Subcellular Targeting of Theranostic Radionuclides

Abstract

The last decade has seen rapid growth in the use of theranostic radionuclides for the treatment and imaging of a wide range of cancers. Radionuclide therapy and imaging rely on a radiolabeled vector to specifically target cancer cells. Radionuclides that emit β particles have thus far dominated the field of targeted radionuclide therapy (TRT), mainly because the longer range (μm-mm track length) of these particles offsets the heterogeneous expression of the molecular target. Shorter range (nm-μm track length) α- and Auger electron (AE)-emitting radionuclides on the other hand provide high ionization densities at the site of decay which could overcome much of the toxicity associated with β-emitters. Given that there is a growing body of evidence that other sensitive sites besides the DNA, such as the cell membrane and mitochondria, could be critical targets in TRT, improved techniques in detecting the subcellular distribution of these radionuclides are necessary, especially since many β-emitting radionuclides also emit AE. The successful development of TRT agents capable of homing to targets with subcellular precision demands the parallel development of quantitative assays for evaluation of spatial distribution of radionuclides in the nm-μm range. In this review, the status of research directed at subcellular targeting of radionuclide theranostics and the methods for imaging and quantification of radionuclide localization at the nanoscale are described.

Keywords: cancer; dosimetry; radioimmunotherapy; radiopharmaceuticals; subcellular targeting; targeted radionuclide therapy.

FIGURE 1

Energy and dose-deposition of various radionuclides. Energy and dose-deposition profiles for various radionuclides in spherical water volumes up to 100 μm in diameter. Calculations for electrons are based on a Monte Carlo method described by Falzone et al. (2017). Energy deposition by α-particles and recoiling daughter nuclei is derived from the NIST (Berger et al., 2005) and SRIM (Ziegler et al., 2010) stopping power data, respectively, and a straight projectile is assumed. (A) Energy deposited (eV) in 1-nm-thick spherical shells as a function of radius (μm) from a point source. The energy-deposition profiles exhibit sinusoidal behavior, apart from the α-emitter ²¹¹At, due to AE emitted from different atomic shells being stopped at different distances. β-emitters (⁶⁷Cu, ¹⁵³Sm, ¹⁶¹Tb, and ¹⁷⁷Lu) eventually overtake the AE-emitters after 10 μm from the point source. ²¹¹At deposits at least an order of magnitude higher energy than other radionuclides for the entire range considered. Its energy-deposition profile is fairly constant from 0.3 μm, where all recoiling daughters are stopped, up to about 30 μm where some α-particles emitted from ²¹¹At start to slow down and eventually come to a halt around 50 μm. (B–D) Absorbed dose in spherical volumes per cumulated activity (Gy/Bq/s) as a function of diameter (μm) with a point source at the center. AE-emitters deposit significantly more dose than β-emitters that have a small AE contribution (⁶⁷Cu and ¹⁷⁷Lu) in organelles with diameter less than 1 μm. For volumes bigger than a cell, β-emitters are more suitable in delivering the desired dose over the entire region. This highlights the need for AE-emitters to be targeted to radiosensitive subcellular organelles for the anticipated therapeutic efficacy. Although ¹⁶¹Tb is a β-emitter, its decay involves a significant contribution of AE so subcellular targeting using this radionuclide would enhance its radiobiological effect. The dose-deposition profile of ²¹¹At shows that it delivers a significantly higher physical dose to a spherical volume of diameter less than 100 μm than the other radionuclides considered.

FIGURE 2

Electron microscopy – microautoradiography of radionuclides in subcellular compartments. EM-MAR images of hypoglossal motoneurons treated with ¹²⁵I-labeled retrogradely transported trophic factors. The location of the radionuclide is revealed by the formation of silver grains. (A) ¹²⁵I-labeled glial cell line-derived neurotrophic factor (GDNF) in a light endosome (arrow). (B) ¹²⁵I-labeled brain-derived neurotrophic factor (BDNF) in a dense endosome (arrow). (C) ¹²⁵I-labeled CT-1 in a lysosome (arrow). (D) ¹²⁵I-labeled GDNF in a heavily labeled multivesicular body (MVB). (E) ¹²⁵I-labeled GDNF in the endoplasmic reticulum. (F) ¹²⁵I-labeled GDNF in the Golgi apparatus with Golgi (G)-associated vesicles (arrows). Scale bars represent 500 nm. Source: Rind et al. (2005). Reproduced with permission from the Journal of Neuroscience. Copyright: Society of Neuroscience.

