Rationale: A high
tumor-to-healthy-tissue uptake ratio of radiolabeled
ligands is an essential prerequisite for safe and effective
peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase
tumor-specific uptake of the radiolabeled
minigastrin analogue [177Lu]Lu-
DOTA-(DGlu)6-
Ala-Tyr-
Gly-Trp-Nle-
Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the
cholecystokinin B receptor (CCKBR) in human
cancers. Methods: A
kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human
epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed
arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected
tumors were used for in vivo validation. Results: Our screen identified the inhibitors of
mammalian target of rapamycin complex 1 (
mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological
mTORC1 inhibition by
RAD001 and
metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of
protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of β-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-
tumor bearing nude mice, 3 or 5 days of
RAD001 pretreatment significantly enhanced
tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [
RAD001/Control] of 1.56 or 1.79, respectively), whereas
metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated
tumors with ratios [
RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated
tumors showed a significant increase in
necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the
tumor vascularization was observed after five-day
RAD001 or
metformin treatment. Conclusions: Our data demonstrates, that increased CCKBR
protein level by
RAD001 pretreatment has the potential to improve
tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging.