RESUMEN
E-selectin is an adhesion molecule expressed on the luminal surface of inflamed blood vessels that mediates hematogenous metastasis by assisting shear-resistant adhesion of circulating tumor cells to the vessel surface under dynamic blood flow. Previously, we developed an E-selectin antagonistic thioaptamer (ESTA) for the prevention of hematogenous metastasis through the blockade of CD44high breast cancer cells (BCa) adhesion to E-selectin-expressing premetastatic endothelial niche. The current study focuses on developing a PEGylated E-selectin targeting thioaptamer with improved pharmaceutical properties. A serial deletion of stem-loops reveled that loop-1 and -2 (ESTA7) are the minimally effective backbone structure necessary to obtain inhibition of the E-selectin/CD44 interaction and shear resistant adhesion of CD44high BCa to E-selectin-expressing human endothelial cells (HMVECs) at a level equal to ESTA. Chemical conjugation of methoxy-polyethylene-glycol (PEG) at the sizes of 5 and 10 kDa did not interfere with ESTA7-mediated shear-resistant adhesion. However, in vivo study demonstrated that only 10 kDa PEG-conjugated ESTA7 (ESTA7-p10) retains the activity to inhibit metastases at a level equal to parental ESTA. Additionally, a single intravenous injection of ESTA7-p10 inhibited the development of lung, brain, and bone metastases of MDA-MB-231, through the blockade of E-selectin. Moreover, PEGylation led to an extension of elimination half-life and increase of AUC, resulting in superior inhibition of metastasis development compared to parental ESTA with a longer interval between dosing in a spontaneous metastasis model. Lastly, repeated intravenous administration of ESTA7-p10 was tolerated in mice, highlighting the potential prophylactic application of ESTA7-p10 for metastasis prevention.
RESUMEN
High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinity aptamers and their associated target proteins in a systematic and accurate way. We created a combinatorial DNA aptamer library that has been modified with thiophosphate substitutions of the phosphate ester backbone at selected 5´dA positions for enhanced nuclease resistance and targeting. Based on morphological assessment, we used image-directed laser microdissection (LMD) to dissect regions of interest bound with the thioaptamer (TA) library and further identified target proteins for the selected TAs. We have successfully identified and characterized the lead candidate TA, V5, as a vimentin-specific sequence that has shown specific binding to tumor vasculature of human ovarian tissue and human microvascular endothelial cells. This new Morph-X-Select method allows us to select high-affinity aptamers and their associated target proteins in a specific and accurate way, and could be used for personalized biomarker discovery to improve medical decision-making and to facilitate the development of targeted therapies to achieve more favorable outcomes.
Asunto(s)
Aptámeros de Nucleótidos/análisis , Biomarcadores de Tumor/análisis , Neoplasias Ováricas/metabolismo , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Biomarcadores de Tumor/química , Biomarcadores de Tumor/metabolismo , Femenino , Humanos , Captura por Microdisección con Láser , Espectrometría de MasasRESUMEN
The medical applications of aptamers have recently emerged. We developed an antagonistic thioaptamer (ESTA) against E-selectin. Previously, we showed that a single injection of ESTA at a dose of 100µg inhibits breast cancer metastasis in mice through the functional blockade of E-selectin. In the present study, we evaluated the safety of different doses of intravenously administered ESTA in single-dose acute and repeat-dose subacute studies in ICR mice. Our data indicated that intravenous administration of up to 500µg ESTA did not result in hematologic abnormality in either study. Additionally, intravenous injection of ESTA did not affect the levels of plasma cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF, IFN-γ, and TNF-α) or complement split products (C3a and C5a) in either study. However, repeated injections of ESTA slightly increased plasma ALT and AST activities, in accordance with the appearance of small necrotic areas in the liver. In conclusion, our data demonstrated that intravenous administration of ESTA does not cause overt hematologic, organs, and immunologic responses under the experimental conditions.