Recent studies have shown that Abs that target the cell-surface enzyme CD73 (ecto-5′-nucleotidase) reduce growth of primary tumors and metastasis in syngenic mice by inhibiting the catalytic activity of CD73, and thus increasing the activity of cytotoxic T lymphocytes. In this article, we report another anticancer mechanism of anti-CD73 Abs and show that an anti-CD73 mAb (AD2) inhibits metastasis formation by a mechanism independent of CD73 catalytic activity and inhibition of primary tumor growth. This mechanism involves clustering and internalization of CD73, but does not require cross-linking of CD73, because both whole IgG anti-CD73 AD2 mAb and Fab′ fragments thereof exhibited this effect. Ex vivo treatment of different breast cancer cell lines with anti-CD73 AD2 mAb before i.v. injection into mice inhibited extravasation/colonization of circulating tumor cells and significantly reduced metastasis development. This effect was also observed when the cancer cell-surface expression of CD73 was significantly reduced by small interfering RNA knockdown. The antimetastatic activity is epitope specific, as another Ab that efficiently binds CD73-expressing live cancer cells did not lead to CD73 internalization and metastasis inhibition. Furthermore, anti-CD73 AD2 mAb inhibited development of metastasis in a spontaneous animal model of human metastatic breast cancer. Our study shows that some anti-CD73 mAbs cause cell-surface clustering of CD73 followed by internalization, thus inhibiting the ability of circulating tumor cells to extravasate and colonize, leading to inhibition of metastasis. Ab-based CD73 cancer therapy should include a combination of Abs that target the catalytic activity of CD73, as well as those with the characteristics described in this article.
Tropoelastin, the precursor of elastin, undergoes a rapid monomer to multimer association in an inverse temperature transition. This association culminates in the rapid formation of stable, optically distinct droplets of tropoelastin. Light scattering and microscope measurements reveal that these droplets are 2-6 microm in diameter. Scanning electron microscopy confirms that the droplets are spherical. Three-dimensional confocal image stacks based on the autofluorescence of tropoelastin reveal that droplets are loaded with hydrated tropoelastin. Droplets are viable intermediates in synthetic elastin macroassembly. Dense clusters of aggregated droplets and partially formed fibers develop when droplets are incubated in the presence of a lysyl oxidase. Lysine-reacting chemical and enzyme-assisted cross-linking conditions generate cross-linked beads due to interactions between multiple, surface-exposed lysine epsilon-amino groups. Droplets represent an efficient mechanism for the bolus delivery during elastogenesis of quantized packages of preaccreted tropoelastin.
Here we describe a novel surface sampling technique termed pressurized liquid extraction surface analysis (PLESA), which in combination with a dedicated high-resolution shotgun lipidomics routine enables both quantification and in-depth structural characterization of molecular lipid species extracted directly from tissue sections. PLESA uses a sealed and pressurized sampling probe that enables the use of chloroform-containing extraction solvents for efficient in situ lipid microextraction with a spatial resolution of 400 μm. Quantification of lipid species is achieved by the inclusion of internal lipid standards in the extraction solvent. The analysis of lipid microextracts by nanoelectrospray ionization provides long-lasting ion spray which in conjunction with a hybrid ion trap-orbitrap mass spectrometer enables identification and quantification of molecular lipid species using a method with successive polarity shifting, high-resolution Fourier transform mass spectrometry (FTMS), and fragmentation analysis. We benchmarked the performance of the PLESA approach for in-depth lipidome analysis by comparing it to conventional lipid extraction of excised tissue homogenates and by mapping the spatial distribution and molar abundance of 170 molecular lipid species across different anatomical mouse brain regions.
Quantum dots (QDs) have long promised to revolutionize fluorescence detection to include even applications requiring simultaneous multi-species detection at single molecule sensitivity. Despite the early promise, the unique optical properties of QDs have not yet been fully exploited in e. g. multiplex single molecule sensitivity applications such as single particle tracking (SPT). In order to fully optimize single molecule multiplex application with QDs, we have in this work performed a comprehensive quantitative investigation of the fluorescence intensities, fluorescence intensity fluctuations, and hydrodynamic radii of eight types of commercially available water soluble QDs. In this study, we show that the fluorescence intensity of CdSe core QDs increases as the emission of the QDs shifts towards the red but that hybrid CdSe/CdTe core QDs are less bright than the furthest red-shifted CdSe QDs. We further show that there is only a small size advantage in using blue-shifted QDs in biological applications because of the additional size of the water-stabilizing surface coat. Extending previous work, we finally also show that parallel four color multicolor (MC)-SPT with QDs is possible at an image acquisition rate of at least 25 Hz. We demonstrate the technique by measuring the lateral dynamics of a lipid, biotin-cap-DPPE, in the cellular plasma membrane of live cells using four different colors of QDs; QD565, QD605, QD655, and QD705 as labels.
Falcarinol (FaOH) and falcarindiol (FaDOH) are cytotoxic and anti-inflammatory polyacetylenic oxylipins, which are commonly found in the carrot family (Apiaceae). FaOH and FaDOH have previously demonstrated a chemopreventive effect on precursor lesions of colorectal cancer (CRC) in azoxymethane (AOM)-induced rats. The purpose of the present study was to elucidate possible mechanisms of action for the preventive effect of FaOH and FaDOH on colorectal precancerous lesions and to determine how this effect was dependent on dose. Gene expression studies performed by RT-qPCR of selected cancer biomarkers in tissue from biopsies of neoplastic tissue revealed that FaOH and FaDOH downregulated NF-κβ and its downstream inflammatory markers TNFα, IL-6, and COX-2. The dose-dependent anti-neoplastic effect of FaOH and FaDOH in AOM-induced rats was investigated in groups of 20 rats receiving a standard rat diet (SRD) supplemented with 0.16, 0.48, 1.4, 7 or 35 µg FaOH and FaDOH g−1 feed in the ratio 1:1 and 20 rats were controls receiving only SRD. Analysis of aberrant crypt foci (ACF) showed that the average number of small ACF (<7 crypts) and large ACF (>7 crypts) decreased with increasing dose of FaOH and FaDOH and that this inhibitory effect on early neoplastic formation of ACF was dose-dependent, which was also the case for the total number of macroscopic neoplasms. The CRC protective effects of apiaceous vegetables are mainly due to the inhibitory effect of FaOH and FaDOH on NF-κB and its downstream inflammatory markers, especially COX-2.
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