The Discovery of RNA Aptamers that Selectively Bind Glioblastoma Stem Cells

Abstract

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. Despite progress in surgical and medical neuro-oncology, prognosis for GBM patients remains dismal, with a median survival of only 14-15 months. The modest benefit of conventional therapies is due to the presence of GBM stem cells (GSCs) that cause tumor relapse and chemoresistance and, therefore, that play a key role in GBM aggressiveness and recurrence. So far, strategies to identify and target GSCs have been unsuccessful. Thus, the development of an approach for GSC detection and targeting would be fundamental for improving the survival of GBM patients. Here, using the cell-systematic evolution of ligand by exponential (SELEX) methodology on human primary GSCs, we generated and characterized RNA aptamers that selectively bind GSCs versus undifferentiated GBM cells. We found that the shortened version of the aptamer 40L, which we have called A40s, costained with CD133-labeled cells in human GBM tissue, suggestive of an ability to specifically recognize GSCs in fixed human tissues. Of note, both 40L and A40s were rapidly internalized by cells, allowing for the delivery of the microRNA miR-34c and the anti-microRNA anti-miR-10b, demonstrating that these aptamers can serve as selective vehicles for therapeutics. In conclusion, the aptamers 40L and A40s can selectively target GSCs. Given the crucial role of GSCs in GBM recurrence and therapy resistance, these aptamers represent innovative drug delivery candidates with a great potential in the treatment of GBM.

Keywords: aptamer; cancer stem cell; delivery; glioblastoma; microRNA.

Graphical abstract

Figure 1

Cell Characterization and SELEX Conditions (A) Growth pattern of glioblastoma cultures established under different cell conditions. Western blot (WB) analysis illustrating different expression markers in stem cell cultures and adherent cell cultures obtained after 2 weeks of culturing in 10% serum. Regarding the other promising sequences, aptamer 5 was not evaluated further since it was not among the most-enriched aptamers of the last six SELEX round media. (B) Panels show representative pictures of primary GBM cells growing as suspension or adherent cell culture. (C) Schematic summary of the selective approach of cell-SELEX.

Figure 2

GSC Aptamer Selection (A) The random regions of all the sequenced aptamers were aligned using ClustalW. The dendrogram shows visual clasterization of similarity among 100 individual sequences cloned after 16 rounds of selection. (B) The enriched pools from rounds 10, 11, 13, 14, 15, and 16 were sequenced by high-throughput sequencing (HTS). sg, SELEX on GBM.

Figure 3

40L Binds GBM Primary Cell Cultures and Cell Lines (A–D) Binding was performed with the most enriched sequences obtained from SELEX at 200 nM on GSC 1 stem (A) or adherent (diff) (B) cells from the same patient and on several GSC lines grown in suspension (C) or under adherent conditions (D). Representative experiments are shown and results are expressed relative to the background binding detected with the starting pool of sequences used for selection. (E and F) Binding of 40L was performed at 200 nM on U251-MG (E) and U87-MG (F) cell line cells in suspension or in adherent culturing conditions. The ability of 40L to be internalized into GSCs is shown. Results are expressed as percentages of the total bound after 30 min of incubation. Representative experiments are shown as mean (±SD) and results are expressed relative to the background binding detected with the starting pool of sequences used for selection.

Figure 4

A40s Aptamer Binding (A) Bidimensional and tridimensional shape predictions of the 40L aptamer are shown; 40L aptamer sequence was shortened in order to have a smaller aptamer with the best properties. The selected portion is shown in the squares. (B and C) Binding assay was performed at 200 nM on GSC lines 83, 1, 74, and 163 grown as stem (B) or as differentiated (C) cells. Representative experiments are shown and results are expressed as mean (±SD) relative to the background binding, detected with an unrelated aptamer of a length similar to that of A40s. (D) Immunofluorescence staining was performed by treating 83, 30p, or 61 differentiated cells with A40s-Alexa488 or scrambled-Alexa488 at 500 nM for 30 min. All images were captured at the same settings, enabling direct comparison of staining patterns. (E) Kd calculation is shown as mean (±SD). Nonlinear binding curve was obtained by treating GSCs with increasing amounts of A40s.

Figure 5

Discrimination of GSCs by A40s in Fixed Human Tissues (A) Distribution of A40s-positive cells in human tissues. (B) The A40s signal perfectly co-localizes with CD133-labeled cells in human tissues. Images are accompanied by H&E staining of the adjacent section.

Figure 6

Aptamer Internalization in GSCs (A and B) 40L (A) and A40s (B) are internalized into GSCs. Results are expressed as percentages of the total bound after 30 min of incubation. (C) Immunofluorescence of 83 stem cells treated with A40s-Alexa488 or scrambled-Alexa488 at 500 nM for 30 min. All images were captured at the same settings, enabling direct comparison of the staining patterns. (D) Secondary structure of the A40s-miR34c chimera as predicted by RNA Structure v.5.7. (E) Non-denaturating gel electrophoresis analysis reveals correct A40s/miR-34c chimera conjugate annealing. (F) Stem or differentiated GBM cells were incubated with A40s/miR-34c chimera for 24 and 48 h. Relative miR-34c levels were assessed by using qRT-PCR. (G) Non-denaturating gel electrophoresis analysis reveals correct A40s-anti-miR-10b chimera conjugate annealing. (H) GSCs were incubated with A40s-anti-miR-10b chimera or negative control for 48 h. Relative miR-10b levels were assessed by qRT-PCR, and the upregulation of one of miR-10b’s targets, BIM, was assessed by WB. In (A), (F), and (H), vertical bars indicate SD values. *p ≤ 0.05, **p ≤ 0.01.