Light has been used to treat diseases for hundreds of years. Convenient and powerful light sources such as lasers make photomedicine a major branch in diseases treatment and detection. Originally, light was often used for local treatment, using photomechanical, photochemical, photothermal reactions and photomodulation as the major mechanisms. More and more investigators have become interested in the systemic effects of light, particularly in its effects on immune systems. Much work has been done to activate and/or enhance the host immune system to combat cancer, either using light as a direct tool or as an adjuvant method. Light has long been used for assisting disease detection and diagnosis. Advances in light technology have made photo-diagnostics ever more precise spatially and temporally. Many techniques facilitate observation of bio-molecule interactions and other biological processes at the cellular level, hence providing opportunities to detect and monitor immune activities. This manuscript will review recent photo-immunological research in treatment of cancer. The recent development of combination therapies involving lasers will be presented. Specifically, the results of cancer treatment using laser photothermal interaction, either with or without additional immunological stimulation will be discussed. The immunological effects of photodynamic therapy (PDT), and of its combination with immunotherapy in cancer treatment will also be discussed. Much interest has been recently concentrated in the immunological responses after laser treatment. Such responses at cellular and molecular levels will be discussed. The effect of these treatment modalities on the distant metastases also showed promise of light induced antitumor immunity. The combination therapy and induced immunological responses appear to be the key for long-term control of tumors.

Cancer is a leading cause of death among modern people largely due to metastatic disease. The ideal cancer treatment should target both the primary tumor and the metastases with minimal toxicity towards normal tissue. This is best accomplished by priming the body's immune system to recognize the tumor antigens so that after the primary tumor is destroyed, distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the IV administration of photosensitizers followed by illumination of the tumor with red light producing reactive oxygen species leading to vascular shutdown and tumor cell death. Anti-tumor immunity is stimulated after PDT due to the acute inflammatory response, generation of tumor-specific antigens, and induction of heat-shock proteins. Combination regimens using PDT and immunostimulating treatments are likely to even further enhance post-PDT immunity. These immunostimulants are likely to include products derived from pathogenic microorganisms that are effectively recognized by Toll-like receptors and lead to upregulation of transcription factors for cytokines and inflammatory mediators. The following cascade of events causes activation of macrophages, dendritic and natural killer cells. Exogenous cytokine administration can be another way to increase PDT-induced immunity as well as treatment with a low dose of cyclophosphamide that selectively reduces T-regulatory cells. Although so far these combination therapies have only been used in animal models, their use in clinical trials should receive careful consideration.

One in 8 women in the United States will develop breast cancer during her lifetime and 40,000 die each year. Deaths are due to tumors that have metastasized despite local control. Photodynamic therapy (PDT) is a promising cancer treatment in which a photosensitizer (PS) accumulates in tumors and is subsequently activated by visible light of an appropriate wavelength. The energy of the light is transferred to molecular oxygen to produce reactive oxygen species that produce cell death and tumor ablation. Mechanisms include cytotoxicity to tumor cells, shutting down of the tumor vasculature, and the induction of a host immune response. The precise mechanisms involved in the PDT-mediated induction of anti-tumor immunity are not yet understood. Potential contributing factors are alterations in the tumor microenvironment via stimulation of proinflammatory cytokines and direct effects of PDT on the tumor that increase immunogenicity. We have studied PDT of 410.4 variant 4T1 tumors growing in the mammary fat pad (orthotopic) in Balb/c mice and which produce metastasis. We have shown that a PDT regimen that produces vascular shutdown and tumor necrosis leads to initial tumor ablation but the tumors recur at the periphery. We studied the combination of PDT with immunostimulating therapies. Low dose cyclophosphamide (CY) is a specific mechanism to deplete the regulatory T cells (CD4+CD25+), these cells play an important role in the immunosuppression activity of tumors. In combination with PDT that produces release of tumor specific antigens, this immunostimulation may lead to generation of cytotoxic CD8 T-lymphocytes that recognize and destroy the tumor. The second alternative therapy is the use of a novel combination of the immunostimulant CpG oligodeoxynucleotides (CpG-ODN) and PDT. CpG-ODN is recognized by Toll-like receptor 9 and directly or indirectly triggers B cells, NK cells, monocyte-macrophages and dendritic cells to proliferate, mature and secrete cytokines, chemokines and immunoglobulins. Both these novel combinations gave significantly enhanced therapeutic benefit not seen with single treatments alone. Tumors grew more slowly and mice lived significantly longer, although cures were rare. We propose that a rational choice of immune stimulant is an ideal addition to PDT regimens.

