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Introduction: One of the factors that limit the efficacy of the drugs, especially in primary brain tumors, is the permeability of the blood-brain barrier (BBB). The low-density lipoprotein receptor (LDL-R) expressed at the BBB mediates the transport of endogenous ligands through the BBB. We developed a new radiolabeled peptide targeting both the human and murine LDLR and able i) to cross the BBB and ii) to target tumors such as glioblastoma that express high levels of the LDLR. The objective of this study was to determine the LDLR targeting properties, the pharmacokinetics of 68Ga radiolabeled RMX-VH in a glioblastoma model that expresses the human LDLR (hLDLR). Methods: The nonclinical studies of 68Ga-RMX-VH were completed in U87MG, A172, U373 glioblastoma cancer cell lines and xenografts mice models. The studies determined in vivo time-dependent accumulation of this agent, in vitro dose-depended cellular uptake, and cellular competition studies. We compared the tumor-specific accumulation of 68Ga-RMX-VH and normal organ distribution in female and male athymic nude mice. Radiotracer, RMX-VH (10-30ug) was labeled with isotope-Ga68 (10-25mCi, ITM GmBH). U87MG and A172- derived xenografts were generated in athymic nude mice (10 weeks) and PET/CT images were acquired using G4 PET/Xray camera (Sofie Biosciences) at 1h, 2h, 3h, and 4h post-injection. The followed up biodistribution studies were done at 30 min, 1h, 2h, and 3 hrs. post-injection. The organs and tumor were collected, weighed, and the tissue radioactivity was measured with Wizard2 Gamma Counter (Perkin-Elmer, Waltham, MA). The percentage of injected dose per gram of tissue (%ID/g) was calculated and decay-corrected. Results: Our studies confirmed the in vitro and in vivo selectivity and specificity of 68Ga-RMX-VH toward LDLR-positive tumors. 68Ga-RMX-VH is a small peptide (MW: 1432.7 g/mol) and renal excretion was expected as a route of agent elimination. The tumor-specific uptake of radiotracer in U87MG xenografts was 1.8%ID/g at 1h and remained unchanged at 3h post-injection. The kidney retention of the agent reached 12.2%ID/g and decreased to 10.2%ID/g. The accumulation of radiotracer in the liver, ovaries, and intestine correlated well with the known normal expression of LDLR. The tumor-to-muscle ratio was 5.97 at 1h; increased to 26.1 and 22.39 at 2h and 3h. The elimination t1/2 of radiotracer was only 18.6 min, with a clearance CL of 502 ml/min. Adsorption from the site of administration is rapid as the Cmax was 5 min. The tumor-specific uptake and normal organ distribution of 68Ga-RMX-VH were lower in male mice than in females. This correlated with differences in lipid and lipoproteins metabolism in males and females. Conclusions: RMX-VH showed favorable hLDLR targeting properties in vitro and in vivo in glioblastoma mice models. Our results suggest that hLDLR may serve as a target for imaging for glioblastoma. The first-in-human exploratory IND study of Ga68-RMX-VH will be initiated in Q2 of 2021. Citation Format: Izabela Tworowska, Leo Garcia Flores II, Xuewei Qu, Cédric Malicet, Nilesh Wagh, David Ranganathan, Jonathan Nowak, Pascaline Lecorche, Jamal Temsamani, Ebrahim S. Delpassand. Radionuclide imaging of low-density-lipoprotein receptor (LDLR)-overexpressing glioblastoma: A preclinical study of Gallium-68 RMX-VH [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P099.