FIGURE 3

The photoresist autoradiography method. (A) Electron beam calibration: (i) 5×5 μm² patterns of varying fluence incident on the PMMA substrate (the laser reflecting off the AFM probe is shown). (ii) AFM image of 5 μm × 5 μm electron beam feature. (iii) Line scan relating depth to electron fluence. (B) Model system consisting of ¹¹¹In-DTPA radiolabeled microspheres: (i) optical image showing the close packing of the microspheres on the PMMA surface, (ii) AFM contour through image of a radiolabeled microsphere pattern, and (iii) 3-D generated profile of the AFM feature. (C) Resist exposed to radionuclide treated cells and isolated cell nuclei, followed by removal of biological material and chemical development of the resist and AFM analysis of the pattern. (D) Demonstration of PAR with ¹¹¹In-DTPA-hEGF treated cells: (i) optical image of radionuclide treated SQ20B (head and neck squamous carcinoma) cells, (ii) AFM image of an ¹¹¹In-DTPA-hEGF treated cell pattern, and (iii) 3-D generated plot of an AFM image of a cell nucleus relating local pattern depth to local fluence based on electron beam calibration.

FIGURE 4

Radioluminescence microscopy. (A) Emission of an intracellular radionuclide can be detected as radioluminescence with a scintillator plate (yellow glow). The optical photons are captured by a high-numerical-aperture objective coupled to a deep-cooled EM-CCD camera. Concurrent fluorescence and brightfield microscopy are enabled by emission and excitation filters used in combination with a light source. (B) An in culture medium immersed scintillator plate in a glass-bottom dish is placed into the inverted microscope. (C) Three GFP-expressing HeLa cells were imaged using fluorescence microscopy. (D) After incubation with ¹⁸F-FDG the focal radioluminescence signal coincided with the fluorescent signal. (E) An example of radioluminescence microscopy. MDA-MB-231 cells were incubated for 1 h with ¹⁸F-FDG and the fluorescent 2-NBDG. Brightfield image (scale bar, 100 μm), radioluminescence (FDG), and fluorescence (2-NBDG) micrographs. The overlay shows co-localized radioluminescence (green) and fluorescence (red). Source: Pratx et al. (2012). Adapted and reproduced with permission from PLoS One.

FIGURE 5

Subcellular targets of Auger electron-emitting theranostic radionuclides. Strategies to reach intracellular targets can broadly be categorized into radioligands that diffuse through the cell membrane by passive/active transport (1) or bind to cell membrane receptors. Membrane receptor-radioligand complexes can be internalized via endocytic pathways (2) or remain surface-bound (3), damaging the cell membrane via hydroxyl radical formation (see text for further explanation). Endocytosed radioligand-receptor can continue to damage endosomes (4), and certain radioligands have the potential to escape endosomal entrapment (5). Cytosolic radioligands can have various fates and targets. Approaches have utilized radioligands that can bind nuclear proteins, such as γH2AX or telomerase (6), or that can directly interact with the DNA (7). Some radioligand strategies involve targeting SSR (8), which can traffic to the nucleus of the cancer cell to exert damage. A more recently explored fate is mitochondrial targeting (9), which can lead to mitochondrial DNA damage and the generation of oxidative stress, resulting in mitochondrial-induced apoptosis. Endosomal escape can also occur for receptor-radioligand complexes, which can travel to the nucleus, as has been found with targeting of the EGFR family (10). Most complexes are unable to escape the endosome and will be sorted out of the cell via large endosomal/lysosomal vesicles. While being processed, radionuclides can continue to do damage endosomal vesicles, and irradiate genomic DNA in case of long track-path radionuclides emitters, such as ¹⁷⁷Lu-/²²⁵Ac-PSMA or ¹⁷⁷Lu-DOTATATE) (11).