Potentiation of ¹⁷⁷Lu-octreotate PRRT by PARP inhibitors in a 3D spheroid model of human-derived neuroendocrine tumor cell lines

Objectives: Peptide receptor radionuclide therapy (PRRT) with ¹⁷⁷Lu-octreotate (LuTate) is one of the most promising targeted therapies for neuroendocrine tumors (NETs), but it rarely achieves cure. Using two human-derived NET cell lines, of gastroenteropancreatic (BON-1) and of bronchopulmonary (NCI-H727) origins, we examined if the pharmacological inhibitors of poly(ADP-ribose) polymerase-1 (PARPi), an enzyme involved in DNA repair, could potentiate the cytotoxic effect of LuTate. To better reproduce the in vivo conditions of tumors, we used 3D spheroid models rather than 2D monolayer cultures.

Methods: Three-dimensional spheroids were made by seeding 1000 cells per well in a 24-well plate previously coated with 1% agarose. The spheroids grew to a diameter of 300-400um by 6 days, and were treated with 5.5 MBq LuTate, 100 µM PARPi (1,5-dihydroxyisoquinoline), LuTate and PARPi combination, or placebo in 0.6 ml of medium (n= 6 per group). The treatment lasted for 5 days and the therapeutic effect was monitored until 15 days as: (a) relative spheroid growth since start of treatment using an inverted phase-contrast microscope at 20X and a digital image processing software; and (b) immunoblotting for key cell growth and death-related proteins in pooled samples derived from 6-12 spheroids per treatment group. The LuTate uptake by spheroids was also monitored.

Results: The combination of LuTate and PARPi was more cytotoxic than either of them alone in both cell lines. While the untreated BON-1 spheroids grew 16.7 folds in volume over 15 days, those treated with LuTate, PARPi, or LuTate + PARPi grew by 5.1, 11.0 and 1.9 folds, respectively. For NCI-H727 spheroids, while the controls exhibited a 11.4-fold growth over 15 days, those treated with LuTate, PARPi, or LuTate + PARPi grew by 4.6, 3.5 and 2.8 folds, respectively. Immunoblotting of spheroids from both cell lines revealed a greater accumulation of γH2AX, indicating greater accumulation of DNA double-strand breaks, and higher levels of activated (cleaved) caspase-3, indicating more DNA damage and cell death, in response to the combination treatment. Concomitantly, there was a greater reduction in the proliferation indices Ki-67 and PCNA under the latter. Notably, no significant increase in LuTate uptake was noted with PARPi treatment, indicating that effects of PARPi were molecularly targeted in response to LuTate-induced DNA damage.

Conclusion: In the in vitro 3D models of NETs from human origin, PARPi enhances tumor growth suppression and cell death by impairing the repair of LuTate-induced DNA damage. Radiosensitization by PARPi thus offers a promising approach for improving the therapeutic efficacy of LuTate-based PRRT for treating NETs. Research support: Mitzi & William Blahd, MD Pilot Research Grant from The Education and Research Foundation for Nuclear Medicine and Molecular Imaging; Pilot Grant from Quebec Bio-Imaging Network; NET Research Grant from CNETS Canada; Scholarship from Fonds de recherche du Québec - Santé to J.M.B.

• Download figure
• Open in new tab
• Download powerpoint