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- Front Matter: Volume 7565
- PDT and Immune Responses
- Photoimmunotherapy: Clinical
- Photoimmunotherapy: Pre-clinical
- Detection of Immune Activities
- Poster Session
Front Matter: Volume 7565
Front Matter: Volume 7565
Show abstract
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.
PDT and Immune Responses
Tumor PDT-associated immune response: relevance of sphingolipids
Show abstract
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.
Photodynamic therapy for cancer and activation of immune response
Show abstract
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.
Can dendritic cells see light?
Show abstract
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.
Photoimmunotherapy: Clinical
Preliminary results of a phase I/II clinical trial using in situ photoimmunotherapy combined with imiquimod for metastatic melanoma patients
Show abstract
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: initial results from a human breast cancer pilot trial
Show abstract
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.
Photoimmunotherapy: Pre-clinical
Effect of laser immunotherapy and surgery on the treatment of mouse mammary tumors
Show abstract
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.
Topical photosan-mediated photodynamic therapy for DMBA-induced hamster buccal pouch premaligant lesions: an in vivo study
Show abstract
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.
Novel applications of diagnostic x-rays in activating photo-agents through x-ray induced visible luminescence from rare-earth particles: an in vitro study
Erkinay Abliz,
Joshua E. Collins,
Joseph S. Friedberg,
et al.
Show abstract
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.
The role of temperature increase rate in combinational hyperthermia chemotherapy treatment
Show abstract
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.
Detection of Immune Activities
Monitoring hepatocellular carcinoma metastasis by in vivo flow cytometer
Yan Li,
Jin Guo,
Guangda Liu,
et al.
Show abstract
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.
A fluorescence-based centrifugal microfluidic system for parallel detection of multiple allergens
Show abstract
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.
Influence of light irradiation modalities on light distribution in human whole blood
Show abstract
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.
Poster Session
Artemisinin induces ROS-mediated caspase3 activation in ASTC-a-1 cells
Show abstract
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 induces concentration-dependent phosphatidylserine (PS) externalization and cell cycle arrest in ASTC-a-1 cells
Show abstract
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.
Bax is not involved in the resveratrol-induced apoptosis in human lung adenocarcinoma cells
Wei-wei Zhang,
Zhi-ping Wang,
Tong-sheng Chen
Show abstract
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.
Involvement of ASK1 activation in apoptosis induced by NPe6-PDT
Show abstract
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.
Growth factor deprivation induces cytosolic translocation of SIRT1
Show abstract
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.
Redistribution of intramitochondrial cardiolipin at the early stage of apoptosis is associated with ROS
Show abstract
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.
Analysis of GFP-FOXO3a nuclear-cytoplasmic shuttling in ASTC-a-1 cells under growth factor stimulation
Show abstract
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.
Study on the best method of SPIO labeling on the cell line ECV304
Show abstract
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.
Two photon microscopy intravital study of DC-mediated anti-tumor response of NK cells
Show abstract
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.