One of the important problems of modern medicine is treatment of infected wounds. There are many diversified expedients of treatment, but none of them obey the modern physician completely. The aim of this study is to develop and test a new combined method of photodynamic ultrasonic therapy (PDUST) for treatment of infected wounds with focus on experimental trials. PDUST is based on a combination of two methods: photodynamic (PD) therapy (PDT) with photosensitizer and low frequency ultrasonic (US) therapy with antibiotic as tools for treatment of wounds and effectively killing bacteria. The main parameters are: US frequency - 26.5 kHz; US tip elongation - 40±20 μm; wavelength of light emitting diodes (LED) array - 660±10 nm; light intensity on biotissue surface - 1-2 mW/cm²; photosensitizer - an aluminum disulfonated phtalocyanine dissolved in a physiological solution in concentration 10 mg/l. The experiments were carried out with 70 male chinchilla rabbits divided into 7 groups, thus the dynamics of wounds healing were studied in different modes of PDUST. The PD and US methods supplement each other and in conjunction provide additive and especially synergetic effects. The experimental data demonstrated advantages of new technology in comparison with conventional methods in cases of treatment of extended suppurative inflammatory and profound wounds. The more detailed study of PDUST method's mechanism, which is based on low intensity of LED light, PD therapy and US influence is required.

Influence of hypericin and synthetic (see manuscript for formula) on haemolysis of human erythrocytes was investigated. It was shown that both hypericin and synthetic porphyrins (TOEPyP and Zn-TOEPyP) did not cause haemolysis in the dark (24 hrs incubation), whereas Ag-TOEPyP leaded to haemolysis already after 40 min incubation, i.e. was cytotoxic. Hypericin (25 μM -125 μM) possessed haemolytic activity upon light exposure (visible spectrum, 30 mW/cm2, 5 min and more). Total haemolysis of erythrocytes was observed at 15 min exposure to light at all the investigated concentrations of hypericin. Dose-dependent haemolytic effect of TOEPyP and Zn-TOEPyP depending on light exposure time was also investigated. TOEPyP demonstrated considerably higher haemolytic activity compared to Zn-TOEPyP. Ag-TOEPyP demonstrated the weakest photodynamic activity. The effect of ascorbic acid on porphyrin-induced haemolysis was also revealed. Ascorbic acid at the concentration of 0.15 μM and above significantly increased haemolysis induced by hypericin, whereas at concentration of 0.75 μM and less it appeared to possess protective property. TOEPyP and Zn-TOEPyP did not demonstrate photodynamic properties in the presence of ascorbic acid (3.75 μM and more). The ascorbic acid at the concentrations below 3.75 μM did not have any influence on erythrocyte haemolysis induced by TOEPyP, while it increased haemolytic effect of Zn-TOEPyP. Thus, the full inhibition of photohaemolysis induced by (see manuscript for formula) by singlet oxygen quenchers - ascorbic acid and tryptophan - was shown. This testifies to the fact, that the photohaemolysis induced by these porphyrins is caused by Type II reactions.

Photodynamic therapy (PDT) is a clinically approved new treatment modality. It has been used for treatment of non-malignant and malignant diseases. Over the last decade its clinical application has gained increasing acceptance around the world after regulatory approvals. PDT offers various treatment options in cancer management and has been used primarily for localized superficial or endoluminal malignant and premalignant conditions. Recently, its application has also been expanded to solid tumors. However, its efficacy for the treatment of malignant tumors remains debatable and its acceptance still variable. Pre-clinical studies demonstrate that, in addition to the direct local cytotoxicity, PDT can induce host immune responses, which may further enhance the therapeutic effects on primary tumor as well as metastasis. Therefore, PDT-induced antitumor immune response might play an important role in successful control of malignant diseases. Furthermore, the antitumor efficacy of PDT might also be enhanced through an effective immunoadjuvant to further expand its usefulness for a possible control of distant metastases. Recent clinical data also indicate that improved clinical outcomes are seen in the combination of PDT and immunomodulation therapy for non-malignant disease. This review will summarize recent progress in developing innovative approaches of PDT combined with immunotherapy for non-malignant and malignant diseases.

