Molecular and biochemical characteristics of inulosucrase InuBK from Alkalihalobacillus krulwichiae JCM 11691.

Information about the inulosucrase of nonlactic acid bacteria is scarce. We found a gene encoding inulosucrase (inuBK) in the genome of the Gram-positive bacterium Alkalihalobacillus krulwichiae JCM 11691. The inuBK open reading frame encoded a protein comprising 456 amino acids. We expressed His-tagged InuBK in culture medium using a Brevibacillus system. The optimal pH and temperature of purified InuBK were 7.0-9.0 and 50-55 °C, respectively. The findings of high-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy, and high-performance size-exclusion chromatography with multiangle laser light scattering showed that the polysaccharide produced by InuBK was an inulin with a molecular weight of 3806, a polydispersity index (PI) of 1.047, and fructosyl chain lengths with 3-27 degrees of polymerization. The size of InuBK was smaller than commercial inulins, and the PI of the inulin that it produced was lower.

Aureolysin of Staphylococcus warneri M accelerates its proteolytic cascade, and participates in biofilm formation.

The aureolysin (Aur) gene of S. warneri M (aurWM) was cloned and sequenced. Analyses of the aurWM-inactivated mutant (S. warneri Mau) suggested that AurWM was probably associated with efficient processing of the PROM protease (homolog of V8/SspA serine protease), whereas considerable amount of mature-PROC protease (homolog of SspB cysteine protease) accumulated without AurWM. Additionally, AurWM appeared to affect biofilm formation in an uncertain suppressive way.

Application of multiple ultra-high-pressure homogenization to the pasteurization process of Japanese rice wine, sake.

Hiire is a pasteurization process in the production of Japanese rice wine (sake), which stabilizes the quality of product; however, it also generates the carcinogen ethyl carbamate (EC). In this study, we investigated the application of ultra-high-pressure homogenization (UHPH) as an alternative sterilization method for sake production. Microbiological analysis revealed that multiple UHPH treatments sterilized hiochi lactobacilli (Lactobacillus fructivorans, L. homohiochii, L. casei, and L. hilgardii) and Saccharomyces cerevisiae. Enzyme activity assays revealed that α-amylase, glucoamylase, and acid-carboxypeptidase activities were reduced to less than 1% of the levels in non-pasteurized sake after four-time UHPH treatment. These results show that UHPH treatment meets the two requirements of the sake sterilization process sterilization and enzyme inactivation. The UHPH-processed sake did not show any significant changes in general properties but had reduced organic acid and aromatic component contents, with ethyl caproate content showing the most significant reduction of approximately 20%. Interestingly, EC was detected in pasteurized sake but not in UHPH-processed sake. These findings indicate that the UHPH technology could be used to inactivate microorganisms and enzymes in sake without generating EC.

Discoidal Porous Silicon Particles: Fabrication and Biodistribution in Breast Cancer Bearing Mice.

Porous silicon (pSi) is emerging as a promising material in the development of nanovectors for the systemic delivery of therapeutic and imaging agents. The integration of photolithographic patterning, typical of the semiconductor industry, with electrochemical silicon etching provides a highly flexible strategy to fabricate monodisperse and precisely tailored nanovectors. Here, a microfabrication strategy for direct lithographic patterning of discoidal pSi particles is presented that enables precise and independent control over particle size, shape, and porous structure. Discoidal pSi nanovectors with diameters ranging from 500 to 2600 nm, heights from 200 to 700 nm, pore sizes from 5 to 150 nm, and porosities from 40 to 90% are demonstrated. The degradation in serum, interaction with immune and endothelial cells in vitro, and biodistribution in mice bearing breast tumors are assessed for two discoidal nanovectors with sizes of 600 nm × 400 nm and 1000 nm × 400 nm. It is shown that both particle types are degraded after 24 h of continuous gentle agitation in serum, do not stimulate cytokine release from macrophages or affect endothelial cell viability, and accumulate up to about 10% of the injected dose per gram tissue in orthotopic murine models of breast cancer. The accumulation of the discoidal pSi nanovectors into the breast tumor mass is found to be up to five times higher than for spherical silica beads with similar diameters.

