Understanding the Radiobiological Mechanisms Induced by 177Lu-DOTATATE in Comparison to External Beam Radiation Therapy

Abstract

Radionuclide Therapy (RNT) with ¹⁷⁷Lu-DOTATATE targeting somatostatin receptors (SSTRs) in neuroendocrine tumours (NET) has been successfully used in routine clinical practice, mainly leading to stable disease. Radiobiology holds promise for RNT improvement but is often extrapolated from external beam radiation therapy (EBRT) studies despite differences in these two radiation-based treatment modalities. In a panel of six human cancer cell lines expressing SSTRs, common radiobiological endpoints (i.e., cell survival, cell cycle, cell death, oxidative stress and DNA damage) were evaluated over time in ¹⁷⁷Lu-DOTATATE- and EBRT-treated cells, as well as the radiosensitizing potential of poly (ADP-ribose) polymerase inhibition (PARPi). Our study showed that common radiobiological mechanisms were induced by both ¹⁷⁷Lu-DOTATATE and EBRT, but to a different extent and/or with variable kinetics, including in the DNA damage response. A higher radiosensitizing potential of PARPi was observed for EBRT compared to ¹⁷⁷Lu-DOTATATE. Our data reinforce the need for dedicated RNT radiobiology studies, in order to derive its maximum therapeutic benefit.

Keywords: 177Lu-DOTATATE; DNA damage; PARP inhibition; external beam radiation therapy; radiobiology; radionuclide therapy.

Figure 1

Effect of ¹⁷⁷Lu-DOTATATE and EBRT on the survival of melanoma (HBL and MM162), multiple myeloma (COLO-677 and EJM) and GEP (MIA-PACA-2 and HT-29) cell lines. (a) Cell survival was assessed 3, 7 and 10 days after 2 Gy EBRT. (b) Cell survival at day 10 after 4 h incubation with 5 MBq of ¹⁷⁷Lu-DOTATATE (purple) or 2 Gy EBRT (pink). Results are expressed as a percentage of the non-treated counterpart (black dotted line) and are represented as mean ± SEM (n = 20 from 5 independent experiments): ** p ≤ 0.01; *** p ≤ 0.001.

Figure 2

Effect of ¹⁷⁷Lu-DOTATATE and EBRT on the cell cycle distribution in melanoma (HBL and MM162), multiple myeloma (COLO-677 and EJM) and GEP (MIA-PACA-2 and HT-29) cell lines. Cell cycle was assessed 10 days after 4 h incubation with 5 MBq of ¹⁷⁷Lu-DOTATATE or 2 Gy EBRT. Results are represented as mean ± SEM (n = 3 from 3 independent experiments).

Figure 3

Effect of ¹⁷⁷Lu-DOTATATE and EBRT on apoptosis induction in melanoma (HBL and MM162), multiple myeloma (COLO-677 and EJM) and GEP (MIA-PACA-2 and HT-29) cell lines. The percentage of total apoptotic cells (Annexin V+ 7-AAD-, Annexin V+ 7-AAD+) was assessed 3 and 10 days after 4 h incubation with 5 MBq of ¹⁷⁷Lu-DOTATATE (purple) or 2 Gy EBRT (pink). Results are represented as mean ± SEM (n = 3 from 3 independent experiments). Only the significant differences are indicated on the graphs: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. The following symbols were used for the comparison between ¹⁷⁷Lu-DOTATATE and EBRT results: # p ≤ 0.05; ## p < 0.01.

Figure 4

Effect of ¹⁷⁷Lu-DOTATATE and EBRT on autophagy induction in melanoma (HBL and MM162), multiple myeloma (COLO-677 and EJM) and GEP (MIA-PACA-2 and HT-29) cell lines. Autophagy was assessed 3 and 10 days after 4 h incubation with 5 MBq of ¹⁷⁷Lu-DOTATATE (purple) or 2 Gy EBRT (pink). Results are expressed as a percentage of the non-treated counterpart (black dotted line) and are represented as mean ± SEM (n = 3 from 3 independent experiments): * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. The following symbols were used for the comparison between ¹⁷⁷Lu-DOTATATE and EBRT results: # p ≤ 0.05; ## p < 0.01.

Figure 5

Effect of ¹⁷⁷Lu-DOTATATE and EBRT on ROS induction in melanoma (HBL and MM162), multiple myeloma (COLO-677 and EJM) and GEP (MIA-PACA-2 and HT-29) cell lines. ROS were assessed immediately (within 15 min) after 4 h incubation with 5 MBq of ¹⁷⁷Lu-DOTATATE (purple) or 2 Gy EBRT (pink) and at day 3. Results are normalized against the non-treated counterpart (black dotted line) (n = 6 from 3 independent experiments). NS: not statistically significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. The following symbols were used for the comparison between ¹⁷⁷Lu-DOTATATE and EBRT results: # p ≤ 0.05; ## p < 0.01; ### p < 0.001.

Figure 6

Effect of ¹⁷⁷Lu-DOTATATE and EBRT on DNA damage repair in melanoma (HBL and MM162), multiple myeloma (COLO-677 and EJM) and GEP (MIA-PACA-2 and HT-29) cell lines. Total DNA damage (pATM+ γH2AX-, pATM- γH2AX+, pATM+ γH2AX+) was assessed at different timepoints after 4 h incubation with 5 MBq of ¹⁷⁷Lu-DOTATATE (purple) or 2 Gy EBRT (pink). Results are represented as mean ± SEM (n = 3 from 3 independent experiments for 15 min, 3.5 h and 10-day time points).

Figure 7

Effect of olaparib and its combination with ¹⁷⁷Lu-DOTATATE and EBRT on survival of melanoma (HBL and MM162), multiple myeloma (COLO-677 and EJM) and GEP (MIA-PACA-2 and HT-29) cell lines. Cell survival was assessed 10 days after 4 h incubation with 5 MBq of ¹⁷⁷Lu-DOTATATE (purple) or 2 Gy EBRT (pink). Olaparib (HBL, MM162 and HT-29: 10⁻⁶ M; MIA-PACA-2 and EJM: 10⁻⁷ M; COLO-677: 10⁻⁸ M) was present in the medium from the day before irradiation until the end of the experiment. Results are expressed as a percentage of the non-treated counterpart (black dotted line) and are represented as mean ± SEM (n = 12 from 3 independent experiments): *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; NS = not statistically significant.

Figure 8

Mean cumulative absorbed dose ± SD to cell for 2 different geometries with a constant volume (sphere and semi-ellipse of 3052 and 3020 µm³ respectively) with contribution in % of the medium (in blue), the internalized fraction during the 4h (in orange) and the 10 days incubation (in grey).

Figure 9

(a) Spherical and (b) semi-ellipsoid cell models used for the simulation set-up (nucleus in green, cytoplasm in red, membrane in blue): a = minor axis; b = major axis; h = height. Ratios are as follows: a/b = 1/3; h/a = 1/2.