Although melanoma accounts for only 4% of skin cancer cases, it causes 79% of all skin cancer deaths. Patients with metastatic melanoma have a poor prognosis, and long term survival is only about 5% [1, 2]. Conventional therapies such as surgery and radiation therapy usually do not cure stage III or stage IV melanoma, while traditional chemotherapy is primarily palliative. Over the last decade we have been developing new methods for treating solid tumors like melanoma, first in animal models and now in humans. We present here preliminary results from a new technique that utilizes a combination of laser stimulation and drug therapy to stimulate brisk immunological responses in cases of advanced melanoma with cutaneous metastases. A high-power, near-infrared diode laser (805 nm) is used to kill tumors in situ and a topical toll-like receptor agonist (imiquimod cream, 5%) is used to intensify the resulting immunological response. This is essentially an in situ, tumor vaccine approach to treating solid tumors.

Laser thermotherapy is interesting from an immunological point of view since it can reduce tumor volume without causing immunosuppression at the same time as it may induce and/or enhance tumor immunity. In a rat liver tumor model, we have demonstrated that laser thermotherapy 1) is superior to surgical resection, 2) gives a strong rejection immunity associated with an immune cellular response of tumor-infiltrating macrophages and CD8 lymphocytes, 3) results in pronounced suppression of the growth of a simultaneous untreated tumor (distant bystander effect), 4) produces an increased anti-tumor lymphocyte proliferative response in tumor-draining and systemic lymph nodes and spleen, and 5) results in increased HSP70 immunoreactivity in tumors and tumor-infiltrating macrophages. Thus, the evidence for a laser-induced immunologic effect in tumor-bearing rats is strong. Some observations suggest that laser thermotherapy may be used for inducing favorable immunologic effects also in patients. Thus, we have shown a laser-induced bystander effect in a patient with malignant melanoma. In patients with breast cancer we have shown that laser thermotherapy induces intratumoral infiltration of immunocompetent cells like CD68 macrophages and CD8 lymphocytes. Laser thermotherapy is likely to be beneficial mainly when tumor burden is small, that is, when treatment is performed with curative intent, either with laser alone or together with surgical resection. For optimal effect, it appears likely that thermotherapy should be combined with other therapies. Most likely, a clinically meaningful effect can only be proven in prospective randomized studies comparing thermotherapy with other methods, particularly surgical resection.

Acute phase response is an effector process orchestrated by the innate immune system for the optimal mobilization of the resources of the organism distant from the local insult site needed in the execution of a host-protecting reaction. Our research has shown that mice bearing tumors treated by photodynamic therapy (PDT) exhibit the three major hallmarks of acute phase response: release of acute phase reactants, neutrophilia, and pituitary/adrenal axis activation. Of particular interest in this study were acute phase proteins that have a pivotal role in the clearance of dead cells, since the occurrence of this process in PDT-treated tumors emerges as a critical event in the course of PDT-associated host response. It is shown that this type of acute phase reactants, including complement proteins (C3, C5, C9, mannose-binding lectin, and ficolin A) and related pentraxins (serum amyloid P component and PTX3), are upregulated following tumor PDT and accumulate in the targeted lesions. Based on the recently accumulated experimental evidence it is definitely established that the acute phase response is manifested in the hosts bearing PDT-treated tumors and it is becoming clear that this effector process is an important element of PDT-associated host response bearing in impact on the eventual outcome of this therapy.

Tumor directed PDT has been shown by a number of pre-clinical studies to enhance a specific anti-tumor immune response, which appears to be critical to long-term tumor growth control by PDT. The PDT enhanced immune response is T cell dependent, however the mechanism behind the potentiation of the immune response by PDT is unknown. Induction of T cell dependent immunity depends upon the presence of activated antigen presenting cells. Therefore we have examined the ability of PDT to stimulate maturation and activation of antigen presenting cells in the PDT-treated tumor bed and tumor draining lymph node. Our studies demonstrate and increase in the number of activated antigen presenting cells in the tumor bed 24h following treatment of EMT6 murine tumors with Photofrin-PDT. Tumor draining lymph nodes also showed increased levels of activated antigen presenting cells within 4h of treatment. The levels peaked at 24h and declined by 48h after PDT. These results demonstrate that PDT-enhanced anti-tumor immunity is accompanied by an increase in antigen presenting cell activity. Therefore it is possible that T cell dependent immunity is enhanced following PDT through enhanced antigen presenting cell activity.