Attenuated Microcirculation in Small Metastatic Tumors in Murine Liver.

Metastatic cancer disease is the major cause of death in cancer patients. Because those small secondary tumors are clinically hardly detectable in their early stages, little is known about drug biodistribution and permeation into those metastatic tumors potentially contributing to insufficient clinical success against metastatic disease. Our recent studies indicated that breast cancer liver metastases may have compromised perfusion of intratumoral capillaries hindering the delivery of therapeutics for yet unknown reasons. To understand the microcirculation of small liver metastases, we have utilized computational simulations to study perfusion and oxygen concentration fields in and around the metastases smaller than 700 µm in size at the locations of portal vessels, central vein, and liver lobule acinus. Despite tumor vascularization, the results show that blood flow in those tumors can be substantially reduced indicating the presence of inadequate blood pressure gradients across tumors. A low blood pressure may contribute to the collapsed intratumoral capillary lumen limiting tumor perfusion that phenomenologically corroborates with our previously published in vivo studies. Tumors that are smaller than the liver lobule size and originating at different lobule locations may possess a different microcirculation environment and tumor perfusion. The acinus and portal vessel locations in the lobule were found to be the most beneficial to tumor growth based on tumor access to blood flow and intratumoral oxygen. These findings suggest that microcirculation states of small metastatic tumors can potentially contribute to physiological barriers preventing efficient delivery of therapeutic substances into small tumors.

Seed- and Soil-Dependent Differences in Murine Breast Tumor Microenvironments Dictate Anti-PD-L1 IgG Delivery and Therapeutic Efficacy.

We sought to determine if Stephen Paget's "seed and soil" hypothesis of organ-preference patterns of cancer metastasis can explain the development of heterogeneity in a tumor microenvironment (TME) as well as immunotherapeutic delivery and efficacy. We established single-cell-derived clones (clones 1 and 16) from parental 4T1 murine breast cancer cells to create orthotopic primary and liver metastasis models to deconvolute polyclonal complexity cancer cells and the difference in TME-derived heterogeneities. Tumor-bearing mice were treated with anti-PD-L1 IgG or a control antibody, and immunofluorescent imaging and quantification were then performed to evaluate the therapeutic efficacy on tumor growth, the delivery of therapy to tumors, the development of blood vessels, the expression of PD-L1, the accumulation of immune cells, and the amount of coagulation inside tumors. The quantification showed an inverse correlation between the amount of delivered therapy and therapeutic efficacy in parental-cell-derived tumors. In contrast, tumors originating from clone 16 cells accumulated a significantly greater amount of therapy and responded better than clone-1-derived tumors. This difference was greater when tumors grew in the liver than the primary site. A similar trend was found in PD-L1 expression and immune cell accumulation. However, the change in the number of blood vessels was not significant. In addition, the amount of coagulation was more abundant in clone-1-derived tumors when compared to others. Thus, our findings reconfirmed the seed- and soil-dependent differences in PD-L1 expression, therapeutic delivery, immune cell accumulation, and tumor coagulation, which can constitute a heterogeneous delivery and response of immunotherapy in polyclonal tumors growing in different organs.

Synergistic Activation of Antitumor Immunity by a Particulate Therapeutic Vaccine.

Success in anticancer immune therapy relies on stimulation of tumor antigen-specific T lymphocytes and effective infiltration of the T cells into tumor tissue. Here, a therapeutic vaccine that promotes proliferation and tumor infiltration of antigen-specific T cells in both inflamed and noninflamed tumor types is described. The vaccine consists of STING agonist 2'3'-cGAMP, TLR9 ligand CpG, and tumor antigen peptides that are loaded into nanoporous microparticles (µGCVax). µGCVax is effective in inhibiting lung metastatic melanoma, primary breast cancer, and subcutaneous colorectal cancer in their respective murine models, including functional cure of HER2-positive breast cancer. Mechanistically, µGCVax potently stimulates type I interferon expression in dendritic cells, and promotes CD8+ and CD103+ dendritic cell maturation and migration to lymph nodes and other lymphatic tissues. Antitumor responses are dependent on TLR9 and interferon α/ß receptor signaling, and to a less extent on STING signaling. These results demonstrate a high potential for µGCVax in mediating antitumor immunity in personalized cancer therapy.

