This PDF file contains the front matter associated with SPIE Proceedings Volume 7565, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Sphingolipids have become recognized as essential effector molecules in signal transduction with involvement in various aspects of cell function and death, immune response and cancer treatment response. Major representatives of sphingolipids family, ceramide, sphingosine and sphingosine-1-phosphate (S1P), have attracted interest in their relevance to tumor response to photodynamic therapy (PDT) because of their roles as enhancers of apoptosis, mediators of cell growth and vasculogenesis, and regulators of immune response. Our recent in vivo studies with mouse tumor models have confirmed that PDT treatment has a pronounced impact on sphingolipid profile in the targeted tumor and that significant advances in therapeutic gain with PDT can be attained by combining this modality with adjuvant treatment with ceramide analog LCL29.

Anti-tumor immunity is stimulated after PDT for cancer due to the acute inflammatory response, exposure and presentation of tumor-specific antigens, and induction of heat-shock proteins and other danger signals. Nevertheless effective, powerful tumor-specific immune response in both animal models and also in patients treated with PDT for cancer, is the exception rather than the rule. Research in our laboratory and also in others is geared towards identifying reasons for this sub-optimal immune response and discovering ways of maximizing it. Reasons why the immune response after PDT is less than optimal include the fact that tumor-antigens are considered to be self-like and poorly immunogenic, the tumor-mediated induction of CD4+CD25+foxP3+ regulatory T-cells (T-regs), that are able to inhibit both the priming and the effector phases of the cytotoxic CD8 T-cell anti-tumor response and the defects in dendritic cell maturation, activation and antigen-presentation that may also occur. Alternatively-activated macrophages (M2) have also been implicated. Strategies to overcome these immune escape mechanisms employed by different tumors include combination regimens using PDT and immunostimulating treatments such as products obtained from pathogenic microorganisms against which mammals have evolved recognition systems such as PAMPs and toll-like receptors (TLR). This paper will cover the use of CpG oligonucleotides (a TLR9 agonist found in bacterial DNA) to reverse dendritic cell dysfunction and methods to remove the immune suppressor effects of T-regs that are under active study.

There are many reports showing that low-level light/laser therapy (LLLT) can enhance wound healing, upregulate cell proliferation and has anti-apoptotic effects by activating intracellular protective genes. In the field of immune response study, it is not known with any certainty whether light/laser is proinflammatory or anti-inflammatory. Increasingly in recent times dendritic cells have been found to play an important role in inflammation and the immunological response. In this study, we try to look at the impact of low level near infrared light (810-nm) on murine bone-marrow derived dendritic cells. Changes in surface markers, including MHC II, CD80 and CD11c and the secretion of interleukins induced by light may provide additional evidence to reveal the mystery of how light affects the maturation of dendritic cells as well how these light-induced mature dendritic cells would affect the activation of adaptive immune response.

In Situ Photoimmunotherapy (ISPI), a newly developed modality for cancer therapy, has been shown to be able to modulate the body's own immune response. This clinical trial was designed to evaluate the safety and therapeutic effect of ISPI, using imiquimod as its immunoadjuvant for metastatic melanoma patients. ISPI consisted of three main components applied directly to the cutaneous metastases: 1) local application of topical imiquimod; 2) injection of indocyanine green; and 3) an 805 nm laser for local irradiation. All patients completed at least one cycle of treatment. All the patients completed at least one cycle of treatments; one patient received 6 cycles. The most common adverse effects were rash, pruritus, pain, fatigue and anorexia. Fever, chills, vomiting and cellulitis were relative rare. No grade 4 toxicity was observed. Complete Response (CR) was observed in 6 patients. Median overall survival of the 11 evaluated patients was 12.2 months. Six of the eleven patients were still alive at the time of this report. Treatment of ISPI using imiquimod is safe and well tolerant. Our preliminary clinical results suggest that this new method can be a promising modality for metastatic melanoma.

Laser Immunotherapy is an experimental treatment modality for late-stage, metastatic tumors, which targets solid primary and/or secondary tumors and utilizes an autologous vaccine-like approach to stimulate immune responses. Specifically, laser immunotherapy combines laser-induced in situ tumor devitalization with an immunoadjuvant for local immunostimulation. Here we report the initial results from a human breast cancer pilot trial with laser immunotherapy. Six stage III and IV cancer patients were treated, all of which were considered to be out of all other options, and preliminary data at the three-month examination are presented. The immediate goal of the trial was to determine the patient tolerance and the toxicity of the therapy, the optimal dose for the alteration of the course of the disease, and the reduction of the tumor burden. Each patient was individually evaluated for toxicity tolerance through physical exams and by appropriate supplemental and routine laboratory tests. Observable tumors in patients were followed with physical examination and radiological evaluations. Treatment efficacy was judged by the size and number of local and distant metastases before and after treatment.