Temperature distribution in tissue can be a crucial factor in laser treatment for inducing immunization responses. In this study, Magnetic Resonance Imaging (MRI) was used to measure thermal temperature distribution in target tissue in laser treatment of metastatic tumors. It is the only feasible method for in vivo, non-invasive temperature distribution measurement. The measurement was conducted using phantom gel and tumor-bearing rats. The thermal couple measurement of target temperature was also was used to calibrate the relative temperature increase. The phantom system was constructed with a dye-enhanced spherical gel embedded in uniform gel phantom, simulating a tumor within normal tissue. Irradiation by an 805-nm laser increased the system temperature. Using an MRI system and proper algorithm processing for small animal studies, a clear temperature distribution matrix was obtained. The temperature profiles of rat tumors, irradiated by the laser with a power in the range of 2-3.5W and injected with a light-absorbing dye, ICG, and an immunoadjuvant, GC, were obtained. The temperature distribution provided in vivo thermal information and future reference for optimizing dye concentration and irradiation parameters to reach the optimum tumor destruction and immunization effects.

Light irradiation can modulate various biological processes. For instance, low-power laser irradiation (LPLI) can induce cell proliferation and differentiation. It has been used to treat diseases of regeneration limitation and to promote wound healing. The biological mechanism of light irradiation remains unclear. Our previous studies have shown that low fluence LPLI induced the proliferation of human lung adenocarcinoma cells (ASTC-a-1) through PKC channel, while high fluence LPLI induced caspase-3 activation and cell apoptosis. The mechanisms of the initiation and regulation of apoptosis are complex and diverse. There are two main pathways to initiate and regulate cell apoptosis, one is the death receptor pathway (receptor/caspase-8/caspase-3), and the other is the mitochondria pathway (mitochondria/ caspase-9/caspase-3). Using fluorescent imaging techniques, we observed a temporal sequence of events during apoptosis induced by high fluence LPLI and PDT. Both the high fluence LPLI and PDT triggers mitochondrial ROS production resulting in dissipation of ΔΨ_m and activation of caspase-3. Our results also show the two treatments do not activate caspase-8. These results suggest that caspase-3 activation induced by high fluence LPLI or PDT is initiated directly from mitochondria ROS generation and dissipation of ΔΨ_m, and independent of the cell death pathway involving caspase-8 activation. Because the progression of the apoptosis induced by high fluence LPLI is the same as that of PDT, we concluded that light is absorbed directly either by endogenous porphyrins or by the cytochromes in mitochondrion, resulting in initial ROS generation. During light irradiation induced apoptosis, apoptotic signals are initiated from mitochondrial ROS production due to photosensitization.

Non-invasive optical techniques offer great potential for observation of mitochondrial metabolism and morphology alternations. Apoptosis can be a result of cancer therapy such as PDT* during which mitochondria are undergoing structural and biochemical changes with different time-courses. Angularly-resolved light scattering is a powerful optical method to study behavior of light interaction with cells and their internal structures. Mitochondria organelles of ~ 1μm size is of the scale for Mie scattering. Fluorescence imaging is another important diagnostic tool to monitor two important endogenous fluorophores of the mitochondrial matrix, NADH (Nicotinamide Adenine Dinucleotide) and FAD (Flavin Adenine Dinucleotide) as metabolic biomarkers. The normalized ratio of these fluorophores called the redox ratio (FP/FP+NADH)¹ is an indicator of metabolism status of tissue independent of the number of mitochondria. The FL5 cells are investigated in normal and apoptotic stages by light scattering, fluorescence imaging and electron microscopy. Apoptosis is induced by substrate withdrawal. Goniometry-based light scattering results, suggest an earlystage apoptosis (8h) causes shrinkage of mitochondria and condensation of cristae whereas a late-stage (24h) is related to swollen mitochondria. These results are consistent with electron microscopy images of mitochondria at the same times. These three techniques provide multiple correlated data: morphological, biochemical and histological changes. Each technique strengthens the hypothesis behind the other data and helps to depict the overall procession of events.

