DNA double strand breaks as predictor of efficacy of the alpha-particle emitter Ac-225 and the electron emitter Lu-177 for somatostatin receptor targeted radiotherapy

Abstract

Rationale: Key biologic effects of the alpha-particle emitter Actinium-225 in comparison to the beta-particle emitter Lutetium-177 labeled somatostatin-analogue DOTATOC in vitro and in vivo were studied to evaluate the significance of γH2AX-foci formation.

Methods: To determine the relative biological effectiveness (RBE) between the two isotopes (as - biological consequence of different ionisation-densities along a particle-track), somatostatin expressing AR42J cells were incubated with Ac-225-DOTATOC and Lu-177-DOTATOC up to 48 h and viability was analyzed using the MTT assay. DNA double strand breaks (DSB) were quantified by immunofluorescence staining of γH2AX-foci. Cell cycle was analyzed by flow cytometry. In vivo uptake of both radiolabeled somatostatin-analogues into subcutaneously growing AR42J tumors and the number of cells displaying γH2AX-foci were measured. Therapeutic efficacy was assayed by monitoring tumor growth after treatment with activities estimated from in vitro cytotoxicity.

Results: Ac-225-DOTATOC resulted in ED50 values of 14 kBq/ml after 48 h, whereas Lu-177-DOTATOC displayed ED50 values of 10 MBq/ml. The number of DSB grew with increasing concentration of Ac-225-DOTATOC and similarly with Lu-177-DOTATOC when applying a factor of 700-fold higher activity compared to Ac-225. Already 24 h after incubation with 2.5-10 kBq/ml, Ac-225-DOTATOC cell-cycle studies showed up to a 60% increase in the percentage of tumor cells in G2/M phase. After 72 h an apoptotic subG1 peak was also detectable. Tumor uptake for both radio peptides at 48 h was identical (7.5%ID/g), though the overall number of cells with γH2AX-foci was higher in tumors treated with 48 kBq Ac-225-DOTATOC compared to tumors treated with 30 MBq Lu-177-DOTATOC (35% vs. 21%). Tumors with a volume of 0.34 ml reached delayed exponential tumor growth after 25 days (44 kBq Ac-225-DOTATOC) and after 21 days (34 MBq Lu-177-DOTATOC).

Conclusion: γH2AX-foci formation, triggered by beta- and alpha-irradiation, is an early key parameter in predicting response to internal radiotherapy.

Figure 1. DNA damage response in AR42J cells following ionizing irradiation.

(A) and (B) Cells were irradiated with 5 Gy and time-dependent DNA damage response was assessed over 24 h using western blot analysis.

Figure 2. Viability of AR42J cells at 48²²⁵Ac-DOTATOC (A) and ¹⁷⁷Lu-DOTATOC (B).

Figure 3. Number of γH2AX foci in AR42J cells at 24 and 48 h after incubation with ²²⁵Ac-DOTATOC (left) and ¹⁷⁷Lu-DOTATOC (right).

(A) shows representative images from all activity levels, (B) shows quantification of γH2AX foci and (C) shows two representative examples for pan nuclear staining after high dose ²²⁵Ac-DOTATOC treatment.

Figure 4. Comparison of cell viability and the number of γH2AX foci in AR42J cells at 48 h after incubation with ²²⁵Ac-DOTATOC and ¹⁷⁷Lu-DOTATOC.

Figure 5. Single-cell gel electrophoresis (alkaline comet assay) of AR42J cells at 24 h and 48 h after incubation with ²²⁵Ac-DOTATOC (A) and ¹⁷⁷Lu-DOTATOC (B).

DNA damage was calculated by the tail moment, the most frequent used parameter of comet features. SubG₁ fraction of AR42J cells at 48 and 72 h after incubation with ²²⁵Ac-DOTATOC (C) and ¹⁷⁷Lu-DOTATOC (D).

Figure 6. Cell-cycle distribution of AR42J cells at 48 h after incubation with ²²⁵Ac-DOTATOC (left) and ¹⁷⁷Lu-DOTATOC (right) (SD <15%).

Figure 7. Immunofluorescence staining of DNA double strand breaks (γH2AX) in AR42J tumors after treatment with 47 kBq ²²⁵Ac-DOTATOC, 30 MBq of ¹⁷⁷Lu-DOTATOC, 1 µg DOTATOC (unlabelled), or PBS.

(A) Representative images, (B) and quantification of γH2AX- positive cells. Scale bar (white) corresponds to 25 µm. (C) Growth delay after treatment with equitoxic doses of ²²⁵Ac-DOTATOC (n = 3, 44kBq/mouse) and ¹⁷⁷Lu-DOTATOC (n = 4, 34 MBq/mouse) versus untreated control (n = 4).