Preclinical evaluation of radiolabeled tissue factor-targeted peptide for theranostics of hepatocellular carcinoma post percutaneous ethanol injection

Rationale: Tissue factor (TF) initiates local blood clotting and infiltration of tumor-associated macrophages, leading to tumor recurrence post-local ablation. Our study addressed inefficient cancer cell killing and immunosuppressive macrophage infiltration after percutaneous ethanol injection (PEI) in hepatocellular carcinoma (HCC). We evaluated the feasibility of ¹⁸F-radiolabeled polypeptide TF-targeted radioligand (tTF) as a PET tracer for assessing tumor response. We also explored the efficacy and safety of ¹⁷⁷Lu-radiolabeled tTF to eradicate residual tumors and tumor-associated macrophages. Methods: TF expression in the locally treated human HCC was assessed. Biodistribution, pharmacokinetics, and TF-targeted specificity of Al¹⁸F-NOTA-tTF were investigated in Kunming (KM) and/or Hepa1-6 mice. Evaluation of FDG/tTF PET imaging, histopathological characteristics, and tumor ablation response was conducted using two incomplete PEI ablation models, with ethanol volumes equivalent to 50% (high-dose (HD) PEI group) or 25% (low-dose (LD) PEI group) of the tumor volume administered. Following PEI, a single dose of ¹⁷⁷Lu-DOTA-tTF was administered on day 1 to assess its efficacy in eradicating residual tumors and immunosuppressive macrophages. Systemic toxicity was evaluated through blood analysis and histological examination of healthy organs. Results: Immunohistochemistry analysis demonstrated elevated TF expression around the ablation margin of residual tissue in human HCC. Radiolabeled tTF exhibited excellent TF-specificity, water solubility, and stability. FDG PET imaging and histological analysis showed tumor recurrence, upregulation of immunosuppressive macrophages, and TF around tumor foci post-treatment in the HD PEI-treated group. Meanwhile, the uptake of ¹⁸F-FDG exhibited a decline, while the uptake of Al¹⁸F-NOTA-tTF showed an increase in both the HD and LD PEI groups, as observed on day 1 and day 6 post-PEI. These results indicated that increased tTF uptake offers a specific and durable avenue for targeted theranostic applications. Following PEI, ¹⁷⁷Lu-DOTA-tTF therapy demonstrated significant tumor suppression and eradication of immunosuppressive macrophages compared to control groups. Safety assessments indicated no significant toxicity in the main organs of tested animals. Conclusions: Al¹⁸F-NOTA-tTF is a promising PET tracer for assessing ablated HCC, while ¹⁷⁷Lu-DOTA-tTF provides an effective tool for inhibiting residual tumor growth and immunosuppressive macrophages post-PEI. Significantly, TF-targeting theranostics may help overcome incomplete cancer cell killing and formation of tumor immunosuppressive microenvironment, offering a promising strategy for effective HCC ablation in future clinical practice.