The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3- interacting domain death agonist (Bid) may contribute to tumor necrosis factor α(TNF-α)-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid/tBid during TNF-α-induced cell death. The TNF-αtriggered Bid cleavage and tBid translocated to mitochondria. Human lung adenocarcinoma cells were co-transfected with both CFP-Bid and 14-3-3-YFP plasmids, and the dynamical interaction between the Bid/tBid and 14-3-3 were performed on laser confocal fluorescence microscope in single living cell during TNF-α-induced cell apoptosis. The Bid distribute equally only in the cytoplasm of healthy cells, and the 14-3-3 protein distribute not only in the cytoplasm but also in the nucleus of healthy cells. Our data showed that the tBid aggregate, but the 14-3-3 protein does not aggregate as the tBid, and the 14-3-3 protein separate from the aggregated tBid, implying that the 14-3-3 proteins do not interact with the aggregated tBid after TNF-αtreatment.

Low-power laser irradiation (LPLI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. Studying the signaling pathways involved in the laser irradiation is important for understanding these processes. The Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that governs proliferation, differentiation and cell survival. Recent studies suggest that Ras/Raf signaling pathway is involved in the LPLI-induced cell proliferation. Protein kinase Cs (PKCs) have been recently presumed to be involved in the regulation of cell proliferation induced by LPLI. In present study, to monitor the direct interaction between Ras and Raf and PKCs activation after LPLI treatment in living cells in real time, Raichu-Ras reporter and C kinase activity reporter (CKAR) were utilized, both of which were constructed based on fluorescence resonance energy transfer (FRET) technique. Our results show that the direct interaction between Ras and Raf is monitored during cell proliferation induced by LPLI (0.8 J/cm²) in serum-starved human lung adenocarcinoma cells (ASTC-a-1) expressing Raichu-Ras reporter using FRET imaging on laser scanning confocal microscope, and that the increasing dynamics of PKCs activity is also monitored during cell proliferation induced by LPLI (0.8 J/cm²) in serum-starved ASTC-a-1 cells expressing CKAR reporter using the similar way. Taken together, LPLI induces the ASTC-a-1 cell proliferation by activated Ras directly interacting with Raf and by specifically activating PKCs.

Transdermal drug delivery system (TDDS), which is one of drug delivery system (DDS) for increasing the effectiveness of drugs, is enhanced absorption of drugs by laser irradiation. The purpose of this study is to investigate the optimum laser parameter for enhancing TDD and to examine the mechanism of TDD enhancement. In this study, hairless mouse skins (in vitro) were irradiated with Er:YAG laser, Nd:YAG laser and free electron laser (FEL), which were set up energy density of 0.5 J/cm²/pulse and exposure time of 5 second. We examined the flux (μg/cm²/h) of lidocaine (C₁₄H₂₂N₂O, FW: 234.38) through the skins using high pressure liquid chromatography (HPLC), observed cross section of the irradiated samples using light microscope, and measured electrical resistance of the surface of skins. The HPLC results demonstrated that the TDD of the irradiated samples was enhanced 200-350 times faster than it of the non-irradiated samples. It of Nd:YAG laser, however, had no enhancement. The observation of cross section and the electrical resistance of skins were found to not remove the stratum corneum (SC), completely. These results show that laser irradiations, which has the strong absorption to skins, enhance TDD dramatically with low invasive.

Reactive oxygen species (ROS) are involved in the pathogenesis of many critical diseases and are also utilized as cytotoxic agents in a variety of treatments for eradication of diseased tissue, including cancer. Oxidative stress ensues when the level of ROS in a system exceeds the antioxidant capacity. Oxidative stress can have local (direct) and long-range (bystander) effects in cells and tissue and this research was carried out to determine the spatial and temporal nature of the photosensitized bystander effect using time-lapse fluorescence microscopy. By initiating photosensitization in only a portion of the microscopic imaging field it was possible to differentiate direct from bystander effects in EMT-6 murine breast cancer cells in 6-well plates. Elevated ROS levels are seen immediately following photodynamic treatment in direct cells with a delayed increase in oxidative stress observed in bystander cells. Cytotoxicity is also seen at earlier times in direct cells and occurs in bystander cells in a delayed fashion. These studies confirm the existence of a bystander effect following photosensitization and implicate mediators capable of diffusing in an intercellular manner from directly photosensitized cells to bystander cells and also implicate increased oxidative stress as a mechanistic factor in generating damage in bystander cells.