PET imaging of hepatocellular carcinoma by targeting tumor-associated endothelium using [68Ga]Ga-PSMA-617

Abstract

Objective: Hepatocellular carcinoma (HCC) is a malignant tumor associated with high morbidity and mortality rates. In many non-prostate solid tumors such as HCC, prostate-specific membrane antigens (PSMA) are overexpressed in tumor-associated endothelial cells. Therefore, the aim of this study was to evaluate the performance of [⁶⁸Ga]Ga-PSMA-617 PET imaging on HCC with different animal models, including cell line-derived xenografts (CDX) and patient-derived xenografts (PDX), and to explore its mechanisms of function.

Methods: [⁶⁸Ga]Ga-PSMA-617 was prepared. The expression level of PSMA in two human hepatocellular cancer cells (HepG2 and HuH-7) was evaluated, and the cellular uptakes of [⁶⁸Ga]Ga-PSMA-617 were assayed. HepG2 and HuH-7 subcutaneous xenograft models, HepG2 orthotopic xenograft models, and four different groups of PDX models were prepared. Preclinical pharmacokinetics and performance of [⁶⁸Ga]Ga-PSMA-617 were evaluated in different types of HCC xenografts models using small animal PET and biodistribution studies.

Results: Low PSMA expression level of HepG2 and HuH-7 cells was observed, and the cellular uptake and blocking study confirmed the non-specificity of the PSMA-targeted probe binding to HepG2 and HuH-7 cells. In the subcutaneous xenograft models, the tumor uptakes at 0.5 h were 0.76 ± 0.12%ID/g (HepG2 tumors) and 0.78 ± 0.08%ID/g (HuH-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). In the orthotopic xenograft models, PET images clearly displayed the tumor locations based on the preferential accumulation of [⁶⁸Ga]Ga-PSMA-617 in tumor tissue versus normal liver tissue, suggesting the possibility of using [⁶⁸Ga]Ga-PSMA-617 PET imaging to detect primary HCC lesions in deep tissue. In the four different groups of HCC PDX models, PET imaging with [⁶⁸Ga]Ga-PSMA-617 provided clear tumor uptakes with prominent tumor-to-background contrast, further demonstrating its potential for the clinical imaging of PSMA-positive HCC lesions. The staining of tumor tissue sections with CD31- and PSMA-specific antibodies visualized the tumor-associated blood vessels and PSMA expression on endothelial cells in subcutaneous, orthotopic tissues, and PDX tissues, confirming the imaging with [⁶⁸Ga]Ga-PSMA-617 might be mediated by targeting tumor associated endothelium.

Conclusion: In this study, in vivo PET on different types of HCC xenograft models illustrated high uptake within tumors, which confirmed that [⁶⁸Ga]Ga-PSMA-617 PET may be a promising imaging modality for HCC by targeting tumor associated endothelium.

Keywords: Diagnosis; Hepatocellular carcinoma (HCC); Positron emission tomography (PET); Prostate-specific membrane antigen (PSMA); Tumor-associated endothelial cells.

Fig. 1

Identification of PSMA expression level in cells and cell uptake study. A and B Prostate-specific membrane antigen (PSMA) expression assay in prostate cancer cells LNCaP (PSMA⁺) and PC-3 (PSMA⁻), and hepatocellular carcinoma cells HepG2 and HuH-7 using Western blot. C Cell uptake of LNCaP, PC-3, HepG2, and HuH-7 cells after 0.5, 1, 2 h of incubation with [⁶⁸Ga]Ga-PSMA-617 (n=4). D Blocking study with inhibitor ZJ-43 (n = 4). E HepG2 and F HuH-7 cell uptakes with or without co-administration of ZJ-43 (n = 4)

Fig. 2

Representative static PET images of [⁶⁸Ga]Ga-PSMA-617 in HCC subcutaneous xenograft models with two different tumor cell lines (HepG2 and HuH-7). White arrows point to the tumors

Fig. 3

Biodistribution of [⁶⁸Ga]Ga-PSMA-617 in HepG2 and HuH-7 subcutaneous xenograft models. A Biodistribution of [⁶⁸Ga] Ga-PSMA-617 in HepG2 subcutaneous xenograft models at 0.5, 1, 2 h post injection with and without co-administration of ZJ-43 as a blocking agent. B Tumor-to-muscle (T/M), tumor-to-blood (T/B), and tumor-to-liver (T/L) ratios at the indicated time points in HepG2 subcutaneous xenograft models. C Biodistribution of [⁶⁸Ga]Ga-PSMA-617 in HuH-7 subcutaneous xenograft models at 0.5, 1, 2 h post injection with and without co-administration of ZJ-43 as a blocking agent. D T/M, T/B, and T/L ratios at the indicated time points in HuH-7 subcutaneous xenograft models. The data are expressed as the mean ± SD (n = 4/group). (***P < 0.001; ****P < 0.0001)

Fig. 4

PET images of the [⁶⁸Ga-PSMA-617 in orthotopic xenograft models and biodistribution results. A Representative static PET imaging and B biodistribution of [⁶⁸Ga]Ga-PSMA-617 in HepG2 orthotopic xenograft models at 0.5 h post injection. C Tumor-to-blood (T/B), tumor-to-muscle (T/M), and tumor-to-liver (T/L) ratios at 0.5 h post injection. D Surgical exploration after PET/CT imaging and biodistribution. The data are expressed as the mean ± SD (n = 4/group). White arrows in A and blue arrow heads in D point to the tumor

Fig. 5

Representative static PET imaging of [⁶⁸Ga]Ga-PSMA-617 in HCC PDX-1, PDX-2, PDX-3, and PDX-4 models. White arrows point to the tumors

Fig. 6

Biodistribution of [⁶⁸Ga]Ga-PSMA-617 in HCC PDX-1, PDX-2, PDX-3, and PDX-4 models at 0.5, 1, and 2 h post injection. The data are expressed as the mean ± SD (n = 4/group). (***P < 0.001)

Fig. 7

T/M, T/B, and T/L ratios at the indicated time points in HCC PDX-1, PDX-2, PDX-3, and PDX-4 models. The data are expressed as the mean ± SD (n = 4/group)

Fig. 8

Histological staining analysis. A Staining of subcutaneous and orthotopic xenografts tissue sections with CD31- and PSMA-specific antibodies. Original magnification, ×400. B Staining of PDX tumor tissue sections with PSMA-specific antibodies indicated clear PSMA expression on the tumor vasculature, but not on the tumor cells. Original magnification, ×400