Eduardo H. Moriyama

Speckle variance detection of microvasculature using swept-source optical coherence tomography

We report on imaging of microcirculation by calculating the speckle variance of optical coherence tomography (OCT) structural images acquired using a Fourier domain mode-locked swept-wavelength laser. The algorithm calculates interframe speckle variance in two-dimensional and three-dimensional OCT data sets and shows little dependence to the Doppler angle ranging from 75 degrees to 90 degrees . We demonstrate in vivo detection of blood flow in vessels as small as 25 microm in diameter in a dorsal skinfold window chamber model with direct comparison with intravital fluorescence confocal microscopy. This technique can visualize vessel-size-dependent vascular shutdown and transient vascular occlusion during Visudyne photodynamic therapy and may provide opportunities for studying therapeutic effects of antivascular treatments without on exogenous contrast agent.

Diblock Copolymer Micelles Deliver Hydrophobic Protoporphyrin IX for Photodynamic Therapy

Polymeric micelles are emerging as an effective drug delivery system for hydrophobic photosensitizers in photodynamic therapy (PDT). The objective of this study was to investigate the formulation of hydrophobic protoporphyrin IX (PpIX) with MePEG5000‐b‐PCL4100 [methoxy poly (ethylene glycol)‐b‐poly (caprolactone)] diblock copolymers and to compare their PDT response to that of free PpIX. The photophysical and photochemical properties of the polymeric PpIX micelles were studied by measuring absorbance and fluorescence spectra, PpIX‐loading efficiency and stability, the micelle particle size and morphology, as well as singlet oxygen luminescence and lifetime. The spherical micelles have a high PpIX‐loading efficiency of 82.4% and a narrow size distribution with a mean diameter of 52.2 ± 6.4 nm. The cellular uptake of PpIX in RIF‐1 cells using PpIX micelles was approximately two‐fold higher than that for free PpIX. Free PpIX and PpIX formulated in micelles exhibited similar subcellular localization in or around the cellular plasma membrane, as demonstrated using fluorescence microscopy. In vitro PDT results showed that the PpIX micelles have markedly increased photocytotoxicity over that with free PpIX, by nearly an order of magnitude at the highest light dose used. The micelles alone had no evident phototoxicity or dark toxicity. These findings suggest that MePEG5000‐b‐PCL4100 diblock copolymer micelles have great potential as a drug delivery system for hydrophobic photodynamic sensitizers.

Hypoxia promotes ligand-independent EGF receptor signaling via hypoxia-inducible factor–mediated upregulation of caveolin-1

Caveolin-1 (CAV1) is an essential structural constituent of caveolae, specialized lipid raft microdomains on the cell membrane involved in endocytosis and signal transduction, which are inexplicably deregulated and are associated with aggressiveness in numerous cancers. Here we identify CAV1 as a direct transcriptional target of oxygen-labile hypoxia-inducible factor 1 and 2 that accentuates the formation of caveolae, leading to increased dimerization of EGF receptor within the confined surface area of caveolae and its subsequent phosphorylation in the absence of ligand. Hypoxia-inducible factor–dependent up-regulation of CAV1 enhanced the oncogenic potential of tumor cells by increasing the cell proliferative, migratory, and invasive capacities. These results support a concept in which a crisis in oxygen availability or a tumor exhibiting hypoxic signature triggers caveolae formation that bypasses the requirement for ligand engagement to initiate receptor activation and the critical downstream adaptive signaling during a period when ligands required to activate these receptors are limited or are not yet available.

In Vivo Study of the Inflammatory Modulating Effects of Low‐level Laser Therapy on iNOS Expression Using Bioluminescence Imaging

