Metastasis‐focused cell‐based SELEX generates aptamers inhibiting cell migration and invasion

Abstract

Metastasis, the capacity of tumour cells to disseminate and grow at distant sites, is the main factor in cancer mortality. Compounds inhibiting migration and invasion of cancer cells are promising candidates for anticancer therapy strategies. We have generated nuclease-resistant RNA ligands (aptamers) recognizing highly metastatic cells with high affinity and specificity, and inhibiting their migratory and invasive potentials. Aptamers were generated by a cell-based subtractive SELEX technology using isogenic cell lines with similar tumorigenic potentials but opposite metastatic aggressiveness. Two aptamers, E37 and E10, bound specifically to the metastatically aggressive cell line and altered the phosphorylation of several tyrosine kinases. Fluorescent microscopy showed intracellular uptake of E37, in contrast to membrane binding of E10. Both aptamers inhibited migration of tumour cells in culture (50 and 85% inhibition with respect to control pool for E10 and E37, respectively) while only E10 inhibited cell invasion (275% with respect to control pool). This proof-of-concept study demonstrates the potential of cell-based SELEX to yield ligands that selectively recognize aggressive metastatic cells and inhibit phenotypes linked to metastatic potential.In cancer, a fatal issue is most often the consequence of metastatic spreading, but very few if any chemotherapies address the metastatic process itself. One reason may be that the process by which tumour cells metastasize is a still imperfectly understood succession of different molecular mechanisms. Another reason is that many of these molecular mechanisms pertain both to metastasis and to ubiquitous cellular activities. For instance, cell migration is an important and obligatory factor for the dissemination of tumour cells, as well as a general feature of many normal cells. Hence, it is far from trivial to target selectively the migration of cancer cells and not that of normal cells. Here, we explored new approaches to selectively target and inhibit specifically abnormal, context-irrelevant, cell migration with the aim to produce molecular binders for the recognition and, possibly, the control of metastatic activity.Among iterative selection methods based on Darwin's survival of the fittest, the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) allows the in vitro evolution of small nucleic acid molecules (aptamers) recognizing a specific molecular target.1,2 Aptamers bind cognate targets with high affinity and specificity, assuming stable three-dimensional structures 3 and often perturb their function. 4 Recently, SELEX has been adapted to the recognition of undefined targets in complex environments, in particular in live cells.5 A high level of molecular discrimination can be obtained using subtractive SELEX protocols, where cells from homologous origin, one expressing the target or phenotype of interest and the other not expressing, are used to alternate negative and positive selection steps. Cellbased SELEX has shown its capacity to track ten...

Evolution of a gastric carcinoma cell-specific DNA aptamer by live cell-SELEX

“…In summary, the results of the present study have demonstrated that the entire live cell-SELEX procedure was simple, but effective in generating cancer cell-specific aptamers, as previously reported (12)(13)(14)(15)(16)(17)(18)(19)(20). By using this method, we have identified a DNA aptamer termed cy-apt 20 with tenable binding affinity and stability to AGS cells.…”

“…A large number of useful aptamers generated by this method are applied in the study of tumor biology, and even in the diagnosis and therapeutics of various types of cancer (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). The encouraging results obtained with aptamers combined with their intrinsic properties and the versatility of the live cell-SELEX procedure have led to the application of this technical method in the development of gastric cancer-specific DNA aptamers.…”

In Vitro Selection of Cell-Internalizing DNA Aptamers in a Model System of Inflammatory Kidney Disease

“…This approach also favors isolation of aptamers that bind to the native folding and/or modification state (e.g., glycosylation, phosphorylation, etc.) of potential target molecules17, 48, 49, 50 over other selection approaches. These aptamers might be employed to identify novel cell surface markers (e.g., receptors), which subsequently become internalized into cells via the endosomal pathway, and to elucidate the mechanisms underlying CKD progression.…”

Selection of Nucleic Acid Aptamers Targeting Tumor Cell-Surface Protein Biomarkers

“…In this respect, aptamers are specific to the molecular signature of a tumor cell type. Such cell-based SELEX have been performed on several tumor cells, including breast cancer cells [141], colorectal cancer cells [142], small-cell lung cancer [143,144], non-small cell lung cancer [145], liver cancer cells [146], leukemia cells [147,148], gastric cancer cells [149], pancreatic ductal adenocarcinoma cells [150], prostate cancer cells [101], metastatic cells [151,152], and tumor initiating cells [153]. For example, J.N.…”