Tumor Site-Dependent Transport Properties Determine Nanotherapeutics Delivery and Its Efficacy.

Insufficient delivery of systemically administered anticancer drugs to tumors can compromise therapeutic efficacy and develop drug delivery-based therapeutic resistance. Nanotherapeutics such as PEGylated liposomal doxorubicin (PLD) are designed to preferentially accumulate in tumors utilizing enhanced permeation and retention effect. However, their antitumor effects and resulting clinical outcomes are modest and heterogeneous among tumors. Here, we aimed to investigate whether the amount and efficacy of PLD delivered to tumors are tumor site dependent. We established orthotopic primary tumor or liver metastases models of murine breast cancer using 4 T1 cells. PLD showed significant therapeutic effects against tumors that grew in primary mammary sites but not in the liver. We found that differences in therapeutic efficacy were not because of the intrinsic biological resistance of cancer cells but rather were associated with tumor site-dependent differences in transport properties, such as the amount of PLD delivery, blood vessel function, relative vascular permeability, and mechanical pressure in tumors. Thus, transport properties in tumor is site dependent and can be used as phenotypic surrogate markers for tumor drug delivery and therapeutic efficacy.

Molecular properties of the putative autolysin Atl(WM) encoded by Staphylococcus warneri M: mutational and biochemical analyses of the amidase and glucosaminidase domains.

The putative autolysin Atl(WM) of Staphylococcus warneri M is a modular protein exhibiting two enzyme activities, an N-terminal side amidase (ami(atlwm)-R1-R2) and a C-terminal side glucosaminidase (R3-glu(atlwm)). Zymographic analysis of the protein overproduced in Escherichia coli showed that both enzymes were active toward 17 Gram-positive bacteria, including staphylococci, lactobacilli, lactococci, enterococci, and micrococci. The purified enzyme core ami(atlwm) (or glu(atlwm)) had the pH and temperature optima of about 7.0 (5.5) and 41 (50) degrees C, respectively. ami(atlwm) was inactivated by EDTA, and was stimulated by such salts as CoCl(2), MnCl(2), CaCl(2), or ZnCl(2). Six mutations within ami(atlwm), (H362A, E421A, H467A, H479, D481A, and Y491D) drastically reduced cell-lytic activity. Comparative analysis with other related amidases suggested that the three residues H362, H467, and D481 likely act as ligands (and/or active sites). The lytic activity of glu(atlwm) markedly declined in four mutants (E1238A, E1238Q, T1239A, and Y1332A). For determination of the putative cell-recognition regions, four domains (R1-R2, R1, R2, and R3) were purified; all the proteins substantially bound to S. warneri M cells from exponential to stationary growth phases, and R1-R2 aggregated the cells. Protein sequencing and immunoblot analysis suggested that the extacellular Atl(WM) might be primarily processed at two specific sites (one between pro and ami(atlwm), and the other between R2 and R3) to yield the mature amidase and glucosaminidase.

Dynamic distortion of visual position representation around moving objects.

The relative visual positions of briefly flashed stimuli are systematically modified in the presence of motion signals (R. Nijhawan, 2002; D. Whitney, 2002). Previously, we investigated the two-dimensional distortion of relative-position representations between moving and flashed stimuli. The results showed that the perceived position of a flash is not uniformly displaced but shifted toward a single convergent point back along the trajectory of a moving object (K. Watanabe & K. Yokoi, 2006, 2007). In the present study, we examined the temporal dynamics of the anisotropic distortion of visual position representation. While observers fixated on a stationary cross, a black disk appeared, moved along a horizontal trajectory, and disappeared. A white dot was briefly flashed at various positions relative to the moving disk and at various timings relative to the motion onset/offset. The temporal emerging-waning pattern of anisotropic mislocalization indicated that position representation in the space ahead of a moving object differs qualitatively from that in the space behind it. Thus, anisotropic mislocalization cannot be explained by either a spatially or a temporally homogeneous process. Instead, visual position representation is anisotropically influenced by moving objects in both space and time.

