This PDF file contains the front matter associated with SPIE Proceedings Volume 8944, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Malignant melanoma is one of the fastest growing cancers and if it cannot be completely surgically removed the prognosis is bleak. Melanomas are known to be particularly resistant to both chemotherapy and radiotherapy. Various types of immunotherapy have however been investigated with mixed reports of success. Photodynamic therapy (PDT) has also been tested against melanoma, again with mixed effects as the melanin pigment is thought to act as both an optical shield and as an antioxidant. We have been investigating PDT against malignant melanoma in mouse models. We have compared B16F10 melanoma syngenic to C57BL/6 mice and S91 Cloudman melanoma syngenic to DBA2 mice. We have tested the hypothesis that S91 will respond better than B16 because of higher expression of immunocritical molecules such as MHC-1, tyrosinase, tyrosinase related protein-2 gp100, and intercellular adhesion molecule-1. Some of these molecules can act as tumor rejection antigens that can be recognized by antigen-specific cytotoxic CD8 T cells that have been stimulated by PDT. Moreover it is possible that DBA2 mice are intrinsically better able to mount an anti-tumor immune response than C57BL/6 mice. We are also studying intratumoral injection of photosensitzers such as benzoporphyrin monoacid ring A and comparing this route with the more usual route of intravenous administration.

Vascular endothelial growth factor A (VEGF-A), commonly known as VEGF, is one of the primary factors that affect tumor angiogenesis. It was found to be expressed in cancer cell lines including oral squamous cell carcinoma. Photodynamic therapy (PDT) is a novel therapeutic modality to treat cancer by using a photosensitizer which is activated by a light source to produce reactive oxygen species and mediates oxygen-independent hypoxic conditions to tumor. Another emerging treatment to cure cancer is the use of interference RNA (e.g. siRNA) to silence a specific mRNA sequence. VEGF-A was found to be expressed in oral squamous cell carcinoma and overexpressed after 24 hour post-PDT by Western blot analysis. Cell viability was found to decrease at 25 nM of transfected VEGF-A siRNA. In vitro combined therapy of PDT and VEGF-A siRNA showed better response as compared with PDT and gene therapy alone. The results suggest that PDT combined with targeted gene therapy has a potential mean to achieve better therapeutic outcome.

T-cell stimulators such as anti-CTLA-4 antibodies enhance immunologic responses to chemotherapy-resistant solid tumors, such as melanoma, advanced breast cancer, ovarian cancer and pancreatic cancer. The efficacy of these new immunotherapy agents can in theory be enhanced substantially by therapies that stimulate new immunologic (T-cell) responses against the tumor. Laser immunotherapy (LIT) with imiquimod and InCVAX are techniques that produce useful responses in patients with advanced melanoma, the prototypical chemotherapy resistant solid tumor. The mechanism of action of these therapies is thought to be immunological, including the development of new T-cell responses. We have therefore been combining LIT using imiquimod and InCVAX treatment with the new T-cell stimulators (ipilimumab) in cases of stage IV melanoma. While still anecdotal, the use of novel combinations of immunologic therapies should provide much improved responses for chemotherapy-resistant solid tumors (such as melanoma) than was previously possible. Newer T-cell stimulating drugs such as the anti-PD-1 antibodies and anti-PD-L1 antibodies will make this general approach to treating chemoresistant advanced tumors even more effective in the future.

The microenvironments of tumors are involved in a complex and reciprocal dialog with surrounding cancer cells. Any novel treatment must consider the impact of the therapy on the microenvironment. Recently, clinical trials with laser immunotherapy (LIT) have proven to effectively treat patients with late-stage, metastatic breast cancer and melanoma. LIT is the synergistic combination of phototherapy (laser irradiation) and immunological stimulation. One prominent cell type found in the tumor stroma is the fibroblast. Fibroblast cells can secrete different growth factors and extracellular matrix modifying molecules. Furthermore, fibroblast cells found in the tumor stroma often express alpha smooth muscle actin. These particular fibroblasts are coined cancer-associated fibroblast cells (CAFs). CAFs are known to facilitate the malignant progression of tumors. A collagen lattice assay with human fibroblast cells is used to elucidate the effects LIT has on the microenvironment of tumors. Changes in the contraction of the lattice, the differentiation of the fibroblast cells, as well as the proliferation of the fibroblast cells will be determined.

