Selection and characterization of DNA aptamer for metastatic prostate cancer recognition and tissue imaging

Abstract

Prostate cancer (PCa) is the second leading cause of death and most prevalent cancer in men. The absence of curative options for castration-resistant metastatic prostate cancer and biomarkers able to discriminate between indolent and aggressive tumors contribute to these statistics. In this study, a DNA aptamer termed DML-7 was successfully selected against human PCa cell line DU145 by using the cell-based systematic evolution of ligands by exponential enrichment (SELEX) method. The selected aptamer DML-7 was found to internalize into target cells in a temperature-dependent manner and exhibit high binding affinity for target cells with dissociation constants in the nanomolar range. Binding analysis further revealed that DML-7 only binds to DU145 and PC-3 cells with metastatic potential, but not to LNCaP or 22Rv1 cells with low or nonmetastatic potential, demonstrating that DML-7 has excellent selectivity for the recognition of the metastatic PCa cells. Clinical tissue imaging further confirmed these results. Therefore, both high binding affinity and specificity to metastatic PCa cells and tissues afford DML-7 with the potential for development into a novel tool for diagnosis and targeted drug delivery against metastatic prostate cancer.

Keywords: SELEX; aptamer; binding affinity; metastasis; prostate cancer.

Figure 1. Monitoring the enrichment of cell-SELEX progression

(A) Schematic representation of the cell-based aptamer selection. (B) Binding of enriched pools to DU145 cells (target cells) and (C) WPMY-1 cells (control cells) from the 4th, 8th, 12th and 18th rounds was monitored by flow cytometry assay. The black curve represents the background fluorescence of untreated cells. Unselected FAM-labeled DNA library was used as negative control.

Figure 2. Binding assay of the selected aptamers

Binding of the selected aptamers to (A) DU145 cells and (B) WPMY-1 cells was analyzed by flow cytometry. The black curve represents the background fluorescence of untreated cells. Unselected FAM-labeled DNA library was used as negative control. (C) Dissociation constant of DML-7 for DU145 cells was determined by flow cytometry.

Figure 3. Binding site of DML-7 to DU145 cells

(A) DU145 and WPMY-1 cells were incubated with Cy5-labeled DML-7, respectively. Fluorescence image, differential interference contrast (DIC) or merged confocal images were shown. (B) After treatment with trypsin, proteinase K or EDTA, the binding of DML-7 to DU145 cells was analyzed by flow cytometry.

Figure 4. Internalization of DML-7 into DU145 cells

(A) DU145 cells were incubated with Cy5-labeled DML-7 and library at (A) 4°C or (B) 37°C, respectively. Fluorescence image, differential interference contrast (DIC) or merged confocal images were shown.

Figure 5. Binding characterization of DML-7 sequence

(A) Secondary structure analysis of DML-7 predicted by NUPACK. (B) Binding of different truncted DML-7 to (B) DU145 and (C) WPMY-1 cells was assayed by flow cytometry.

Figure 6. Specificity of DML-7 to other cell lines

FAM-labeled DML-7 was incubated with (A) PCa cell lines with different origins, (B) epithelial cancer cell lines and (C) hematopoietic cell lines, respectively. Binding ability was analyzed by flow cytometry.

Figure 7. Fluorescence images of tissue section stained with FAM-labeled DML-7

Typical fluorescence image in normal prostate tissues (−, no fluorescence signal), nonmetastatic PCa tissues (+, weak fluorescence signal) and metastatic PCa tissues (++, moderate fluorescence signal; +++, strong fluorescence signal) was shown.