Mutational and biochemical analyses of the endolysin Lys(gaY) encoded by the Lactobacillus gasseri JCM 1131T phage phi gaY.

The Lys(gaY) of Lactobacillus gasseri JCM 1131(T) phage phigaY endolysin was purified to homogeneity using the Escherichia coli/His.Tag system. Zymographic and spectrophotometric assays showed that Lys(gaY) lysed over 20 heated Gram-positive bacterial species as the substrates, including lactobacilli, lactococci, enterococci, micrococci, and staphylococci. The enzymatic activity had the pH and temperature optima of about 6.5 and 37 degrees C, respectively. Amino-acid substitution analysis revealed that 13 residues of Lys(gaY) were involved in cell-lytic activity: in the beta/alpha(gaY) domain, G10, D12, E33, D36, H60, Y61, D96, E98, V124, L132, and D198; in the SH3b(gaY) domain, Y272 and W284. In addition, deletion analysis demonstrated that the beta/alpha(gaY) domain of N-terminal 216 residues is the core enzyme portion, although the cell-lytic ability is lower than that of Lys(gaY). These mutational experiments suggested that beta/alpha(gaY) (in which two acidic residues of D12 and E98 likely act as catalytic residues) is responsible for cell-lytic activity, and SH3b(gaY) promotes beta/alpha(gaY) possibly through cell-wall binding function. The purified His-tagged SH3b(gaY) domain containing 94 residues from S217 to K310 (i) bound to Gram-positive bacteria susceptible to Lys(gaY), (ii) induced aggregation of exponentially growing cells of L. gasseri JCM 1131(T), L. casei IAM 1045, Lactococcus lactis C2, L. lactis MG 1363, and Enterococcus hirae IAM 1262 by forming thread-like chained cells, (iii) inhibited lytic activity of Lys(gaY), and (iv) impeded autolysis of L. gasseri JCM 1131(T) in buffer systems. A truncated protein HDeltaSH3b(gaY) lacking in N-terminal 21 residues (from S217 to E237) of SH3b(gaY) and an amino-acid substituted protein HSH3b(gaY)G (W284G) lost the activities of HSH3b(gaY), showing that the N-terminal region and W284 probably play important roles in the SH3b(gaY) function(s).

Object-based anisotropic mislocalization by retinotopic motion signals.

The relative visual positions of briefly flashed stimuli are systematically modified in the presence of motion signals. We have recently shown that the perceived position of a spatially extended flash stimulus is anisotropically shifted toward a single convergent point back along the trajectory of a moving object without a significant change in the perceived shape of the flash [Watanabe, K., & Yokoi, K. (2006). Object-based anisotropies in the flash-lag effect. Psychological Science, 17, 728-735]. In the previous experiment, the moving stimulus moved in both retinotopic and environmental coordinates. In the present study, we examined whether the anisotropic mislocalization depends on retinotopic or object motion signals. When the retinal image of a moving stimulus was rendered stationary by smooth pursuit, the anisotropic pattern of mislocalization was not observed. In contrast, when the retinal image of a stationary stimulus was moved by eye movements, anisotropic mislocalization was observed, with the magnitude of the mislocalization comparable to that in the previous study. In both cases, there was little indication of shape distortion of the flash stimulus. These results demonstrate a clear case of object-based mislocalization by retinotopic motion signals; retinotopic--not object--motion signals distort the perceived positions of visual objects after the shape representations are established.

Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model.