Laser immunotherapy (LIT) is an innovative cancer modality that uses laser irradiation and immunological stimulation to treat late-stage, metastatic cancers. The current mode of operation in LIT is through interstitial laser irradiation. Although LIT is still in development, recent clinical trials have shown that it can be used to successfully treat patients with late-stage breast cancer and melanoma. Cyclophosphamide is a chemotherapy drug that suppresses regulatory T cells when used in low doses. In this study tumor-bearing rats were treated with LIT using an 805-nm laser with a power of 2.0 W and low-dose cyclophosphamide. Glycated chitosan was used as an immunological stimulant. The goal was to observe the effects of different doses of cyclophosphamide in addition to LIT on the survival of the tumor-bearing rats.

Laser immunotherapy (LIT) is a cancer treatment with promising results in animal models and some `no other option' patients. The therapy elicits an immune response that targets the primary tumor and, perhaps more importantly, otherwise untreatable metastases. We develop and analyze a mathematical model that includes key elements of the host immune response set in motion by LIT. We use Latin Hypercube Sampling (LHS) to EFFICIENTLY sample model parameters from a high-dimensional parameter space and get a broad sense of model dynamics. Depending on a variety of tumor, therapy, and immune parameters, a variety of patient outcomes ranging from tumor clearance to patient death are possible.

Rapamycin (RAPA) as a unique tolerance-promoting therapeutic drug is crucial to successful clinical organ transplantation. DC (Dendritic cells) play a critical role in antigen presentation to T cells to initiate immune responses involved in tissue rejection. Although the influence of RAPA on DC differentiation and maturation had been reported by some research groups, it is still controversial and unclear right now. In addition, it is also lack of study on investigating the role of DC in DTH reaction via intravital optical imaging. Herein, we investigated the effect of rapamycin on phenotype and function of bone marrow monocyte-derived DC both in vitro and in vivo. In vitro experiments by flow cytometry (FACS) showed that DC displayed decreased cell size and lower expression levels of surface molecule CD80 induced by RAPA; Furthermore, the phagocytic ability to OVA of DC was inhibited by RAPA started from 1 h to 2 h post co-incubation, but recovered after 4 h; In addition, the capacity of DC to activate naïve OT-II T cell proliferation was also inhibited at 3 day post co-incubation, but had no effect at 5 day, the data indicated this effect was reversible when removing the drug. More importantly, the DC-T interaction was monitored both in vitro and in intravital lymph node explant, and showed that RAPA-DC had a significant lower proportion of long-lived (>15min) contacts. Thus, RAPA displayed immunosuppressive to phenotypic and functional maturation of DC, and this phenomenon induced by RAPA may favorable in the clinical organ transplantation in future.

We use two-photon microscopy to monitor the infection process of marine zooplankton, Cafeteria roenbergensis (C.roenbergensis), by Cafeteria roenbergensis virus (CroV), a giant DNA virus named after its host. Here, we image C.roenbergensis in culture by two-photon excited NADH autofluorescence at video-rate (30 frame/s), and the movement of C.roenbergensis is recorded in live videos. Moreover, CroV is stained with DNA dye SYBR gold and recorded simultaneously with this two-photon microscope. We observed the initial infection moment with this method. The result demonstrates the potential use of two-photon microscopy to investigate the fast dynamic interaction between C.roenbergensis with virus CroV. After catching this initial moment, we will freeze the sample in liquid nitrogen for cryo-electron microscopy (EM) study to resolve the virus-host interaction at molecular level. The long-term goal is to study similar fast moving pathogen-host interaction process which could lead to important medical applications.

The objective of this research is to demonstrate an in-line phase contrast tomosynthesis prototype operated under high x-ray tube voltage, and a phantom study was conducted to characterize the potentials of this system. The prototype is based on an in-line phase contrast system accompanying with digital tomosynthesis imaging mechanism; and the tube voltage is operated at 120 kVp. A phantom study was conducted by using a custom-designed fish bone phantom to demonstrate the ability of this imaging system in edge enhancement and noise suppression. As the result, edge enhancement could be observed on the in-plane slices by plotting and comparing the intensity profiles with DTS images. As employing phase retrieval method onto the original angular projections could dramatically improve the image quality in edge enhancement, 3D imaging box was preliminarily constructed by using reconstructed in-plane slices acquired with PAD phase retrieval. As expected, high-energy in-line phase contrast tomosynthesis imaging system shows its potentials in edge enhancement and noise suppression by introducing phase retrieval method. Dose studies and perfecting photon energies and phantom designs will be our future interest.