Abstract This study was designed to demonstrate that bioluminescence imaging (BLI) can be used as a new tool to evaluate the effects of low-level laser therapy (LLLT) during in vivo inflammatory process. Here, the efficacy of LLLT in modulating inducible nitric oxide synthase (iNOS) expression using different therapeutic wavelengths was determined using transgenic animals with the luciferase gene under control of the iNOS gene expression. Thirty transgenic mice, FVB/N-Tg(iNOS-luc)Xen, were allocated randomly to one of four experimental groups treated with different wavelengths (λ = 635, 785, 808 and 905 nm) or a control group (nontreated). Inflammation was induced by intra-articular injection of zymosan A in both knee joints. Laser treatment (25 mW cm−2, 200 s, 5 J cm−2) was applied to the knees 15 min after inflammation induction. Measurements of iNOS expression were performed at various times (0, 3, 5, 7, 9 and 24 h) by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The results showed a significant increase in BLI signal after irradiation with 635 nm laser when compared to the nonirradiated animals and the other LLLT-treated groups, indicating wavelength dependence of LLLT effects on iNOS expression during the inflammatory process, and thus demonstrating an action spectrum of iNOS gene expression following LLLT in vivo that can be detected by BLI. Histological analysis was also performed and demonstrated the presence of fewer inflammatory cells in the synovial joints of mice irradiated with 635 nm compared with nonirradiated knee joints.

Proof-of-principle demonstration of a Mueller matrix decomposition method for polarized light tissue characterization in vivo

We demonstrate the first in vivo use of a Mueller matrix decomposition method for polarization-based characterization of tissue. Collagenase is injected into a region of dermal tissue in a dorsal skin window chamber in a nude mouse to alter the structure of the extracellular matrix. Mueller matrices for polarized light transmitted through the window chamber in the collagenase-treated region, as well as a distal control region, are measured. From the measured matrices, the individual constituent polarization properties of the tissue are extracted through polar matrix decomposition. Large decreases in birefringence and depolarization are seen in the collagenase-treated region due to the destruction of collagen, showing the potential for this method to monitor the organization and structural anisotropy of tissue. This study represents the first in vivo demonstration of a Mueller matrix decomposition method for polarimetric tissue characterization.

Continuous Docetaxel Chemotherapy Improves Therapeutic Efficacy in Murine Models of Ovarian Cancer

Ovarian cancer is known as the silent killer for being asymptomatic until late stages. Current first-line treatment consists of debulking surgery followed by i.v. chemotherapeutics administered intermittently, which leads to insufficient drug concentrations at tumor sites, accelerated tumor proliferation rates, and drug resistance, resulting in an overall median survival of only 2 to 4 years. For these reasons, more effective treatment strategies must be developed. We have investigated a localized, continuous chemotherapy approach in tumor models of human and murine ovarian cancers using the antineoplastic agent docetaxel. We show here that continuous docetaxel therapy is considerably more efficacious than intermittent therapy, resulting in a greater decrease in tumor burden and ascites fluid accumulation. Immunohistochemical analyses show that continuous chemotherapy abrogates tumor cell proliferation and angiogenesis to the tumor microenvironment, leading to greater tumor cell death than intermittent docetaxel therapy. Overall, our results show greater therapeutic advantages of continuous over intermittent chemotherapy in the treatment of ovarian cancer. Mol Cancer Ther; 9(6); 1820–30. ©2010 AACR.

Efficacy assessment of sustained intraperitoneal paclitaxel therapy in a murine model of ovarian cancer using bioluminescent imaging

We evaluated the pre-clinical efficacy of a novel intraperitoneal (i.p.) sustained-release paclitaxel formulation (PTXePC) using bioluminescent imaging (BLI) in the treatment of ovarian cancer. Human ovarian carcinoma cells stably expressing the firefly luciferase gene (SKOV3Luc) were injected i.p. into SCID mice. Tumour growth was evaluated during sustained or intermittent courses of i.p. treatment with paclitaxel (PTX). In vitro bioluminescence strongly correlated with cell survival and cytotoxicity. Bioluminescent imaging detected tumours before their macroscopic appearance and strongly correlated with tumour weight and survival. As compared with intermittent therapy with Taxol®, sustained PTXePC therapy resulted in significant reduction of tumour proliferation, weight and BLI signal intensity, enhanced apoptosis and increased survival times. Our results demonstrate that BLI is a useful tool in the pre-clinical evaluation of therapeutic interventions for ovarian cancer. Moreover, these results provide evidence of enhanced therapeutic efficacy with the sustained PTXePC implant system, which could potentially translate into successful clinical outcomes.

Oncolytic targeting of renal cell carcinoma via encephalomyocarditis virus.

