Identification of a PET Radiotracer for Imaging of the Folate Receptor-α: A Potential Tool to Select Patients for Targeted Tumor Therapy

Abstract

The aim of this study was to identify a folate receptor-α (FRα)-selective PET agent potentially suitable for the selection of patients who might profit from FRα-targeted therapies. The 6R and 6S isomers of ¹⁸F-aza-5-methyltetrahydrofolate (MTHF) were assessed regarding their binding to FRα and FRβ, expressed on cancer and inflammatory cells, respectively, and compared with ¹⁸F-AzaFol, the folic acid-based analog. Methods: FR selectivity was investigated using FRα-transfected (RT16) and FRβ-transfected (D4) CHO cells. The cell uptake of ¹⁸F-folate tracers was investigated, and receptor-binding affinities were determined with the nonradioactive analogs. In vitro autoradiography of the ¹⁸F-folate tracers was performed using RT16 and D4 tissue sections. Biodistribution studies and PET/CT imaging of the radiotracers were performed on mice bearing RT16 and D4 xenografts. Results: The uptake of ¹⁸F-6R-aza-5-MTHF was high when using RT16 cells (62% ± 10% of added activity) but much lower when using D4 cells (5% ± 2%). The FRα selectivity of ¹⁸F-6R-aza-5-MTHF was further demonstrated by its approximately 43-fold higher binding affinity to FRα (half-maximal inhibitory concentration [IC₅₀], 1.8 ± 0.1 nM) than to FRβ (IC₅₀, 77 ± 27 nM). The uptake of ¹⁸F-6S-aza-5-MTHF and ¹⁸F-AzaFol was equal in both cell lines (52%-70%), with similar affinities to FRα (IC₅₀, 2.1 ± 0.4 nM and 0.6 ± 0.3 nM, respectively) and FRβ (0.8 ± 0.2 nM and 0.3 ± 0.1 nM, respectively). The autoradiography signal obtained with ¹⁸F-6R-aza-5-MTHF was 11-fold more intense for RT16 than for D4 tissue sections. Biodistribution data showed high uptake of ¹⁸F-6R-aza-5-MTHF in RT16 xenografts (81% ± 20% injected activity per gram [IA]/g 1 h after injection) but significantly lower accumulation in D4 xenografts (7.3% ± 2.1% IA/g 1 h after injection), which was also visualized using PET. The uptake of ¹⁸F-6S-aza-5-MTHF and ¹⁸F-AzaFol was similar in RT16 (53% ± 10% IA/g and 45% ± 2% IA/g, respectively) and D4 xenografts (77% ± 10% IA/g and 52% ± 7% IA/g, respectively). Conclusion: This study demonstrated FRα selectivity for ¹⁸F-6R-aza-5-MTHF but not for ¹⁸F-6S-aza-5-MTHF or ¹⁸F-AzaFol. This characteristic, together with its favorable tissue distribution, makes ¹⁸F-6R-aza-5-MTHF attractive for clinical translation to enable detection of FRα-positive cancer while preventing undesired accumulation in FRβ-expressing inflammatory cells.

Keywords: 18F; 5-MTHF; FRα selectivity; PET; folate receptor (FR).

Graphical abstract

FIGURE 1.

Chemical structures of ¹⁸F-AzaFol, ¹⁸F-6R-aza-5-MTHF, and ¹⁸F-6S-aza-5-MTHF, as well as folic acid, 6S-5-MTHF, and 6R-5-MTHF (). Stereochemical nomenclature of corresponding isomers of nonfluorinated and fluorinated 5-MTHFs are inversed because of change in substituents’ priority at stereogenic center.

FIGURE 2.

(A) Western blot analysis of FRα expression in RT16 cell lysates and FRβ expression in D4 cell lysates (top panel). β-Actin staining as loading control (bottom panel). (B) Cell uptake and internalization of ¹⁸F-6R-aza-5-MTHF, ¹⁸F-6S-aza-5-MTHF, and ¹⁸F-AzaFol in RT16 and D4 cells after 3 h of incubation at 37°C. Unspecific binding of radiotracers (blocked) was determined by coincubation of cells with folic acid (100 μM). Results are presented as average ± SD (n = 3).

FIGURE 3.

Signal intensities (normalized digital light units [DLU] per area [mm²]) of ¹⁸F-6R-aza-5-MTHF, ¹⁸F-6S-aza-5-MTHF, and ¹⁸F-AzaFol (set as 100%) quantified on the basis of RT16 (left) or D4 (right) autoradiography images. Bottom panel demonstrates FR blockade performed with excess folic acid.

FIGURE 4.

Graph representing uptake of ¹⁸F-6R-aza-5-MTHF (red), ¹⁸F-6S-aza-5-MTHF (blue), and ¹⁸F-AzaFol (green) in RT16 and D4 xenografts and in kidneys at 1 and 3 h after injection of ¹⁸F-folate radiotracers (5 MBq/mouse). p.i. = after injection.

FIGURE 5.

PET/CT images of mice bearing RT16 and D4 xenografts 1 h (A–C) and 3 h (D–F) after injection of ¹⁸F-folate radiotracers (5 MBq/mouse) shown as maximum-intensity projections.