Development Of A Probe Targeting Both Glypican-3 And PSMA For Hepatocellular Carcinoma PET Imaging

Part Ⅰ.Prostate-Specific Membrane Antigen Expression in Hepatocellular Carcinoma,Intrahepatic Cholangiocarcinoma,and Liver CirrhosisOBJECTIVE:Primary liver cancer includes three subtypes:hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICC),and combined hepatocellular carcinoma.Patients with primary liver cancer experiencing poor prognosis and high mortality,so accurate diagnosis of liver cancer and improved management of metastases are both key strategies to reduce the death toll from liver cancer.Prostate-specific membrane antigen(PSMA)expression in the tumor-associated neovasculature of non-prostate malignancies including liver cancer has been reported recently,but conclusive evidence of PSMA expression based on the pathological type of liver cancers remains limited.This large-scale retrospective study was performed to study the expression of PSMA in HCC,ICC and liver cirrhosis.METHODS:Immunohistochemistry(IHC)was used to detect PSMA expression in a total of 446 paraffin-embedded liver biopsy specimens(213 HCC,203 ICC,and 30 liver cirrhosis).Correlation between the PSMA expression and clinicopathologic information was also assessed.RESULTS:PSMA was expressed primarily in the endothelium of neovascular systems associated with tumors.The positive rate of PSMA staining in HCC was significantly higher than that in ICC(86.8%vs.79.3%;P=0.04),meanwhile,only 6.6%in liver cirrhosis(P<0.001;P<0.001).HCC had more score of 3 in a 4-point scoring system(0～3 points)than ICC(89/213,41.8%vs.35/203,17.2%;P<0.001).High PSMA expression was prone to be found in high-grade and advanced-stage liver cancer.CONCLUSION:This study provides the evidence of differential PSMA expression in HCC,ICC and liver cirrhosis,suggesting neovascular PSMA may be used as a promising marker to differentiate HCC from liver cirrhosis and a prognostic marker for anti-tumor angiogenesis therapy for HCC.Part Ⅱ.Development of a 68Ga and 18F Labeled Probe Targeting both Glypican-3 and PSMA for Hepatocellular Carcinoma PET ImagingOBJECTIVE:To construct a dual-target probe consisting of TJ12P2 peptide and PSMA inhibitor,which can target GPC3 and PSMA respectively,for PET imaging of HCC.METHODS:TJ12P2 and PSMA inhibitor were first modified with PEG4 and then conjugated to BCN-NOTA.The resulting precursor(NOTA-TJ12P2-PSMA)was purified and labeled with 68Ga and 18F respectively.The radiochemical purity of each molecular probe and the stability of each molecular probe in serum and PBS was detected respectively;LogP of each molecular probe was detected.In vitro cell uptake assay and blocking assay were conducted to verify whether NOTA-TJ12P2-PSMA specifically bound to GPC3 and PSMA.Animal PET imaging and blocking studies were performed in mice bearing Huh7 xenograft models(n=3).In vivo and in vitro properties of TJ12P2,PSMA monomer and heterodimer NOTA-TJ12P2-PSMA labeled with 68Ga were compared.The molecular probe 68Ga-TJ12P2-PSMA was compared with 18F-TJ12P2-PSMA for targeting Huh7 tumor.The biodistribution of each molecular probe in Huh7 tumor xenograft mice(n=4)was detected.The expression levels of GPC3 and PSMA in tumors of Huh7 tumor-bearing mice were checked via immunohistochemistry.RESULTS:The heterodimeric probe was successfully synthesized and radiolabeled with 68Ga and 18F respectively.Molecular probes 68Ga-TJ12P2-PSMA,68Ga-TJ12P2,68Ga-PSMA,18F-TJ12P2-PSMA were successfully prepared.All the molecular probes demonstrated high stability in vitro.The uptake of 68Ga-TJ12P2-PSMA by GPC3 expressing cell(Huh7)and PSMA expressing cell(C4-2)increased gradually over time,and reached plateau at 2 h.Its uptake was blocked by the corresponding blocking agent,indicating that the molecular probe was specific binding to GPC3 and PSMA.In static PET imaging studies,Huh7 tumor could be clearly visualized and exhibited higher uptake at 60 min post injection(p.i.)of 68Ga-TJ12P2-PSMA than that of either 68Ga-TJ12P2 or 68Ga-PSMA alone(1.75 ± 0.16,1.25 ± 0.07,1.07 ± 0.06%ID/g,t=4.95,P=0.007;t=18.9,P<0.0001).Compared with TJ12P2 and PSMA monomer,68Ga-TJ12P2-PSMA had better tumor uptake,and the tumor to muscle ratio(TMR)was better than that of monomeric counterpart(4.86 ± 0.02,4.21 ± 0.01,3.96 ± 0.08,t=50.35,P<0.0001;t=18.9,P<0.0001).High specificity was shown in blocking studies using TJ12P2,PSMA(2-PMPA),TJ12P2+PSMA(TJ12P2+2-PMPA),TJ12P2-PSMA,this applies to 68Ga-TJ12P2-PSMA and the corresponding probe labeled with 18F(68Ga:0.55±0.03,0.76± 0.12,0.35 ± 0.09,0.41 ± 0.11%ID/g;18F:0.60 ± 0.05,0.78 ± 0.08,0.43 ± 0.06,0.39 ±0.09%ID/g).TJ12P2 had better blocking effect than PSMA(t=2.94,P=0.042;t=3.30,P=0.029).18F-TJ12P2-PSMA showed better TMR than 68Ga-TJ12P2-PSMA when delayed to 90 min(4.31 ± 0.10,3.80 ± 0.17;t=4.48,P=0.01).The biodistribution was consistent with PET imaging,and the ratio of tumor to background(muscle,blood and liver)was better than that of monomeric counterpart(P<0.05).Immunohistochemistry confirmed that Huh7 tumor expressed GPC3 and PSMA.CONCLUSION:The heterodimer 68Ga/18F-TJ12P2-PSMA targeting both GPC3 and PSMA was successfully synthesized and used for HCC imaging.The molecular probe has ideal labeling rate,radiochemical purity and stability.It showed good tumor targeting ability,which is better than the corresponding monomeric counterpart in vivo and in vitro.Its promising in vivo performance makes it an ideal candidate for future clinical translation.