Laser immunotherapy using laser photothermal therapy and immunological stimulation could achieve tumor-specific immune responses, as indicated by our previous pre-clinical and preliminary clinical studies. To further study the effect of laser immunotherapy, we conducted an investigation combining laser immunotherapy and surgery. After laser immunotherapy, treated tumors were surgically removed at different time points. The survival rates of treated mice were compared among different groups. Furthermore, the cured mice were rechallenged to test the immunity induced by laser immunotherapy. Our results showed that the mice treated with surgical removal one week after laser immunotherapy had the highest survival rate (77%). When the tumors were removed immediately after laser immunotherapy treatment, the survival rate was 57%. Most cured mice withstood tumor rechallenges, indicating an induction of tumor immunity by laser immunotherapy. The differentiations between different surgery groups indicate that the treated tumors have contributed to the immunological responses of the hosts.

One of the best strategies to prevent the occurrence of oral cancer is to eliminate oral precancers and block their further malignant transformation. Previous studies showed that photosan-mediated photodynamic therapy (photosan-PDT) is very effective for human head and neck cancers. To avoid the systemic photodynamic toxicity of photosan, this study was designed to use a topical photosan-PDT for treatment of DMBA-induced hamster buccal pouch precancerous lesions. Twelve 10-week-old male Syrian golden hamsters were used in this study. DMBA was applied to the left buccal pouches thrice a week for 8 to 10 weeks and mineral oil was painted on the right buccal pouches thrice a week for 8 to 10 weeks as the normal controls. Six hamsters were euthanized for tissue harvest. Precancerous lesions of moderate to severe dysplasia were consistently induced and proven by histological examination. These induced precancerous lesions in the remaining 6 hamsters were used for testing the efficacy of topical photosan-PDT. Before PDT, fluorescence spectroscopy was used to determine when protoporphyrine IX (PpIX) reached its peak level in the lesional epithelial cells after topical application of photosan-gel. We found that PpIX reached its peak level in precancerous lesions about 13.5 min after topical application of photosan-gel. The precancerous lesions in 4 hamsters were treated with topical photosan-PDT using the 635-nm LED light once or twice a week. Complete regression of the precancerous lesions was found after 2-4 PDT treatments by visual and histological examination. Our findings indicate that topical photosan-PDT is a very effective treatment modality for DMBA-induced hamster buccal pouch precancerous lesions.

Photodynamic agents such as Photofrin II (Photo II) utilized in photodynamic therapy (PDT) possess a remarkable property to become preferentially retained within the tumor's micro-environment. Upon the photo-agent's activation through visible light photon absorption, the agents exert their cellular cytotoxicity through type II and type I mechanistic pathways through extensive generation of reactive oxygen species (ROS): singlet oxygen ¹O₂, superoxide anion O₂ -, and hydrogen peroxide H₂O₂, within the intratumoral environment. Unfortunately, due to shallow visible light penetration depth (~2mm to 5mm) in tissues, the PDT strategy currently has largely been restricted to the treatments of surface tumors, such as the melanomas. Additional invasive strategies through optical fibers are currently utilized in getting the visible light into the intended deep seated targets within the body for PDT. In this communication, we report on a novel strategy in utilizing "soft" energy diagnostic X-rays to indirectly activate Photo II through X-ray induced luminescence from Gadolinium oxysulfide (20 micron dimension) particles doped with Terbium: Gd₂O₂S:Tb. X-ray induced visible luminescence from Gd₂O₂S:Tb particles was spectroscopically characterized and the ROS production levels from clinically relevant concentration (10 μg/ml) of Photo II was quantified through changes in the Vitamin C absorbance. ROS kinetics through X-ray induced luminescence was found to be similar to the ROS kinetics from red He-Ne laser exposures used in the clinics. Taken together, in-vitro findings herein provide the basis for future studies in determining the safety and efficacy of this non-invasive X-ray induced luminescence strategy in activating photo-agent in deep seated tumors.