Although gemcitabine has been approved as the first-line chemotherapeutic reagent for pancreatic cancer, its response rate is low and average survival duration is still only marginal. Because epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) modulate tumor progression, we hypothesized that inhibition of phosphorylation of all three on tumor cells, tumor-associated endothelial cells, and stroma cells would improve the treatment efficacy of gemcitabine in an orthotopic pancreatic tumor model in nude mice and prolong survival. We implanted L3.6pl, a human pancreatic cancer cell, in the pancreas of nude mice. We found that tumor-associated endothelial cells in this model highly expressed phosphorylated EGFR, VEGFR, and PDGFR. Oral administration of AEE788, a dual tyrosine kinase inhibitor against EGFR and VEGFR, decreased phosphorylation of EGFR and VEGFR. PDGFR phosphorylation was inhibited by STI571. Although i.p. injection of gemcitabine did not inhibit tumor growth, its combination with AEE788 and STI571 produced >80% inhibition of tumor growth and prolonged survival in parallel with increases in number of tumor cells and tumor-associated endothelial cell apoptosis, decreased microvascular density, decreased proliferation rate, and prolonged survival. STI571 treatment also decreased pericyte coverage on tumor-associated endothelial cells. Thus, inhibiting phosphorylation of EGFR, VEGFR, and PDGFR in combination with gemcitabine enhanced the efficacy of gemcitabine, resulting in inhibition of experimental human pancreatic cancer growth and significant prolongation of survival.

Dual inhibition of epidermal growth factor receptor and vascular endothelial growth factor receptor phosphorylation by AEE788 reduces growth and metastasis of human colon carcinoma in an orthotopic nude mouse model.

We studied growth factors and their receptors in tumor cells and tumor-associated endothelial cells as the therapeutic targets in colon cancer. Immunohistochemical analysis of 13 surgical specimens of human colon adenocarcinoma revealed that both tumor cells and tumor-associated endothelial cells in 11 of the 13 specimens expressed the epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), EGF receptor (EGFR), phosphorylated EGFR (pEGFR), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and phosphorylated VEGFR (pVEGFR). HT29 human colon cancer cells growing orthotopically in the cecum of nude mice expressed a high level of EGF, EGFR, pEGFR, VEGF, VEGFR, and pVEGFR. Double-immunofluorescence staining found that tumor-associated mouse endothelial cells also expressed pEGFR and pVEGFR. Tumors in mice treated for 5 weeks with oral AEE788 (an inhibitor of EGFR and VEGFR tyrosine kinase) as a single agent or with CPT-11 alone were smaller (>50%) than those in control mice. Mice treated with the combination of AEE788 and CPT-11 had significantly smaller tumors (P < 0.01) and complete inhibition of lymph node metastasis. AEE788 alone or in combination with CPT-11 inhibited pEGFR, pVEGFR, and phosphorylated Akt expression on tumor-associated endothelial cells as well as on tumor cells. The combination therapy also significantly decreased microvessel density and tumor cell proliferation and increased the level of apoptosis in both tumor cells and tumor-associated endothelial cells. Collectively, these data suggest that the dual inhibition of EGFR and VEGFR signaling pathways in tumor cells and tumor-associated endothelial cells in combination with chemotherapy can provide a new approach to the treatment of colon cancer.

Integrated Microfluidic System for Gene Silencing and Cell Migration.

Metastasis involves the phenotype transition of cancer cells to gain invasiveness, and the following migration at the tumor site. Here an integrated microfluidic chip to study this process is presented by combining on-chip delivery of siRNA for gene silencing and cell migration assay. The major advantage of the integrated chip is the simple input of cells and gene transfection materials, and the ultimate output of migration ability. The reverse-fishbone structure and 0.7× phosphate-buffered saline solution are the optimized parameters for improved delivery efficiency. Using the chip, it is validated that cofilin plays an essential role in regulating cancer cell migration. The integrated chip may provide a simple and effective platform for biologists to easily check the role of specific genes in metastasis.

Tumor progression effects on drug vector access to tumor-associated capillary bed.

