SSTR2-Targeted Theranostics in Hepatocellular Carcinoma

Abstract

Background: While the clinical use of radiolabeled somatostatin analogs is well established in neuroendocrine tumors, there is growing interest in expanding their application to other somatostatin receptor 2 (SSTR2)-expressing cancers. This study investigates the potential utility of SSTR2-targeted theranostics in hepatocellular carcinoma (HCC).

Methods: SSTR2 expression in HCC cell lines and clinical samples was evaluated using qRT-PCR, Western blot analysis, and a public dataset. ⁶⁷Ga-DOTATATE uptake was measured, ¹⁷⁷Lu-DOTATATE cytotoxicity was assessed, and ⁶⁸Ga-DOTATATE tumor targeting was evaluated in HCC animal models and a patient via PET/CT imaging.

Results: SSTR2 expression was confirmed in HCC cell lines and clinical samples. Radioligand uptake studies demonstrated SSTR2-mediated ⁶⁷Ga-DOTATATE uptake. ¹⁷⁷Lu-DOTATATE treatment reduced cell proliferation and enhanced the anti-tumor efficacy of the multikinase inhibitor sorafenib. ⁶⁸Ga-DOTATATE PET/CT scans successfully identified tumors in HCC animal models and spinal metastases in a patient with HCC.

Conclusion: These findings provide evidence that SSTR2-based theranostics could have significant implications for the detection and treatment of HCC.

Keywords: SSTR2; hepatocellular carcinoma; theranostics.

Figure 1

SSTR2 amplification and expression in HCC. (A) SSTR2 is amplified and/or overexpressed in a significant proportion of HCC cases, based on data from the TCGA HCC dataset [20]. (B) Comparative analysis of SSTR2 protein expression in HCC patients versus other solid tumor types, derived from the Human Protein Atlas dataset (https://www.proteinatlas.org), accessed on 9 January 2024. (C,D) IHC staining for SSTR2 in an HCC patient (Patient ID #2766) compared with a prostate adenocarcinoma patient (Patient ID #3304), who shows negative SSTR2 staining. Images are from the Human Protein Atlas, with staining conducted using anti-SSTR2 antibody (Sigma, #HPA007264).

Figure 2

(A) Relative mRNA expression levels of SSTR2 in HCC cell lines measured by qRT-PCR. Data represent the mean ± SD from three independent experiments, each conducted in triplicate. Gene expression was normalized to GAPDH for each cell line and further normalized to HepG2 cells. (B) Western blot analysis of SSTR2 protein expression in HCC cell lines, with comparison to NCI-H69 small cell lung cancer cells. β-actin was used as a loading control. (C) Uptake of ⁶⁷Ga-DOTATATE in HCC cell lines relative to NCI-H69 and BON-1 cells, with receptor blocking by 100X octreotide. Data were analyzed using two-way ANOVA followed by Šídák’s multiple comparisons test, with statistical significance indicated by * p < 0.05 and **** p < 0.0001. ns indicates not significant. (D) Anti-proliferative effects of ¹⁷⁷Lu-DOTATATE in HCC cell lines assessed by WST-1 cell proliferation assay (Sigma), with NCI-H69 cells serving as a benchmark. Data represent the mean ± SD from three independent experiments, each performed in triplicate.

Figure 3

(A) Anti-proliferative effects of the anti-angiogenic agents lenvatinib, sorafenib, and cabozantinib on HCC cell lines. Cells were treated with increasing concentrations of these drugs (in µM) for 48 h, and cell proliferation was assessed using the WST-1 assay. (B,C) Synergistic interaction between ¹⁷⁷Lu-DOTATATE and sorafenib in SNU449 cells. Co-treatment with sorafenib (0.01–5 µM) and ¹⁷⁷Lu-DOTATATE (0.1, 0.5, and 1 megabecquerels (MBq)/mL) for 48 h resulted in an IC₅₀ shift and synergistic effects, as determined by Bliss synergy analysis. Cell viability was measured using the WST-1 assay.

Figure 4

(A) Maximum-intensity projection showing tumor-specific ⁶⁸Ga-DOTATATE uptake in subcutaneous models of Huh7 and SNU449. Mice were i.v. injected with 7.4 MBq of ⁶⁸Ga-DOTATATE into the tail vein and PET/CT imaging was performed at 1 h p.i. (B) Quantification of radioactive biodistribution in subcutaneous models of Huh7, SNU449, and HCT116. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test, with statistical significance indicated by * p < 0.05 and **** p < 0.0001. ns indicates not significant. (C) Corresponding IHC analysis showing SSTR2 expression in FFPE tumor sections from the mice with the HCC tumors. The tumor from the HCT116-bearing mice was used as the negative control. The scale bar is 200 μm.

Figure 5

A ⁶⁸Ga-DOTATATE PET/CT scan showing SSTR2 positivity in spinal metastases from a patient with HCC. The blue arrow indicates the location of the spinal metastases originating from the liver.