⁶⁸Ga/¹⁷⁷Lu-PSMA-617 Theragnostic Probe For Hepatocellular Carcinoma Imaging And Therapy

Objective:Hepatocellular carcinoma（HCC）is one of the malignant tumors associated with high morbidity and mortality rates.Prostate-specific membrane antigen（PSMA）is overexpressed in tumor associated endothelial cells of many non-prostate solid tumors.The aim was to evaluate the performance of ⁶⁸Ga-PSMA-617 PET imaging on HCC diagnosis with different animal models including patient-derived xenograft（PDX）tumors and to explore its mechanism.Methods:⁶⁸Ga-PSMA-617 was prepared.Human HCC cells Hep G2 and Hu H-7 were cultured.The expression level of PSMA in Hep G2 and Hu H-7 was evaluated by Western blot,and the cellular uptakes of ⁶⁸Ga-PSMA-617 were assayed.Hep G2 and Hu H-7subcutaneous xenograft models,Hep G2 orthotopic xenograft models,and four different groups of PDX models were prepared.⁶⁸Ga-PSMA-617 was injected into tumor-bearing mice via tail veins for PET imaging and biodistribution studies to verify its targeting specificity in vivo.Preclinical pharmacokinetics and performance of ⁶⁸Ga-PSMA-617 was evaluated in different types of HCC xenografts models using small-animal PET and biodistribution studies.Results:Low PSMA expression level of Hep G2 and Hu H-7 cells was observed,and the blocking study confirmed the non-specificity of the PSMA-targeted probe binding to Hep G2and Hu H-7 cells.In the subcutaneous xenograft models,the tumor uptake at 0.5 h was 0.76±0.12%ID/g（Hep G2 tumors）,and 0.78±0.08%ID/g（Hu H-7 tumors）respectively,which were significantly higher than those of the blocking groups（0.23±0.04%ID/g and 0.20±0.04%ID/g,respectively）,confirming the specific imaging with ⁶⁸Ga-PSMA-617.In the orthotopic xenograft models,PET images clearly displayed the tumor locations based on the preferential accumulation of ⁶⁸Ga-PSMA-617 in tumor tissue versus normal liver tissue,suggesting the possibility of ⁶⁸Ga-PSMA-617 PET imaging for detecting primary HCC lesions in deep-tissue.In four different groups of HCC PDX models,PET imaging with⁶⁸Ga-PSMA-617 provided clear tumor uptakes with prominent tumor-to-background contrast,further demonstrating its potential for clinical imaging of PSMA-positive HCC lesions.Staining of tumor tissue sections with CD31-and PSMA-specific antibodies visualized the tumor-associated blood vessels and the PSMA expression on endothelial in subcutaneous,orthotopic tissues,and PDX tissues.Conclusion:In this study,in vivo PET studies on different types of HCC xenograft models preliminarily confirmed that ⁶⁸Ga-PSMA-617 might be a promising radiotracer for PET imaging of HCC by targeting tumor-associated endothelium.Object: Hepatocellular carcinoma（HCC）is one of the malignant tumors associated with high morbidity,mortality rates and unfavorable clinical course.Limited treatment options of patients with HCC lead to negative survival results,thus alternative therapy is needed.Prostate-specific membrane antigen（PSMA）is expressed in the tumor-associated endothelium of non-prostate solid tumors including HCC,making it a potential therapeutic target for HCC.In order to explore new treatment methods and to improve the therapeutic effect,in this study,we explored the feasibility of 177Lu-PSMA-617 and 177Lu-EB-PSMA-617 for in vivo radioligand therapy in PSMA-positive HCC xenograft models.Methods: 177Lu-PSMA-617 and 177Lu-EB-PSMA-617 were prepared.Labelling efficiency and radiochemical purity were determined using HPLC.Human HCC cells Hep G2 were cultured,and Hep G2 subcutaneous xenograft models were prepared.After the intravenous injection of 177Lu-PSMA-617,177Lu-EB-PSMA-617 or 177 Lu Cl3（37 MBq）in saline,tumorbearing mice were investigated using a small animal SPECT/CT scanner.Biodistribution studies were carried out to verify targeting specificity and pharmacokinetics in vivo.For radiotherapy study,tumor-bearing mice were randomized into 6 groups: 37 MBq 177LuPSMA-617,18.5 MBq 177Lu-PSMA-617,7.4 MBq 177Lu-EB-PSMA-617,37 MBq 177 Lu Cl3,18.5 MBq 177 Lu Cl3 and saline.Tumor volume,body weight,and survival rate were monitored every 2 days until the end of the study.After the end of treatment,the mice were euthanized and tumors were weighed,the blood and major organs were obtained for evaluating systematic toxicity.Results: 177Lu-PSMA-617 and 177Lu-EB-PSMA-617 were successfully prepared with high purity and stability.SPECT images and biodistribution studies showed that the tumor uptake of 177Lu-EB-PSMA-617 was higher than that of 177Lu-PSMA-617.177Lu-EB-PSMA-617 exhibited significantly higher blood retention than 177Lu-PSMA-617（20.66 ± 0.84% ID/g vs.0.43 ± 0.11% ID/g,at 1 h p.i.,P<0.0001）.The tumor uptake of 177Lu-EB-PSMA-617 was retained over time（6.27 ± 2.00 % ID/g at 24 h p.i.,and 4.61 ± 0.15 % ID/g at 48 h p.i.）,whereas the tumor uptake of 177Lu-PSMA-617 significantly decreased over time.After injection of 177 Lu Cl3,tumor and bone showed high uptake at 1,4,24,48 h（6.22 ± 0.46 % ID/g for tumor,and 22.52 ± 4.30 % ID/g for bone,at 48 h p.i.）