Disturbing the Redox Balance Using Buthionine Sulfoximine Radiosensitized Somatostatin Receptor-2 Expressing Pre-Clinical Models to Peptide Receptor Radionuclide Therapy with 177Lu-DOTATATE

Abstract

Peptide receptor radionuclide therapy with ¹⁷⁷Lu-DOTATATE improves the outcome of patients with somatostatin receptor (SSTR)-expressing neuroendocrine tumours. Nevertheless, stable disease has been the main response pattern observed, with some rare complete responses. Lu-177 exerts about two-thirds of its biological effects via the indirect effects of ionizing radiation that generate reactive oxygen species, eventually leading to oxidative damage and cell death. This provides a rationale for targeting the antioxidant defence system in combination with ¹⁷⁷Lu-DOTATATE. In the present study, the radiosensitizing potential and the safety of depleting glutathione (GSH) levels using buthionine sulfoximine (BSO) during ¹⁷⁷Lu-DOTATATE therapy were assessed in vitro and in vivo using a xenograft mouse model. In vitro, the combination resulted in a synergistic effect in cell lines exhibiting a BSO-mediated GSH decrease. In vivo, BSO neither influenced ¹⁷⁷Lu-DOTATATE biodistribution nor induced liver, kidney or bone marrow toxicity. In terms of efficacy, the combination resulted in reduced tumour growth and metabolic activity. Our results showed that disturbing the cell redox balance using a GSH synthesis inhibitor increased ¹⁷⁷Lu-DOTATATE efficacy without additional toxicity. Targeting the antioxidant defence system opens new safe treatment combination opportunities with ¹⁷⁷Lu-DOTATATE.

Keywords: 177Lu-DOTATATE; BSO; antioxidant defenses; glutathione; multiple myeloma; peptide receptor radionuclide therapy; radiosensitization.

Figure 1

Timeline of the in vivo experiments. Female athymic nude mice were subcutaneously injected in the right flank with EJM multiple myeloma cells. Mice were randomized into four treatment groups: (1) control (IV saline injection), (2) BSO (IV saline injection and BSO via drinking water), (3) ¹⁷⁷Lu-DOTATATE (a single IV injection of 30 MBq) and (4) ¹⁷⁷Lu-DOTATATE + BSO (a single IV injection of 30 MBq and BSO via drinking water). Mice had a baseline and a post-treatment ¹⁸F-FDG PET/CT to assess metabolic tumour response. Tumour growth was also followed using a calliper. Another group of mice receiving ¹⁷⁷Lu-DOTATATE alone or in combination with BSO was sacrificed on days 1, 3 and 7 p.i. for ex vivo biodistribution studies and early glutathione quantification. All animals were continuously monitored for body weight. IV = intravenous, p.o. = per os.

Figure 2

Effect of BSO and its combination with ¹⁷⁷Lu-DOTATATE on the survival of melanoma (HBL and MM162), multiple myeloma (COLO-677 and EJM) and GEP (MIA-PACA-2 and HT-29) cell lines. Cells were exposed to 5 MBq of ¹⁷⁷Lu-DOTATATE for 4 h. BSO (10⁻⁷M) was present in the medium from the day before irradiation until cell survival assessment on day 10. Results are expressed as a percentage of the non-treated counterpart and are represented as mean ± SEM (n = 12 from 3 independent experiments). The black dotted line represents 100%. *** p ≤ 0.001; ** p ≤ 0.01; NS = non-statistically significant.

Figure 3

Total glutathione in melanoma (HBL and MM162), multiple myeloma (COLO-677 and EJM) and GEP (MIA-PACA-2 and HT-29) cell lines exposed to BSO. Total glutathione quantification was performed using cell lines exposed to 10⁻⁷ M BSO for 18 h. Results are expressed as a percentage of the non-treated counterpart (black dotted line) and are represented as mean ± SEM (n = 6 from 2 independent experiments). *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; NS = non-statistically significant.

Figure 4

Biodistribution of ¹⁷⁷Lu-DOTATATE at 24, 72 and 168 h p.i. in EJM xenograft mice. Mice in both groups were intravenously injected with 30 MBq of ¹⁷⁷Lu-DOTATATE on day 0. Mice in the combination group (¹⁷⁷Lu-DOTATATE + BSO) additionally received 10 mM BSO via drinking water, starting on day-1. Results are expressed as the mean of the % injected activity per gram of tissue (%IA/g) ± SD of 4 mice per group.