Over the last decade, the benefits of drug vectors to treat cancer have been well recognized. However, drug delivery and vector distribution differences in tumor-associated capillary bed at different stages of disease progression are not well understood. To obtain further insights into drug vector distribution changes in vasculature during tumor progression, we combined intra-vital imaging of metastatic tumors in mice, microfluidics-based artificial tumor capillary models, and Computational Fluid Dynamics (CFD) modeling. Microfluidic and CFD circulation models were designed to mimic tumor progression by escalating flow complexity and chaoticity. We examined flow of 0.5 and 2µm spherical particles, and tested the effects of hematocrit on particle local accessibility to flow area of capillary beds by co-circulating red blood cells (RBC). Results showed that tumor progression modulated drug vector distribution in tumor-associated capillaries. Both particles shared 80-90% common flow area, while 0.5 and 2µm particles had 2-9% and 1-2% specific flow area, respectively. Interestingly, the effects of hematocrit on specific circulation area was opposite for 0.5 and 2µm particles. Dysfunctional capillaries with no flow, a result of tumor progression, limited access to all particles, while diffusion was shown to be the only prevailing transport mechanism. In view of drug vector distribution in tumors, independent of formulation and other pharmacokinetic aspects, our results suggest that the evolution of tumor vasculature during progression may influence drug delivery efficiency. Therefore, optimized drug vectors will need to consider primary vs metastatic tumor setting, or early vs late stage metastatic disease, when undergoing vector design.

Macrophage Polarization Contributes to the Anti-Tumoral Efficacy of Mesoporous Nanovectors Loaded with Albumin-Bound Paclitaxel.

Therapies targeted to the immune system, such as immunotherapy, are currently shaping a new, rapidly developing branch of promising cancer treatments, offering the potential to change the prognosis of previously non-responding patients. Macrophages comprise the most abundant population of immune cells in the tumor microenvironment (TME) and can undergo differentiation into functional phenotypes depending on the local tissue environment. Based on these functional phenotypes, tumor-associated macrophages (TAMs) can either aid tumor progression (M2 phenotype) or inhibit it (M1 phenotype). Presence of M2 macrophages and a high ratio of M2/M1 macrophages in the TME are clinically associated with poor prognosis in many types of cancers. Herein, we evaluate the effect of macrophage phenotype on the transport and anti-cancer efficacy of albumin-bound paclitaxel (nAb-PTX) loaded into porous silicon multistage nanovectors (MSV). Studies in a coculture of breast cancer cells (3D-spheroid) with macrophages and in vivo models were conducted to evaluate the therapeutic efficacy of MSV-nAb-PTX as a function of macrophage phenotype. Association with MSV increased drug accumulation within the macrophages and the tumor spheroids, shifting the inflammation state of the TME toward the pro-inflammatory, anti-tumorigenic milieu. Additionally, the treatment increased macrophage motility toward cancer cells, promoting the active transport of therapeutic nanovectors into the tumor lesion. Consequently, apoptosis of cancer cells was increased and proliferation decreased in the MSV-nAb-PTX-treated group as compared to controls. The results also confirmed that the tested system shifts the macrophage differentiation toward an M1 phenotype, possessing an anti-proliferative effect toward the breast cancer cells. These factors were further incorporated into a mathematical model to help analyze the synergistic effect of the macrophage polarization state on the efficacy of MSV-nAb-PTX in alleviating hypovascularized tumor lesions. In conclusion, the ability of MSV-nAb-PTX to polarize TAM to the M1 phenotype, causing (1) enhanced penetration of the drug-carrying macrophages to the center of the tumor lesion and (2) increased toxicity to tumor cells may explain the increased anti-cancer efficacy of the system in comparison to nAb-PTX and other controls.

Gemcitabine enhances the transport of nanovector-albumin-bound paclitaxel in gemcitabine-resistant pancreatic ductal adenocarcinoma.

