) responsible for the transient production of intracellular ROS. In addition, the two PDTs also increased in situ transglutaminase 2 (TG2) activity, with a higher stimulation by HDP, and this increase in activity was prevented by Trolox, BAPTA-AM, and TG2-siRNA. LDP-induced apoptotic cell death was strongly inhibited by Trolox and TG2-siRNA and moderately suppressed by BAPTA-AM. However, HDP-mediated necrotic cell death was partially inhibited by BAPTA-AM but not by TG2-siRNA. Thus, these results demonstrate that LDP and HDP induced apoptotic and necrotic cell death by differential signaling mechanisms involving intracellular Ca(2+) , ROS, and TG2.

Induction of Immune Mediators in Glioma and Prostate Cancer Cells by Non-Lethal Photodynamic Therapy

BackgroundPhotodynamic therapy (PDT) uses the combination of photosensitizing drugs and harmless light to cause selective damage to tumor cells. PDT is therefore an option for focal therapy of localized disease or for otherwise unresectable tumors. In addition, there is increasing evidence that PDT can induce systemic anti-tumor immunity, supporting control of tumor cells, which were not eliminated by the primary treatment. However, the effect of non-lethal PDT on the behavior and malignant potential of tumor cells surviving PDT is molecularly not well defined.Methodology/Principal FindingsHere we have evaluated changes in the transcriptome of human glioblastoma (U87, U373) and human (PC-3, DU145) and murine prostate cancer cells (TRAMP-C1, TRAMP-C2) after non-lethal PDT in vitro and in vivo using oligonucleotide microarray analyses. We found that the overall response was similar between the different cell lines and photosensitizers both in vitro and in vivo. The most prominently upregulated genes encoded proteins that belong to pathways activated by cellular stress or are involved in cell cycle arrest. This response was similar to the rescue response of tumor cells following high-dose PDT. In contrast, tumor cells dealing with non-lethal PDT were found to significantly upregulate a number of immune genes, which included the chemokine genes CXCL2, CXCL3 and IL8/CXCL8 as well as the genes for IL6 and its receptor IL6R, which can stimulate proinflammatory reactions, while IL6 and IL6R can also enhance tumor growth.ConclusionsOur results indicate that PDT can support anti-tumor immune responses and is, therefore, a rational therapy even if tumor cells cannot be completely eliminated by primary phototoxic mechanisms alone. However, non-lethal PDT can also stimulate tumor growth-promoting autocrine loops, as seen by the upregulation of IL6 and its receptor. Thus the efficacy of PDT to treat tumors may be improved by controlling unwanted and potentially deleterious growth-stimulatory pathways.

Rationale of Combined PDT and SDT Modalities for Treating Cancer Patients in Terminal Stage: The Proper Use of Photosensitizer

