Abstract
The present study used a spontaneous cell-based SELEX method (Systemic Evolution of Ligands by EXponential Enrichment) to produce DNA aptamers that specifically bind to cell surface proteins or biomarkers produced by primary cultured mouse tumor endothelial cells (mTECs). In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as metastasis. To combat angiogenesis, an appropriate diagnosis and a molecular-level understanding of the different cancer types are now a high priority. The novel DNA aptamer AraHH001, developed in this study, binds specifically to mTECs with high affinity in the nano-molar range, but does not bind to normal skin endothelial cells (skin-ECs). The selected DNA aptamer was also found to bind to cultured human tumor endothelial cells (hTECs), isolated from a clinical patient with a renal carcinoma. The aptamer AraHH001 showed significant anti-angiogenesis activity by inhibiting tube formation by mTECs on matrigel. Interestingly, a confocal laser scanning microscopy examination of in vitro cellular uptake revealed that AraHH001 was assimilated by mTECs, and became co-localized in acidic compartments, as detected by labeling with Lysotracker Red. Therefore, the development of a specific DNA aptamer that binds to mTECs, as reported here for the first time, holds great promise not only as a therapeutic aptamer but also as a targeted molecular probe that appears to play a major role in angiogenesis, and for the development of a targeted new drug delivery system.
Highlights
Angiogenesis-dependent tumor growth was first reported by Folkman in 1971 [1]
The focus of our current project involves the use of isolated primary cultured mouse tumor endothelial cells (mTECs) as the positive target, and primary cultured normal skinECs [4,5,6] and OS-RC-2 cell lines for negative selection
The binding affinity of the ssDNA pools to mTECs was clearly enhanced with an increase in the number of selection cycles, proving that the ssDNA libraries with higher binding affinity to the target cells were enriched
Summary
Angiogenesis-dependent tumor growth was first reported by Folkman in 1971 [1]. Preventing or inhibiting angiogenesis, which is associated with the increased vascularity necessary for tumor progression and metastasis, is a challenging issue in combating cancer. Tumor blood vessels differ from their normal counterparts, in that they are more permeable and, the thickness of the basement membrane is uneven. This suggests that tumor endothelial cells may express surface markers that are different from those found on normal cells. Since progressive tumor growth and metastasis depend on angiogenesis, inhibiting angiogenesis by targeting tumor endothelial cells represents a promising strategy for cancer treatment [2,3]
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