11C-radiolabeled aptamer for imaging of tumors and metastases using positron emission tomography- computed tomography

Abstract

Identification of primary tumors and metastasis sites is an essential step in cancer diagnostics and the following treatment. Positron emission tomography-computed tomography (PET/CT) is one of the most reliable methods for scanning the whole organism for malignancies. In this work, we synthesized an ¹¹C-labeled oligonucleotide primer and hybridized it to an anti-cancer DNA aptamer. The ¹¹C-aptamer was applied for in vivo imaging of Ehrlich ascites carcinoma and its metastases in mice using PET/CT. The imaging experiments with the ¹¹C-aptamer determined very small primary and secondary tumors of 3 mm² and less. We also compared ¹¹C imaging with the standard radiotracer, 2-deoxy-2-[fluorine-18]fluoro-D-glucose (¹⁸F-FDG), and found better selectivity of the ¹¹C-aptamer to metastatic lesions in the metabolically active organs than ¹⁸F-FDG. ¹¹C radionuclide with an ultra-short (20.38 min) half-life is considered safest for PET/CT imaging and does not cause false-positive results in heart imaging. Its combination with aptamers gives us high-specificity and high-contrast imaging of cancer cells and can be applied for PET/CT-guided drug delivery in cancer therapies.

Keywords: 11C radiolabeling; DNA aptamers; Ehrlich ascites carcinoma; PET/CT; in vivo imaging; metastasis; radiopharmaceuticals.

Graphical abstract

Figure 1

Finding the 3D structure of the truncated AS-14 aptamer (A) The secondary structure of AS-14t. (B) (1) Experimental SAXS data (dark blue dots) fitted by the theoretical SAXS curve (a light blue line). (2) Pair distance distribution function p (r) of AS-14t. The maximal value of r is the D_max (the size of the molecule). (C) Comparing the modeled 3D structures with the experimental SAXS model. Dark blue sticks are from molecular modeling; blue color spheres are from SAXS data.

Figure 2

Synthesis of the ¹¹C-labeled aptamer (A) Synthesis of the ¹¹CH₃-labeled primer. (B) The primary sequence of the ¹¹CH₃ primer. (C) Secondary structure of ¹¹CH₃-AS-14. (D) The tertiary structure of ¹¹СH₃- AS-14 (green ribbon, AS-14 aptamer; cyan ribbon, ¹¹CH₃ primer).

Figure 3

Degradation and binding analyses of ¹¹C-labeled oligonucleotides (A) Agarose gel electrophoresis of the ¹¹C-labeled primer after synthesis. (B) Agarose gel electrophoresis of the FAM-labeled primer AS-14 in mouse serum at different times. (C) Dependence of the sample's radioactivity on the content of ¹¹CH₃ primer or ¹¹CH₃-AS-14, where sample (1) corresponds to 0.6 nmol of ¹¹CH₃ primer; (2) 0.6 nmol of ¹¹CH₃ primer hybridized with 0.6 nmol of AS-14; (3) 0.3 nmol of ¹¹CH₃ primer; (4) 0.3 nmol of ¹¹CH₃ primer hybridized with 0.3 nmol of AS-14 (¹¹CH₃-AS-14); (5) 0.6 nmol of ¹¹CH₃ primer hybridized with 0.6 nmol of AS-14 (¹¹CH₃-AS-14) incubated with Ehrlich cells; (6) 0.3 nmol of ¹¹CH₃ primer hybridized with 0.3 nmol of AS-14 (¹¹CH₃-AS-14) incubated with Ehrlich cells; (7) 0.6 nmol of ¹¹CH₃ primer incubated with Ehrlich cells; (8) 0.3 nmol of ¹¹CH₃ primer incubated with Ehrlich cells; (9) Ehrlich cells only. (D) Correlation of the radioactivity of the samples versus the number of ascites cells or hepatocytes bound with ¹¹CH₃-AS-14 or the ¹¹CH₃-unrelated aptamer. Errors bars are one SD of three measurements.

Figure 4

Tumor and metastases localization analysis using the ¹¹CH₃-AS-14 probe and PET/CT (A–C) The representative PET/CT images of mice with metastases (A) were confirmed by autopsy (B) and histological analyses of metastatic tumor lesions (C) in different organs: intestines (2); liver (3); heart (4); and lung spleen (5). Cytological characteristics of the tumor tissue (C). Hematoxylin-eosin staining. Magnification С1, ×400; С2, ×200; C3, ×30; С4 and 5, ×100. Arrows and dashed circles indicate the tumor sites in different organs at PET/CT, autopsy, and corresponding tissue sections.

Figure 5

Comparative PET/CT imaging using ¹¹CH₃-AS-14 with ¹⁸F-FDG (A–D) Images with ¹¹CH₃-AS-14 (A1 and 2) and ¹⁸F-FDG (B1 and 2) on the same mice scanned on the next day. Accuracy of PET/CT results was confirmed by autopsy (C) and histological analyses of metastatic tumor lesions (D) in different organs: thyroid gland (1); stomach (2); liver (3); kidney (4); intestines (5); muscle (6); lung (7); pancreas (8); rib bone marrow (9). Arrows and dashed circles indicate the tumor sites in different organs at PET/CT, autopsy, and corresponding tissue sections.

Figure 6

Specific recognition of the tumor sites by ¹¹CH₃-AS-14 proved by control experiments (A–F) The representative PET/CT analysis of mice with tumor metastasis (I) using the ¹¹CH₃ primer (A) and ¹¹CH₃-unrelated aptamer (B) as probes. Metastasis formation was confirmed by autopsy (A2, B3). PET/CT images of physiological distribution and excretion of ¹¹CH₃-AS-14 (C) and ¹⁸F-FDG (E) in healthy mice (II). PET/CT image of a healthy mouse without the injected radiopharmaceutical (D). Histological examination with the staining of tissue sections with hematoxylin-eosin (F). Arrows indicate the tumor sites in different organs at PET/CT and autopsy.