We read with interest the recent article by Wang et al, reporting the clinical application of SF1 (Sonoflora 1)—a sensitizer that can be activated by light and ultrasound. SF1 is an analog of chlorophyll in that its macrocycle backbone is porphyrin-based and the center of the porphyrin ring consists of a metal ion. The chlorophyll derivative has light absorption peaks at 402 and 636 nm. A very unusual combination of this investigational agent and various prototype light sources and ultrasound devices was used in the treatment of three patients who suffered from late stages of breast cancer with systemic metastases to multiple organs. The combination of photosensitizer and light illumination is commonly known as photodynamic therapy (PDT) and the combination of drug or sonosensitizer and acoustic activation as sonodynamic therapy (SDT). In theory, the direct tumoricidal effects of these modalities are mediated by cytotoxic agents generated through photochemical or sonochemical reactions inside tumor tissue. The narrow gap between potential benefit and potential risk is determined by intrinsic cellular sensitivity and the sensitizer ratio between tumor and normal tissue. The synergistic effects of sensitizer and low-power ultrasound have been examined in many in vitro studies and to a lesser extent in in vivo models. Although the sonosensitization-induced cytotoxicity might be attributed to the sonochemical reaction, such as sensitizer-mediated energy transfer and free radical generation, data suggest that the mechanism of sonosensitization is probably not governed by a universal mechanism, but may be influenced by multiple factors including the nature of the biological model, sensitizer distribution, ultrasound parameters and acoustic energy distribution. Nevertheless, to date, the correlation between ultrasound parameters and biological effects has not been fully established in preclinical investigation (in vitro or in vivo). There is no convincing data that shows that ultrasound used in this way is effective in the treatment of primary tumor and multiple metastases. Therefore, this article raises a few basic questions. First of all, without those critical safety and efficacy information, it is unjustifiable to test the unproven prototypes in humans, particularly those in terminal stages for any purposes. The authors state that acoustic activation was delivered after intravenous administration of the chlorophyll derivative in two different modes—local and whole body ultrasound activation. A handheld ultrasound transducer was used for two patients, which was associated with a side effect of pain. Another patient was placed in a water tub to receive whole body ultrasound treatment. It was expected that ultrasound could reach deep-seated tumor(s) and sensitizers that are beyond the reach of external light of 630 nm. Although no details were provided in terms of the number of transducers in the water tub, the geometric distribution of acoustic energy within the body, and its accessibility to the multiple tumor sites (e.g., breast, lung, bones, liver, neck, axillary lymph nodes, and abdominal lymph nodes), the authors indicated that high acoustic power (2 W/cm at 1 MHz for 20 minutes) was used in both settings without providing justifications on the selection of those parameters. It is also questionable that sufficient acoustic energy will reach the deep-seated metastases in lung and bone tissues. Second, single high-dose PDT and metronomic PDT (i.e., to deliver photosensitizer and/or light doses at low rates over an extended period) have distinct and complementary therapeutic goals. In contrast to the conventional PDT regimen of single dose drug administration (intravenous or oral), the authors used a repeated lingual delivery of the chlorophyll derivative (a total dose of 30 or 60 mg) for 2 to 3 consecutive days followed by light plus ultrasound treatment daily for 3 days starting at day 3 or day 4 after the onset

Photodynamic therapy selectively enhances liposomal doxorubicin uptake in sarcoma tumors to rodent lungs

In specific conditions, photodynamic therapy (PDT) can enhance the distribution of macromolecules across the endothelial barrier in solid tumors. It was recently postulated that tumor neovessels were more responsive to PDT than the normal vasculature. We hypothesized that Visudyne(R)-mediated PDT could selectively increase liposomal doxorubicin (Liporubicin) uptake in sarcoma tumors to rodent lungs while sparing the normal surrounding tissue. Sarcoma tumors were generated subpleurally in the left lower lung lobe of 66 Fischer rats. Ten days following sarcoma implantation, tumors underwent different pre-treatment schemes: no PDT (controls), low-dose PDT (0.0625 mg/kg Visudyne(R), 10 J/cm(2) and 35 mW/cm(2)) and high-dose PDT (0.125 mg/kg Visudyne(R), 10 J/cm(2) and 35 mW/cm(2)). Liporubicin was then administered and allowed to circulate for 1, 3, or 6 hours. At the end of each treatment scheme, we assessed the uptake of Liporubicin in tumor and lung tissues by high-performance liquid chromatography and fluorescence microscopy. In all PDT-treated groups, there was a significant enhancement of Liporubicin uptake in tumors compared to controls after 3 and 6 hours of drug circulation. In addition, Liporubicin distribution within the normal lung tissue was not affected by PDT. Thus, PDT pre-treatment significantly enhanced the ratio of tumor-to-lung drug uptake compared to controls. Finally, fluorescence microscopy revealed a well-detectable Liporubicin signaling throughout PDT-treated tumors but not in controls. PDT is a tumor-specific enhancer of Liporubicin distribution in sarcoma lung tumors which may find a translation in clinics.

The molecular basis of prostate cancer cell escape from protoporphyrin IX-based photodynamic therapy