Accurate background estimation to isolate the fluorescence signals is an important issue for quantitative X-ray fluorescence (XRF) analysis of gold nanoparticles (GNPs). Though a good estimation can be obtained experimentally through acquiring the background spectrum of water solution, it inevitably leads to unnecessary second exposure in reality. Thus, several numerical methods such as trapezoidal shape estimation, interpolation by polynomial fitting and SNIP (Statistics sensitive Nonlinear Iterative Peak-Clipping) algorithm are proposed to achieve this goal. This paper aims to evaluate the estimation results calculated by these numerical methods through comparing with that acquired using the experimental way, in term of mean squared error (MSE). Four GNP/water solutions with various concentrations from 0.0% to 1.0% by weight are prepared. Then, ten spectra are acquired for each solution for further analysis, under the identical condition of using pencil beam x-ray and single spectrometer. Finally, the experimental and numerical methods are performed on these spectra within the optimally determined energy window and their statistical characteristics are analyzed and compared. These numerical background estimation methods as well as the evaluation methods can be easily extended to analyze the fluorescence signals of other nanoparticle biomarkers such as gadolinium, platinum and Barium in multiple biomedical applications.

Laser immunotherapy (LIT), a novel technique with a local intervention to induce systemic antitumor effects, was developed to treat metastatic cancers. The pre-clinical studies of LIT have shown its unique characteristics in generating a specific antitumor immunity in treating metastatic tumors in rats and mice. For late-stage, metastatic breast cancer patients, who were considered to be out of other available treatment options, we conducted a small Phase II clinical trial using LIT starting in 2009 in Lima, Peru. This Phase II study was closed in December of 2012, as acknowldged by the Ministry of Health (MOH) of Peur letter 438-2014-OGITT/INS dated March 5^th, 2014. Ten patients were enrolled and received LIT in one or multiple 4-week treatment cycles. At the study closing date, four patients were alive and two of them remained cancer free. Here, following the successful conclusion of our Phase II study, we report the clinical effects of LIT on metastatic breast cancer patients. Specifically, we present the overall status of all the patients three years after the treatment and also the outcomes of two long-term surviving patients.

In our previous work, we constructed a multifunction nano system SWNT-GC, which can synergize photothermal and immunological effects. To further improve the application of this system, we study the cytotoxicity of SWNT-GC and investigate the effects on malignant tumor therapy. Here, we selected the optimal concentration of GC and SWNTs for the stable SWNT-GC construction. No cytotoxicity was observed under the dose used in the experiments. Using mouse melanoma tumor model, Laser+SWNT-GC treatment resulted in a significant mice survival rate, there were no long-term survivors under other treatment. It is providing a promising treatment modality for the malignancy.

Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

In this study, we presented a method combining optical coherence tomography with wavelet differentiation method to quantify the elastic properties of tissue. Based on the optimized kernel size for 2D normalized cross-correlation, the wavelet differentiation method was used to estimate the tissue strains. The influences of the dilation parameter of wavelet on calculations of axial strain had been investigated. Experimental results suggest that the dilation parameter of 8 was selected in strain calculation for best quality of axial strain images. The method based on wavelet differentiation shows great potential for optical coherence tomography elastography. In addition, elastic properties images of thyroid with suspected cysts were depicted to distinguish benign lesions qualitatively. Thus, elastic properties imaging based on optical coherence elastography shows great promise for the detailed characterization of lesions and preliminary diagnosis of human thyroid diseases.

Melanoma, a malignant tumor of melanocytes, is the most serious type of skin cancer in the world. It accounts for about 80% of deaths of all skin cancer. For cancer detection, circulating tumor cells (CTCs) serve as a marker for metastasis development, cancer recurrence, and therapeutic efficacy. Melanoma tumor cells have high content of melanin, which has high light absorption and can serve as endogenous biomarker for CTC detection without labeling. Here, we have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of melanoma cancer by counting CTCs of melanoma tumor bearing mice in vivo. To test in vivo PAFC’s capability of detecting melanoma cancer, we have constructed a melanoma tumor model by subcutaneous inoculation of highly metastatic murine melanoma cancer cells, B16F10. In order to effectively distinguish the targeting PA signals from background noise, we have used the algorithm of Wavelet denoising method to reduce the background noise. The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the blood stream. Compared with fluorescence-based in vivo flow cytometry (IVFC), PAFC technique can be used for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs).