Apoptosis is a fundamental host defence mechanism against invading microbes. Inactivation of NF‐κB attenuates encephalomyocarditis virus (EMCV) virulence by triggering rapid apoptosis of infected cells, thereby pre‐emptively limiting viral replication. Recent evidence has shown that hypoxia‐inducible factor (HIF) increases NF‐κB‐mediated anti‐apoptotic response in clear‐cell renal cell carcinoma (CCRCC) that commonly exhibit hyperactivation of HIF due to the loss of its principal negative regulator, von Hippel–Lindau (VHL) tumour suppressor protein. Here, we show that EMCV challenge induces a strong NF‐κB‐dependent gene expression profile concomitant with a lack of interferon‐mediated anti‐viral response in VHL‐null CCRCC, and that multiple established CCRCC cell lines, as well as early‐passage primary CCRCC cultured cells, are acutely susceptible to EMCV replication and virulence. Functional restoration of VHL or molecular suppression of HIF or NF‐κB dramatically reverses CCRCC cellular susceptibility to EMCV‐induced killing. Notably, intratumoural EMCV treatment of CCRCC in a murine xenograft model rapidly regresses tumour growth. These findings provide compelling pre‐clinical evidence for the usage of EMCV in the treatment of CCRCC and potentially other tumours with elevated HIF/NF‐κB‐survival signature.

Imaging the Modulation of Adenoviral Kinetics and Biodistribution for Cancer Gene Therapy

See page 841 To explore systemic utilization of Epstein-Barr virus (EBV)-specific transcriptionally targeted adenoviruses, three vectors were constructed to examine kinetics, specificity, and biodistribution: adv.oriP.luc, expressing luciferase under EBV-specific control; adv.SV40luc, expressing luciferase constitutively; and adv.oriP.E1A.oriP.luc, a conditionally replicating adenovirus, expressing both luciferase and E1A. Bioluminescence imaging (BLI) was conducted on tumor-bearing severe combined immunodeficient (SCID) mice (C666-1, EBV-positive human nasopharyngeal cancer) treated intravenously (i.v.) with 3 × 108 infectious units (ifu) of the adenoviral vectors. At 72 hours, adv.oriPluc demonstrated an 8.4-fold higher tumor signal than adv.SV40luc; adv.oriP.E1A.oriP.luc was 26.7-fold higher; however, a significant liver signal was also observed, necessitating further action to improve biodistribution. Several compounds were examined to this end, including norepinephrine, serotonin, clodronate liposomes, and STI571, to determine whether any of these measures could improve adenoviral biodistribution. Each of these interventions was assessed using BLI in mice i.v. injected with adv.oriP.luc. STI571 achieved the highest increase in tumor-to-liver ratio (TLR; 6.6-fold), which was associated with a 59% reduction in tumor interstitial fluid pressure (IFP) along with a decrease in platelet-derived growth factor-β receptor (PDGFβR) activation. This study reports the favorable modulation by STI571 of the biodistribution of adenoviral vectors, providing a potential approach to improving therapeutic outcome.

Oxygen-independent degradation of HIF-α via bioengineered VHL tumour suppressor complex

Tumour hypoxia promotes the accumulation of the otherwise oxygen‐labile hypoxia‐inducible factor (HIF)‐α subunit whose expression is associated with cancer progression, poor prognosis and resistance to conventional radiation and chemotherapy. The oxygen‐dependent degradation of HIF‐α is carried out by the von Hippel–Lindau (VHL) protein‐containing E3 that directly binds and ubiquitylates HIF‐α for subsequent proteasomal destruction. Thus, the cellular proteins involved in the VHL–HIF pathway have been recognized as attractive molecular targets for cancer therapy. However, the various compounds designed to inhibit HIF‐α or HIF‐downstream targets, although promising, have shown limited success in the clinic. In the present study, we describe the bioengineering of VHL protein that removes the oxygen constraint in the recognition of HIF‐α while preserving its E3 enzymatic activity. Using speckle variance–optical coherence tomography (sv–OCT), we demonstrate the dramatic inhibition of angiogenesis and growth regression of human renal cell carcinoma xenografts upon adenovirus‐mediated delivery of the bioengineered VHL protein in a dorsal skin‐fold window chamber model. These findings introduce the concept and feasibility of ‘bio‐tailored’ enzymes in the treatment of HIF‐overexpressing tumours.