Abstract Introduction Photodynamic therapy (PDT) uses the combination of a photosensitizing drug and light to cause selective damage to solid tumors. For early or localized disease, PDT can be a curative therapy with many advantages over available alternatives. For more advanced or disseminated disease, curative therapy is usually not possible with current technologies of light application, nevertheless it can improve quality of life and lengthen survival. More recently, there has been increasing evidence that PDT can also induce systemic anti-tumor immunity, indicating that PDT can become a rational therapeutic option, even if not all tumor cells are primarily eliminated by PDT. One prerequisite to minimize tumor relapse in these settings is to elucidate how tumor cells can cope with the PDT-induced damage. Material and methods Here we have characterized the rescue response of human PC-3 prostate cancer cells exposed in vitro to sublethal PDT after 5-aminolevulinic acid-induced protoporphyrin IX sensitization at the transcriptome level using Affymetrix HG U133 Plus 2.0 oligonucleotide microarrays. Cells were irradiated with laser light at a wavelength of 635 nm adjusted to an irradiance of 100 mW/cm 2 with irradiations of 1.5 and 3 J/cm 2 . Results The early response was characterized by the up-regulation of early response genes like FOS , JUN , EGR1 , ATF3 , DUSP , heat shock protein genes as well as histone genes, therefore resembling the early response of tumor cells to high dose PDT but without signs of irreversible cell damage. Twenty-four hours after PDT the cells still express high level of early response genes but 235 additional probe sets/genes were now significantly up-regulated (≥3 x ) that were not up-regulated 4 h after PDT. The most prominently up-regulated genes belong to gene families encoding the aldo-keto reductases, fibroblast growth factors, and HSP40-related proteins. In terms of a possible anti-tumor immune response it is noteworthy that also a multitude of chemokine and interleukin genes were up-regulated by the tumor cells upon PDT. Most of them are involved in granulocyte attraction and activation and some are also important for angiogenesis. Conclusion In conclusion, the global molecular characterization of the rescue response to PDT of tumor cells indicates that PDT rather favors an anti-tumor immune response than tumor immune escape reactions. Therefore, combining PDT and immunotherapy seem to be an attractive direction for the establishment of novel multimodal tumor therapies.

5.09] The molecular basis of tumor cell escape from protoporphyrin IX-based photodynamic therapy

Objective Photodynamic therapy (PDT) uses a combination of a photosensitizing drug and light to cause selective damage to solid tumors. For early or localized disease, PDT can be a curative therapy with many advantages over available alternatives. For more advanced or disseminated disease curative therapy is usually not possible with current technologies of light application, however it can improve quality of life and lengthen survival. More recently there is increasing evidence that PDT can also induce systemic anti-tumor immunity, indicating that PDT could become a rational therapeutic option, even if not all tumor cells are primarily eliminated by PDT. One prerequisite to minimize tumor relapse in these settings is to elucidate how tumor cells can cope with the PDT-induced damage. Material and methods Here we have characterized the rescue response of human prostate cancer and glioblastoma cells exposed in vitro to sub-lethal PDT after 5-aminolevulinic acid-induced protoporphyrin IX sensitization at the transcriptome level using Affymetrix U133 2.0 oligonucleotide microarrays. Cells were irradiated with laser light at a wavelength of 635 nm adjusted to an irradiance of 100 mW/cm 2 with fluences varying between 0.5 and 3 J/cm 2 . Results The early response was characterized by the up-regulation of early response genes like FOS, JUN, EGR1, ATF3, DUSP, heat-shock protein genes as well as histone and metallothionein genes, therefore resembling the early response of tumor cells to high-dose PDT but without signs of irreversible cell damage. Twenty-four hours after PDT the cells still expressed high level of early response genes but 241 additional probe sets/genes were significantly up-regulated (5 x ) compared to those that were not up-regulated (⩽2 x ) 4 h after PDT. The most prominently up-regulated genes belong to gene families encoding the aldo-keto reductases, fibroblast growth factors and Hsp40-related proteins. In terms of a possible anti-tumor immune response it is noteworthy that also a multitude of chemokines and interleukins were up-regulated by the tumor cells upon PDT. Most of them are involved in granulocyte attraction and activation and some are also important for angiogenesis. Conclusion In conclusion, the global molecular characterization of the rescue response to PDT of tumor cells indicated that PDT rather favors an anti-tumor immune response than tumor immune escape reactions. Therefore combining PDT and immunotherapy seem to be an attractive direction for the establishment of novel multimodal tumor therapies.