Figure 5

¹⁷⁷Lu-DOTATATE time–activity curves of the liver and kidneys of EJM xenograft mice. Mice in both groups were intravenously injected with 30 MBq of ¹⁷⁷Lu-DOTATATE on day 0. Mice in the combination group (¹⁷⁷Lu-DOTATATE + BSO) additionally received 10 mM BSO via drinking water, starting on day-1. Solid lines represent the single exponential curve fitting. Dashed lines represent 95% confidence bands. n = 1 to 6 mice per group.

Figure 6

Effect of BSO on total glutathione levels in the liver and kidneys. Mice were intravenously injected with 30 MBq of ¹⁷⁷Lu-DOTATATE on day 0. BSO (10 mM) was administered via drinking water starting on day-1. Total glutathione levels in the liver and kidneys after 2, 4, 8 and 14–18 days of continuous BSO exposure. Results are expressed as the mean ± SD of 2 to 5 mice per group (pooled data from mice sacrificed between days 14 and 18). ** p ≤ 0.01; * p ≤ 0.05; NS = non-statistically significant.

Figure 7

Effect of BSO on total glutathione levels in EJM tumours. Mice were intravenously injected with 30 MBq of ¹⁷⁷Lu-DOTATATE on day 0. BSO (10 mM) was administered via drinking water starting on day-1. Total glutathione levels in tumours after 2 days (A) and 14–18 days (B) of continuous BSO exposure. Results are expressed as the mean ± SD of 2 to 5 mice per group (pooled data from mice sacrificed between days 14 and 18). *** p ≤ 0.001; ** p ≤ 0.01; NS = non-statistically significant.

Figure 8

Effect of ¹⁷⁷Lu-DOTATATE and its combination with BSO on bone marrow. Mice were intravenously injected with 30 MBq of ¹⁷⁷Lu-DOTATATE on day 0. Mice in the combination group (¹⁷⁷Lu-DOTATATE + BSO) additionally received 10 mM BSO via drinking water starting on day-1. BM (from both femurs) of 3 to 4 mice per group, sacrificed 29 to 38 days post-¹⁷⁷Lu-DOTATATE injection, were analysed for apoptosis (A) or cytological assessment (B–D). (A) Apoptotic cells were classified into early-apoptotic (Annexin V+/7AAD−) and late-apoptotic/necrotic cells (Annexin V+/7AAD+). Results are expressed as mean ± SEM. The relative proportions of major BM lineages were evaluated: erythroid (B), granulocytic (C) and lymphoid lineages (D). Results are represented as mean ± SD. The dotted lines represent lower and upper normal ranges. NS = non-statistically significant.

Figure 9

Representative H&E-stained slides of mice kidneys. (A) Control, (B) BSO (C), ¹⁷⁷Lu-DOTATATE and (D) ¹⁷⁷Lu-DOTATATE + BSO. Magnification 20×. No morphological changes were observed. G = glomeruli.

Figure 10

Representative H&E-stained slides of mice liver. (A) Control, (B) BSO, (C) ¹⁷⁷Lu-DOTATATE and (D) ¹⁷⁷Lu-DOTATATE + BSO. Magnification 20×. No morphological changes were observed.

Figure 11

Effect of ¹⁷⁷Lu-DOTATATE alone and in combination with BSO on EJM tumours. Mice bearing EJM multiple myeloma tumours were treated on day 28 post-tumour cell inoculation with 30 MBq of ¹⁷⁷Lu-DOTATATE alone or in combination with 10 mM BSO via drinking water, starting on day 27, for 3 weeks. Tumours were measured 3 times per week in 8 to 10 mice per group. (A) Tumour growth over time, represented as mean relative tumour volume ± SEM. (B) Box plot distribution of relative tumour volume (calliper measurement) on day 45. Only significant differences are indicated, where * p ≤ 0.05. (C) Tumour doubling time was obtained by fitting an exponential growth equation from tumour growth measures. (D) Box plot distribution of changes in tumour volume before and after treatment, measured on the CT part of the PET/CT, represented as a fractional increase in tumour volume (ΔVol_T). Only significant differences are indicated, where * p ≤ 0.05.

Figure 12

Effect of ¹⁷⁷Lu-DOTATATE alone and in combination with BSO on EJM tumours ¹⁸F-FDG metabolic activity. Mice bearing EJM multiple myeloma tumours were treated on day 28 post-tumour cells inoculation with 30 MBq of ¹⁷⁷Lu-DOTATATE alone or in combination with 10 mM BSO via drinking water, starting on day 27, for 3 weeks. Box plot distribution showing the changes in ¹⁸F-FDG uptake before and after treatment (day 45 post-inoculation) represented as fractional increase in total lesion glycolysis (ΔTLG). Only significant differences are indicated, where * p ≤ 0.05.