.For in vivo radiotherapy study,37 MBq 177Lu-PSMA-617,18.5 MBq 177Lu-PSMA-617,7.4 MBq 177Lu-EB-PSMA-617,and 18.5 MBq 177 Lu Cl3 treated groups significantly suppress tumor growth,compared to the saline group,and the median survival was 40 d,44d,43 d,48d,30 d,respectively.No major organ toxicity was observed in safety and tolerability evaluation.However,37 MBq 177 Lu Cl3 showed toxicity on mice,and the mice died on 10d–14d after the therapy studies began.Conclusions: 177Lu-PSMA-617,177Lu-EB-PSMA-617 and relatively low dose of 177 Lu Cl3 significantly suppressed tumor growth and prolonged the survival time of tumor-bearing mice,with no obvious toxicity.These results justify that 177Lu-PSMA-617,177Lu-EBPSMA-617 and 177 Lu Cl3 at safe and well-tolerated doses would be new route for the treatment of HCC.Objective: Imaging diagnosis of hepatocellular carcinoma（HCC）still remains challenging,mainly due to a limited ability in early disease detection.Nuclear imaging can reflect the changes of molecular level and achieve an early,sensitive,and accurate diagnosis.But the current positron emitting radiotracers 18F-FDG also showed intense accumulation in normal liver tissue,affecting the diagnosis of HCC lesions.68Ga-PSMA-617 might be a promising clinical application HCC detecting by targeting tumor-associated endothelium.This study screened patients with clinical suspicion or confirmed hepatocellular carcinoma to perform 68Ga-PSMA-617 PET/MR multiparametric functional imaging and compared with pathological results to explore its ability to screen patients with PSMA positive hepatocellular carcinoma,and to evaluate its value in the diagnosis and staging of hepatocellular carcinoma.Methods: Four patients with histologically proven HCC were recruited from September to October 2021,and each patient underwent 68Ga-PSMA-617 PET/MR examination.MR imaging findings assessed the location,extension,and tumor invasion into surrounding tissues of the HCC lesion.Region of interest（ROI）is outlined on the fusion image to semiquantitatively calculate the tumor radioactive uptake in the fusion image,and the maximum standard uptake value（SUVmax）were measured.To avoid bias,at least 2 groups of experienced nuclear medicine physicians independently analyzed the 68Ga-PSMA-617 PET/MR images.Image interpretation included visual analysis and quantitative assessment,and the results were discussed to reach a consensus.The efficacy of 68Ga-PSMA-617 PET/MR in the preoperative diagnosis of hepatocellular carcinoma and its effect on clinical staging were evaluated and compared with pathological results.PSMA expression in tumor tissue of patients was detected by immunohistochemical staining.Results: A total of 6 lesions were detected.Pathologically,out of the 6 lesions,5 lesions were hepatocellular carcinoma and one lesion was intrahepatic cholangiocarcinoma（ICC）.The tumor tissues of HCC showed significant MR signal changes with heterogeneous signal on T1-weighted images（T1WI）,mild to moderate hyper-intense signal on T2-weighted images（T2WI）,and hyper-intense signal on diffusion-weighted imaging（DWI）.The tumor tissue of ICC showed significant MR signal changes with moderate hyper-intense signal on T1 WI and T2 WI,and hyper-intense signal on DWI.PET results showed that the uptake of 68Ga-PSMA-617 was heterogeneous in HCC lesions.The PSMA positive tumor tissues often showed intense radioactive uptake,while the PSMA negative tumor tissues showed slightly increased uptake or similar to that of the normal liver tissue.PET imaging findings corresponded well with immunohistochemical staining results,indicating that 68Ga-PSMA-617 PET/MR imaging is helpful in screening PSMA positive HCC patients.Conclusion: The uptake of 68Ga-PSMA-617 in HCC lesions is closely related to the PSMA expression level of the lesions,and 68Ga-PSMA-617 PET/MR imaging helps to screen PSMA positive HCC patients,which might be a useful strategy to guide clinical decisionmaking and the clinical management of HCC.