Immune Response Against Angiosarcoma Following Lower Fluence Rate Clinical Photodynamic Therapy

Tumor response to photodynamic therapy (PDT) is dependent on treatment parameters used. In particular, the light fluence rate may be an important determinant of the treatment outcome. In this clinical case report, we describe the response of angiosarcoma to PDT carried out using different fluence rates and drug and light doses. A patient with recurrent multifocal angiosarcoma of the head and neck was recruited for PDT. A new generation chlorin-based photosensitizer, Fotolon, was administered at a dose of 2.0 to 5.7 mg/kg. The lesions were irradiated with 665 nm laser light for a light dose of 65 to 200 J/cm2 delivered at a fluence rate of 80 or 150 mW/cm2. High dose PDT carried out at a high fluence rate resulted in local control of the disease for up to a year; however, the disease recurred and PDT had to be repeated. PDT of new lesions carried out at a lower fluence rate resulted in tumor eradication. More significantly, it also resulted in spontaneous remission of neighboring and distant untreated lesions. Repeat PDT carried out on a recurrent lesion at a lower fluence rate resulted in eradication of both treated and untreated lesions despite the lower total light dose delivered. Immunohistochemical examination of biopsy samples implies that PDT could have activated a cell-mediated immune response against untreated lesions. Subsequent histopathological examination of the lesion sites showed negative for disease. Our clinical observations show that lower fluence rate PDT results in better outcome and also indicate that the fluence rate, rather than the total light dose, is a more crucial determinant of the treatment outcome. Specifically, lower fluence rate PDT appears to activate the body's immune response against untreated lesions.

Retinal Angiomatous Proliferation Reactivation 6 Months after High-Dose Intravitreal Acetonide Triamcinolone and Photodynamic Therapy

To describe the incidence of retinal angiomatous proliferation (RAP) reactivation after combined treatment with a high-dose intravitreal triamcinolone acetonide (IVTA) and photodynamic therapy (PDT) at 1-year follow-up. All patients had undergone a full ophthalmic examination. High-dose IVTA (20 mg) was injected via pars plana. Four to 7 days later, PDT was delivered. Fourteen eyes of 13 patients were included. Eight lesions (57%) reopened and needed retreatment with combined therapy at 6 months follow-up. At 1-year follow up, the lesion was obliterated in nine cases (64.2%) and best-corrected visual acuity improved from 0.87 logMar (range, 0.7-1) to 0.79 logMar (range, 0.5-1). Combined therapy using high-dose IVTA and PDT is beneficial in stabilizing RAP. However, a high incidence of RAP reactivation has been observed at 6 months, even with a high-dose IVTA injection.

Apoptosis and Autophagy After Mitochondrial or Endoplasmic Reticulum Photodamage†

Photodynamic therapy (PDT) can cause lethal photodamage by both direct and indirect mechanisms. Direct modes of cell death relate to nonspecific necrosis and the initiation of signaling pathways that elicit apoptosis, autophagy or both. In this report, effects of low-dose and high-dose PDT are explored, comparing sensitizers that localize in the endoplasmic reticulum (the porphycene termed CPO) or mitochondria (mesochlorin). To explore the role of autophagy, two cell lines were examined--the murine L1210 leukemia and an Atg7 knockdown derivative of L1210. The Atg7 gene is central to the process of autophagy. High-dose PDT with either sensitizer resulted in a substantial loss of the Bcl-2 protein. As Bcl-2 regulates both apoptosis and autophagy, loss of this protein can lead to initiation of either or both processes. Low-dose PDT with either sensitizer resulted in the initiation of apoptosis in the L1210/Atg7- cell line and a 20% loss of viability. In contrast, the same PDT dose led to the rapid appearance of autophagic cells in the L1210 line, less apoptosis and only a 5% loss of viability. These results are consistent with autophagy serving as a pro-survival response via the recycling of damaged organelles. At a higher PDT dose more apoptosis was again seen in the L1210/Atg7- line, but both cell lines exhibited comparable cytotoxicity in colony formation assays. We conclude that autophagy offers protection from the phototoxic effects of low-dose PDT, but can serve as an alternate death mode when the PDT dose is increased.