The mechanism for improved therapeutic efficacy of the combination therapy with nanoparticle albumin-bound paclitaxel (nAb-PTX) and gemcitabine (gem) for pancreatic ductal adenocarcinoma (PDAC) has been ascribed to enhanced gem transport by nAb-PTX. Here, we used an orthotopic mouse model of gem-resistant human PDAC in which increasing gem transport would not improve the efficacy, thus revealing the importance of nAb-PTX transport. We aimed to evaluate therapeutic outcomes and transport of nAb-PTX to PDAC as a result of (1) encapsulating nAb-PTX in multistage nanovectors (MSV); (2) effect of gem on caveolin-1 expression. Treatment with MSV/nAb-PTX + gem was highly efficient in prolonging animal survival in comparison to other therapeutic regimens. MSV/nAb-PTX + gem also caused a substantial increase in tumor PTX accumulation, significantly reduced tumor growth and tumor cell proliferation, and increased apoptosis. Moreover, gem enhanced caveolin-1 expression in vitro and in vivo, thereby improving transport of nAb-PTX to PDAC. This data was confirmed by analysis of PDACs from patients who received gem-based neo-adjuvant chemotherapy. In conclusion, we found that nAb-PTX treatment of gem-resistant PDAC can be enhanced by (1) gem through up-regulation of caveolin-1 and (2) MSV through increasing accumulation of nAb-PTX in the tumor.

Nanoplasmonic Quantification of Tumor-derived Extracellular Vesicles in Plasma Microsamples for Diagnosis and Treatment Monitoring.

Tumour-derived extracellular vesicles (EVs) are of increasing interest as a resource of diagnostic biomarkers. However, most EV assays require large samples, are time-consuming, low-throughput and costly, and thus impractical for clinical use. Here, we describe a rapid, ultrasensitive and inexpensive nanoplasmon-enhanced scattering (nPES) assay that directly quantifies tumor-derived EVs from as little as 1 µL of plasma. The assay uses the binding of antibody-conjugated gold nanospheres and nanorods to EVs captured by EV-specific antibodies on a sensor chip to produce a local plasmon effect that enhances tumour-derived EV detection sensitivity and specificity. We identified a pancreatic cancer EV biomarker, ephrin type-A receptor 2 (EphA2), and demonstrate that an nPES assay for EphA2-EVs distinguishes pancreatic cancer patients from pancreatitis patients and healthy subjects. EphA2-EVs were also informative in staging tumour progression and in detecting early responses to neoadjuvant therapy, with better performance than a conventional enzyme-linked immunosorbent assay. The nPES assay can be easily refined for clinical use, and readily adapted for diagnosis and monitoring of other conditions with disease-specific EV biomarkers.

Antivascular therapy for orthotopic human ovarian carcinoma through blockade of the vascular endothelial growth factor and epidermal growth factor receptors.

PURPOSE: We determined whether the administration of the tyrosine kinase inhibitor, AEE788, which targets the epidermal growth factor receptor and the vascular endothelial growth factor receptor, alone or in combination with paclitaxel, can inhibit progressive growth of human ovarian carcinoma in the peritoneal cavity of female nude mice. EXPERIMENTAL DESIGN: Western blot analysis and immunohistochemical analysis identified the optimal dose and schedule of AEE788 therapy. In several different experiments, paclitaxel-sensitive and paclitaxel-resistant human ovarian carcinoma cells were injected into the peritoneal cavity of nude mice. Seven days later, treatment with saline (control), AEE788 alone, paclitaxel alone, or a combination of AEE788 and paclitaxel began and continued for 45 days when the mice were necropsied. In independent survival experiments, the mice were necropsied when they became moribund. RESULTS: Oral administration of AEE788 inhibited phosphorylation of the epidermal growth factor receptor and vascular endothelial growth factor receptor for up to 48 hours. Treatment with AEE788 plus paclitaxel significantly reduced tumor weight and increased survival of mice implanted with paclitaxel-sensitive cell lines compared with control mice or mice treated with AEE788 alone or paclitaxel alone. In mice implanted with paclitaxel-resistant cells, the combination therapy also significantly reduced tumor weight but did not prolong survival. The combination therapy induced apoptosis of both tumor cells and tumor-associated endothelial cells. CONCLUSIONS: The administration of AEE788 and paclitaxel inhibits the progression of human ovarian carcinoma in the peritoneal cavity of female nude mice, in part, by inducing apoptosis of tumor-associated endothelial cells.