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./nMethods: 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./nResults: 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./nConclusions: 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.

中文翻译：

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mTORC1的药理抑制作用可增加放射性标记的小胃泌素类似物[177Lu] Lu-PP-F11N的CCKBR特异性肿瘤吸收

理由：放射性标记配体的高肿瘤与健康组织摄取比率是安全有效的肽受体放射性核素治疗（PRRT）的必要前提。在本研究中，我们寻找增加放射性标记的小胃泌素类似物[ ¹⁷⁷ Lu] Lu-DOTA-（DGlu）₆ -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH _2的肿瘤特异性摄取的新机会。（[ ¹⁷⁷ Lu] Lu-PP-F11N），其靶向人类癌症中的胆囊收缩素B受体（CCKBR）。/n方法：采用激酶抑制剂文库进行筛选，然后进行增殖和内在化分析，以鉴定可增加摄取量的化合物。 [ ¹⁷⁷Lu] Lu-PP-F11N在CCKBR转染的人表皮样癌A431细胞和天然表达CCKBR的大鼠胰腺腺泡AR42J细胞中发挥作用。Western印迹（WB）分析验证了信号通路和CCKBR水平的抑制作用，而基于细胞的分析则分析了抑制蛋白的募集。A431 / CCKBR异种移植小鼠模型的生物分布和SPECT成像以及解剖的肿瘤的组织学分析用于体内验证。/n结果：我们的筛选确定了雷帕霉素复合物1（mTORC1）的哺乳动物靶标抑制剂[ ¹⁷⁷ Lu] Lu-PP-F11N的细胞摄取。RAD001和二甲双胍抑制mTORC1的药理作用可增加[ ¹⁷⁷Lu] Lu-PP-F11N在A431 / CCKBR和AR42J细胞中。对RAD001处理的细胞的蛋白裂解物进行分析后发现，CCKBR的水平升高（2.2倍），并且抑制了S6的磷酸化。PP-F11N诱导募集β-arrestin1/ 2和ERK1 / 2磷酸化。在带有A431 / CCKBR肿瘤的裸鼠中，RAD001预处理3天或5天可显着提高[ ¹⁷⁷ Lu] Lu-PP-F11N的肿瘤特异性摄取（[RAD001 /对照]的比分别为1.56或1.79），而二甲双胍治疗无明显差异。SPECT / CT图像的定量证实了[ ^177]的更高摄取RAD001处理的肿瘤中的Lu] Lu-PP-F11N的平均浓度和最大浓度之比[RAD001 / Control]分别达到3.11和3.17。RAD001治疗的肿瘤的HE染色和IHC显示坏死显着增加（对照为1.4％，坏死面积为10.6％），增殖和有丝分裂活性细胞（80％对照vs Ki67阳性细胞的61％）减少（对照组为1.08％，而有丝分裂率为0.75％）。RAD001或二甲双胍治疗5天后未观察到肿瘤血管形成的显着差异。/n结论：我们的数据表明，通过RAD001预处理增加CCKBR蛋白水平有可能改善肿瘤的摄取[ ^177]Lu] Lu-PP-F11N并为开发旨在提高目标受体水平，提高PRRT和核成像效果的分子策略提供了概念验证。