Photodynamic therapy of diseased bone

Photodynamic therapy (PDT) defines the light activation of a photosensitizing compound, leading to the generation of cytotoxic, reactive oxygen species. We are investigating the unique application of PDT for diseased bone. Bioluminescent, human breast cancer cells (MT-1) were injected intracardially into athymic rats. At 2-3 weeks following injection rats developed diffuse bone metastases that were visible using an in vivo bioluminescence imaging system. Metastatic lesions within vertebrae and long bones were treated with differing regimens of 690nm laser light (25-150J) following injection of benzoporphyrin-derivative monoacid (BPD-MA) and the tumour response assayed histologically upon sacrifice. Subsequent large animal studies involved light propagation studies in porcine vertebrae and BPD-MA-PDT treatment of large primary osteosarcomas in canine. Canine were subject to high dose PDT (500J/cm). A second rat model involved bacterial infection in bone. Bioluminescent Staphylococcus aureus (S. aureus) were grown as biofilms onto wires, implanted into rat tibia and exposed to 5-aminolevulinic acid (ALA)-PDT delivered percutaneously with 635nm laser light (75J/cm(2)). Response to therapy was monitored as changes in bioluminescence signal and colony forming assay upon sacrifice. PDT (150J) proved effective in ablating metastatic lesions within rat femur and lumbar vertebrae. Porcine studies confirmed the average depth of light penetration into trabecular bone as 0.16±0.04cm with an average incident fluence rate of 4.3mW/cm(2), however, the necrotic/non-necrotic interface extended 0.6cm out from the treatment fiber. In canine a single, high dose treatment created well-circumscribed margins of effect encompassing the entire 3-4cm osteosracoma. Finally, ALA-PDT proved effective against S. aureus biofilms in bone, although recurrent infection did occur in some. Results support the application of PDT to the treatment of primary or metastatic lesions within bone. Secondly, ALA-PDT may be useful as a treatment for osteomyelitis with repeat or chronic treatment regimens.

Photodynamic therapy with 5-aminolevulinic acid induces distinct microcirculatory effects following systemic or topical application

In photodynamic therapy (PDT) the photosensitiser 5-aminolaevulinic acid (ALA) can be used by systemic or topical application. Previous experiments showed that the photodynamic effects might not be mediated solely by porphyrins localized in the parenchyma, but also by porphyrins in the microvasculature. Therefore, the microcirculatory effects of PDT following systemic versus topical application of ALA have been investigated. Amelanotic melanomas were implanted in the dorsal skin fold chamber of Syrian Golden hamsters. ALA was injected i.v. for systemic PDT before irradiation, whereas ALA was applied to the chambers for topical PDT before irradiation with an incoherent lamp. FITC-labelled erythrocytes were injected to determine red blood cell velocity (RBCV) and functional vessel density (FVD). Twenty-four hours after PDT tissue was taken for histology and immunohistochemistry to reveal the degree of apoptosis and to show the accumulation of leukocytes. FVD or RBCV was not altered significantly by systemic or topical low-dose PDT (10 J cm(-2)), whereas a significant reduction of RBCV and FVD was detected after high-dose PDT (100 J cm(-2)) following systemic or topical application of ALA. Systemic PDT with 100 J cm(-2) stopped the flow only in the tumor center, whereas topical PDT with 100 J cm(-2) lead to a breakdown of RBCV in all chamber areas. Two hours and 24 h after systemic high-dose PDT, perfused microvessels and capillaries could be detected in normal tissue and tumor periphery, in contrast to topical high-dose PDT leading to a shut down of FVD 24 h after irradiation in all areas of the chamber tissue. Histological staining revealed a more pronounced intracellular oedema and swelling of cells after topical high-dose PDT than systemic high-dose PDT. These results indicate that topical high-dose PDT with ALA has a more pronounced effect on microcirculation as compared to systemic high-dose PDT in this model.

Cell death and growth arrest in response to photodynamic therapy with membrane-bound photosensitizers

Photodynamic therapy (PDT) is a treatment for cancer and for certain benign conditions that is based on the use of a photosensitizer and light to produce reactive oxygen species in cells. Many of the photosensitizers currently used in PDT localize in different cell compartments such as mitochondria, lysosomes, endoplasmic reticulum and generate cell death by triggering necrosis and/or apoptosis. Efficient cell death is observed when light, oxygen and the photosensitizer are not limiting (“high dose PDT”). When one of these components is limiting (“low dose PDT”), most of the cells do not immediately undergo apoptosis or necrosis but are growth arrested with several transduction pathways activated. This commentary will review the mechanism of apoptosis and growth arrest mediated by two important PDT agents, i.e. pyropheophorbide and hypericin.