Hyperthermia in combination with chemotherapy has been widely used in cancer treatment. Our previous study has shown that rapid rate hyperthermia in combination with chemotherapy can synergistically kill cancer cells whereas a sub-additive effect was found when a slow rate hyperthermia was applied. In this study, we explored the basis of this difference. For this purpose, in vitro cell culture experiments with a uterine cancer cell line (MES-SA) and its multidrug resistant (MDR) variant MES-SA/Dx5 were conducted. P-glycoprotein (P-gp) expression, Caspase 3 activity, and heat shock protein 70 (HSP 70) expression following the two different modes of heating were measured. Doxorubicin (DOX) was used as the chemotherapy drug. Indocyanine green (ICG), which absorbs near infrared light at 808nm (ideal for tissue penetration), was chosen for achieving rapid rate hyperthermia. Slow rate hyperthermia was provided by a cell culture incubator. Two sets of thermal doses were delivered by either slow rate or rapid rate hyperthermia. HSP70 expression was highly elevated under low dose slow rate incubator hyperthermia while maintained at the baseline level under the other three treatments. Caspase3 level slightly increased after low dose slow rate incubator hyperthermia while necrotic cell death was found in the other three types of heat treatment. In conclusion, when given at the same thermal dose, slow rate hyperthermia is more likely to induce thermotolerance. Meanwhile, hyperthermia showed a dose dependent capability in reversing P-gp mediated MDR; when MDR is reversed, the combinational treatment induced extensive necrotic cell death. During this process, the rate of heating also played a very important role; necrosis was more dramatic in rapid rate hyperthermia than in slow rate hyperthermia even though they were given at the same dose.

A recently developed "in vivo flow cytometer" and optical imaging are used to assess liver tumor cell spreading and the circulation kinetics of liver tumor cells. The in vivo flow cytometer has the capability to detect and quantify the number and flow characteristics of fluorescently labeled cells in vivo continuously. The depletion rate of circulating tumor cells provides insights in early cancer metastasis. It is useful to understand the molecular mechanisms of liver tumor metastasis. A real-time quantitative monitoring of circulating liver tumor cells by the in vivo flow cytometer will help assess the effectiveness of the potential therapeutic interventions.

This paper reports a robust polymer based centrifugal microfluidic analysis system that can provide parallel detection of multiple allergens in vitro. Many commercial food products (milk, bean, pollen, etc.) may introduce allergy to people. A low-cost device for rapid detection of allergens is highly desirable. With this as the objective, we have studied the feasibility of using a rotating disk device incorporating centrifugal microfluidics for performing actuationfree and multi-analyte detection of different allergen species with minimum sample usage and fast response time. Degranulation in basophils or mast cells is an indicator to demonstrate allergic reaction. In this connection, we used acridine orange (AO) to demonstrate degranulation in KU812 human basophils. It was found that the AO was released from granules when cells were stimulated by ionomycin, thus signifying the release of histamine which accounts for allergy symptoms ^[1-2]. Within this rotating optical platform, major microfluidic components including sample reservoirs, reaction chambers, microchannel and flow-control compartments are integrated into a single bio-compatible polydimethylsiloxane (PDMS) substrate. The flow sequence and reaction time can be controlled precisely. Sequentially through varying the spinning speed, the disk may perform a variety of steps on sample loading, reaction and detection. Our work demonstrates the feasibility of using centrifugation as a possible immunoassay system in the future.

To compare the differences of the light distributions in blood with different light irradiation modalities, Monte Carlo simulation was applied. The light distribution of He-Ne laser (632.8nm) in human whole blood was simulated. The diameter of the optic fiber used was 400μm. We referred to the work done by other researchers to determine tissue optical parameters of blood. For the same output laser power of 5mW, our results showed that the highest power density could be more than 6000mW/cm² using flat end optic fiber. But when using optical fiber coupled with cylindrical light diffuser (the length for light emission =3mm), the highest power density was less than 210mW/cm². Increasing the length of light emission could further reduce the highest power density. In summary, the optical fiber coupled with cylindrical light diffuser is a good modality for in vivo light irradiation in whole blood, which can decrease the light intensity and make it more uniformed distributed in blood. It is of great importance to choose the suitable light emission length of optic fiber coupled with cylindrical light diffuser for clinical applications.

Artemisinin (ART), an antimalarial phytochemical from the sweet wormwood plant or a naturally occurring component of Artemisia annua, has been shown a potential anticancer activity by apoptotic pathways. In our report, cell counting kit (CCK-8) assay showed that treatment of human lung adenocarcinoma (ASTC-a-1) cells with ART effectively increase cell death by inducing apoptosis in a time- and dose-dependent fashion. Hoechst 33258 staining was used to detect apoptosis as well. Reactive oxygen species (ROS) generation was observed in cells exposed to ART at concentrations of 400 μM for 48 h. N-acetyl-L-cysteine (NAC), an oxygen radical scavenger, suppressed the rate of ROS generation and inhibited the ART-induced apoptosis. Moreover, AFC assay (Fluorometric assay for Caspase3 activity) showed that ROS was involved in ART-induced caspase3 acitvation. Taken together, our data indicate that ART induces ROS-mediated caspase3 activation in a time-and dose-dependent way in ASCT-a-1 cells.

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Different concentrations of taxol can trigger distinct effects on both the cellular microtubule network and biochemical pathways. Apoptosis induced by low concentrations (5-30 nM) of taxol was associated with mitotic arrest, alteration of microtubule dynamics and/or G2/M cell cycle arrest, whereas high concentrations of this drug (0.2-30 μM) caused significant microtubule damage, and was found recently to induce cytoplasm vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. In present study, cell counting kit (CCK-8) assay, confocal microscope, and flow cytometry analysis were used to analyze the cell death form induced by 35 nM and 70 μM of taxol respectively in human lung adenocarcinoma (ASTC-a-1) cells. After treatment of 35 nM taxol for 48 h, the OD450 value was 0.80, and 35 nM taxol was found to induce dominantly cell death in apoptotic pathway such as phosphatidylserine (PS) externalization, G2/M phase arrest after treatment for 24 h, and nuclear fragmentation after treatment for 48 h. After 70 μM taxol treated the cell for 24 h, the OD450 value was 1.01, and 70 μM taxol induced cytoplasm vacuolization programmed cell death (PCD) and G2/M phase as well as the polyploidy phase arrest in paraptotic-like cell death. These findings imply that the regulated signaling pathway of cell death induced by taxol is dependent on taxol concentration in ASTC-a-1 cells.

Resveratrol (RV) is a natural plant polyphenol widely present in foods such as grapes, wine, and peanuts. Previous studies indicate that RV has an ability to inhibit various stages of carcinogenesis and eliminate preneoplastic cells in vitro and in vivo. However, little is known about the molecular mechanism of RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cell. In this report, we analyzed whether Bax translocation from cytoplasm to mitochondria during RV-induced apoptosis in single living cell using onfocal microscopey. Cells were transfected with GFP-Bax plasmid. Cell counting kit (CCK-8) assay was used to assess the inhibition of RV on the cells viability. Apoptotic activity of RV was detected by Hoechst 33258 and propidium iodide (PI) staining. Our results showed that RV induced a dose-dependent apoptosis in which Bax did not translocate to mitochondrias.

Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and initiate apoptotic pathway. Apoptosis signal-regulating kinase (ASK1) is an important regulator of apoptosis in response to various stresses, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, lipopolysaccharide (LPS) and calcium influx. In this study, we investigated the molecular mechanisms of apoptosis induced by NPe6-PDT in ASTC-a-1 cells. The results showed that the activities of ASK1 increased in response to NPe6-PDT. Over-expression of wild-type or activated mutant of ASK1 could obviously decrease cell viability and increase cell death; while inhibition of ASK1 significantly decreased cell apoptosis. These results suggested that ASK1 plays an important role in apoptosis induced by NPe6-PDT.

Sirtuin type 1 (SIRT1), a NAD+-dependent histone deacetylases, plays a critical role in cellular senescence, aging and longevity. In general, SIRT1 is localized in nucleus and is believed as a nuclear protein. Though overexpression of SIRT1 delays senescence, SIRT1-protein levels decline naturally in thymus and heart during aging. In the present studies, we investigated the subcellular localization of SIRT1 in response to growth factor deprivation in African green monkey SV40-transformed kidney fibroblast cells (COS-7). Using SIRT1-EGFP fluorescence reporter, we found that SIRT1 localized to nucleus in physiological conditions. We devised a model enabling cell senescence via growth factor deprivation, and we found that SIRT1 partially translocated to cytosol under the treatment, suggesting a reduced level of SIRT1's activity. We found PI3K/Akt pathway was involved in the inhibition of SIRT1's cytosolic translocation, because inhibition of these kinases significantly decreased the amount of SIRT1 maintained in nucleus. Taken together, we demonstrated that growth factor deprivation induces cytosolic translocation of SIRT1, which suggesting a possible connection between cytoplasm-localized SIRT1 and the aging process.

