Perfluorobutane-enhanced US Targeting M2 Tumor-associated Macrophages for Predicting Programmed Cell Death-1 Response in Hepatocellular Carcinoma.

Purpose To evaluate the potential of perfluorobutane-enhanced US as a predictive biomarker for anti-programmed cell death (PD)-1 treatment response by targeting alternatively activated phenotype (M2)-polarized tumor-associated macrophages (TAMs) in hepatocellular carcinoma (HCC). Materials and Methods This study was conducted from June 2021 to February 2025. Male animal models included 4-week-old C57BL/6 mice for HCC models and 2-3-month-old New Zealand white rabbits for VX2 liver tumor models. The phagocytic capacity of M2-polarized TAMs for perfluorobutane microbubbles was evaluated in vitro using transmission microscopy to confirm selective microbubble uptake. In vivo, HCC models were established, including mice with primary HCC induced by Akt/N-ras oncogenes, an orthotopic intrahepatic transplantation mouse model, and a VX2 metastatic tumor rabbit model. Targeted imaging of M2-TAMs in these models was performed using perfluorobutane microsphere-based contrast-enhanced US, and the presence of hyperenhanced rims during the postvascular phase was analyzed to assess their association with M2-TAM accumulation. In addition, an orthotopic liver transplant mouse model was developed, in which contrast-enhanced US was used to depict hyperenhanced rims and analyze their correlation with M2-TAM distribution and the efficacy of anti-PD-1 therapy. Results In vitro studies demonstrated predominant uptake of perfluorobutane microbubbles by M2-TAMs, with 92% (46 of 50) of U937-derived M2 macrophages engulfing the microspheres compared with 0% of M1 macrophages ( < .001). In vivo, the observation of hyperenhanced rims during the postvascular phase of contrast-enhanced US was associated with the presence of M2-TAMs. In orthotopic liver transplant mouse models receiving anti-PD-1 treatment, tumor progression differed between the hyperenhanced rim-positive and rim-negative groups ( < .05). Conclusion Perfluorobutane microbubble-based contrast-enhanced US, which targets M2-TAMs, presents a promising method for predicting the response to anti-PD-1 therapy in HCC. Contrast-enhanced Ultrasound, Tumor-associated Macrophages, Anti-PD-1 Treatment, Hepatocellular Carcinoma © RSNA, 2025.