KillerRed is a unique red fluorescent protein exhibiting excellent phototoxic properties. It has the ability to produce reactive oxygen species (ROS), for killing tumor cells in vitro upon laser irradiation and has the potential to act as a photosensitizer in the application of tumor therapy. Here, we investigated the effects of KillerRed-based photodynamic therapy (PDT) on tumor growth in vivo and examined the subsequent tumor metabolic states including the changes of pyridine nucleotide (PN) and flavoprotein (Fp), two important metabolic coenzymes of tumor cells. Results showed that the tumor was scabbed in response to 561 nm laser irradiation at 80 mV for 3 min, and the tumor growth had been significantly inhibited by KillerRed-based PDT treatment compared to control groups. More importantly, a home-made cryo-imaging redox scanner was used to measure intrinsic fluorescence and exogenous KillerRed fluorescence signals in tumors. The flavoprotein was remarkable elevated and the PN was seldom increased with concomitant photobleaching of KillerRed fluorescence after irradiation, suggesting that flavoprotein and PN were oxidized in the course of KillerRed-based PDT.

With the help of the specific absorption spectrum of carbon nanotubes, we achieved selective photothermal tumor cell destruction, particularly using a near-infrared laser to reduce potential damage to untargeted tissues. Combined with immunological stimulation, using a novel adjuvant, we also observed the anti-tumor immune responses when treating animal tumors using the laser-nano treatment. In fact, the local application of laser-nano-immunotherapy appeared to result in a systemic curative effect. In our mechanistic study, we found that the laser-nano-immuno treatment can activate antigen-presenting cells, such as dendritic cells (DCs). More importantly, the uptake and presentation of antigens by these antigen presenting cells were significantly enhanced, as shown by the strong binding of tumor cells and DCs as well as the proliferation of T cells caused by the DCs after the DCs had been incubated with laser-nano-immuno treated tumors. These cellular observations provide evidence that a systemic anti-tumor immune response was induced by the combination of laser and nanotechnology.

Fluorescence in situ Hybridization technology is a commonly used tool to detect chromosome aberrations, which are often pathologically significant. Since manual FISH analysis is a tedious and time-consuming procedure, reliable and robust automated image acquisition and analysis are in demand. Under high magnification objective lenses such as 60x and 100x, the depth of field will often be too small and the FISH probes may not always lie in the same focal plane. A statistical variance based automated FISH analysis method is developed in order to address this problem. On a stack of slices at consecutive image planes with a step size d, the statistical variance alone the z-axis is calculated to form a 2-D matrix. Since pixels shift dramatically to high intensity at FISH probe location, the probes will manifest high peak values in the matrix. A computer-aided detection scheme based on top-hat transform is applied to the matrix to detect FISH probe signals. This study demonstrates a simple and robust method for FISH probe detection as well as a way of 2- D representation of 3-D data.

We use a mathematical model to describe and predict the population dynamics of tumor cells, immune cells, and other immune components in a host undergoing laser immunotherapy treatment against metastatic cancer. We incorporate key elements of the treatment into the model: a function describing the laser-induced primary tumor cell death and parameters capturing the role and strength of the primary immunoadjuvant, glycated chitosan. We focus on identifying conditions that ensure a successful treatment. In particular, we study the patient response (i.e., anti-tumor immune dynamics and treatment outcome) in two different but related mathematical models as we vary quantitative features of the immune system (supply, proliferation, death, and interaction rates). We compare immune dynamics of a `baseline' immune model against an `augmented' model (with additional cell types and antibodies) and in both, we find that using strong immunoadjuvants, like glycated chitosan, that enhance dendritic cell activity yields more promising patient outcomes.

It has been known for a long time that microglial activation plays an important role in the pathology of neurodegenerative diseases. Once activated, they have macrophage-like capabilities, which can be detrimental by producing proinflammatory and neurotoxic factors including cytokines, reactive oxygen species (ROS) and nitric oxide that directly or indirectly cause neurodegeneration. Therefore, the regulation of microglial-induced neuroinflammation is considered a useful strategy in searching for neuroprotective treatments. In this study, our results showed that low power laser irradiation (LPLI) (20 J/cm²) could suppress microglial-induced neuroinflammation in LPS-activated microglia. We found that LPLI-mediated neuroprotection was achieved by activating tyrosine kinases Src, which led to MyD88 tyrosine phosphorylation, thus impairing MyD88-dependent proinflammatory signaling cascade. Our research may provide a feasible therapeutic approach to control the progression of neurodegenerative diseases.