Cardiolipin is a unique and ubiquitous diphosphatidylglycerol phospholipid, located exclusively in inner membrane of mitochondria and particularly intermembrane contact sites. Cardiolipin is essential for mitochondrial to maintain its functions. Numerous mitochondrial proteins and processes require the presence of cardiolipin. Recent researches gradually confirm that cardiolipin participates in several mitochondria-dependent apoptotic steps: interactions between cardiolipin and cytochrome c, Bid and caspase-8 have now been established. These functions are associated with the redistribution of cardiolipin in mitochondria. However, the exact mechanism of the redistribution, which happens at the early stage of apoptosis, is still controversial. In this study, we used 10-N-nonyl-3, 6-bis (dimethylamino) acridine (10-N-nonyl acridine orange), a specific probe for cardiolipin to monitor changes of cardiolipin redistribution during apoptosis. We demonstrated that during apoptosis cardiolipin moves to the outer leaflet of mitochondrial inner membrane from the inner leaflet, where it used to be riched in. We also found that ROS (reactive oxygen species) may have association with the redistribution of cardiolipin.

FOXO transcription factors are important regulators of cell survival in response to a variety of stimuli, among which are hypoxic stress, oxidative stress, and growth factor deprivation. Subcellular localization of FOXO proteins plays a major role in the regulation of their activity. In this study, using confocal imaging of the cells transfected with GFP-FOXO3a and fluorescence recovery after photobleaching technique, we visualized the dynamic nuclear translocation of GFP-FOXO3a in ASTC-a-1 cells under growth factor stimulus. In healthy cells, GFP-FOXO3a was well-distributed in the cytoplasm or widespread distributed in the cytoplasm and the nucleus but the cytoplasm was significantly more than the nucleus. Deprivation of growth factor, we monitored the nuclear localization of GFP-FOXO3a and the dynamic translocation of it from cytoplasm to nucleus. Interestingly, upon stimulation with growth factor in cells again, we visualized the dynamic nuclear exclusion of GFP-FOXO3a and cytoplasm distribution rapidly. In conclusion, these results demonstrated that FOXO3a can reversible shuttling between cytoplasm and nucleus upon stimulation with growth factor.

Studies the superparamagnetic iron oxide (SPIO) label ECV304 method and test its effect on cell physiological activity .We use different concentration PLL-SPIO incubate ECV304 cells, Prussian blue dye was taken to examine SPIO mark efficiency, DCF dyeing and PI & Annexin the VFITC double dyeing was carried out to analyze cell apoptosis. The result shows that 25μg/ml gave the most efficient labeling concentration, there is also no toxic effect on cells in this concentration. This outcome can be used in MRI of living body cell transplant tracking.

Recent studies have demonstrated that dendritic cells (DCs) play a crucial role in the activation of Natural Killer cells (NKs) that are responsible for anti-tumor innate immune responses. The focus of this report is on the role of pathogen associated molecular pattern (PAMP) activated-DCs in inducing NK cell-mediated anti-tumor responses. Mice transplanted sub-cute (s.c.) with AK7 cells, a mesothelioma cell line sensitive to NK cell responses, are injected with fluorescent NK cells and DC activation is then induced by s.c. injection of Lipopolysaccharide (LPS). Using 4 dimensional tracking we follow the kinetic behavior of NK cells at the Draining Lymph-Node (DLN). As control, noninflammatory conditions are also evaluated. Our data suggest that NK cells are recruited to the DLN where they can interact with activated-DCs with a peculiar kinetic behavior: short lived interactions interleaved by rarer longer ones. We also found that the changes in the NK dynamic behavior in inflammatory conditions clearly affect relevant motility parameters such as the instantaneous and average velocity and the effective diffusion coefficient. This observation suggests that NK cells and activated-DCs might efficiently interact in the DLN, where cells could be activated. Therefore the interaction between activated-DCs and NK cells in DLN is not only a reality but it may be also crucial for the start of the immune response of the NKs.