CXCR4-targeted ultrasound microbubbles for imaging and enhanced chemotherapy/Immunotherapy in liver cancer

Ultrasound molecular imaging is an innovative imaging modality that combines ultrasound with molecular probes to observe live biological processes at the cellular and molecular levels. C-X-C chemokine receptor type 4 (CXCR4) is a specific target in liver tumors and plays a crucial role in promoting tumor growth, invasion, metastasis, and angiogenesis. This study pioneered the use of CXCR4-targeted ultrasound molecular imaging for visualized antitumor therapy and investigated the potential of CXCR4-targeted microbubbles (MBs) in sensitizing liver tumor treatment. CXCR4-targeted MBs demonstrated high ligands conjugation efficiency to vascular endothelial cells (99.77 ± 0.15 %) and significantly inhibited the migration and invasion of Hepa1-6 cells. Molecular CEUS imaging results indicated that the MBs carrying LFC131 peptides facilitated site-specific recognition in BALB/c mice bearing Hep G2 tumors. After the 2-week of chemotherapy, ultrasound molecular imaging signals were significantly reduced in liver cancer when using CXCR4-targeted MBs compared to the SonoVue group which were corroborated by quantitative immunohistochemical grading of CXCR4 expression. In liver cancer immunotherapy, the anti-PD-L1 mAb + CXCR4-targeted MBs group yielded a remarkable tumor inhibition rate (94.6 %) with increased CD8+ T-cell infiltration and decreased FOXP3+ regulatory T cells. Bulk RNA-seq analysis and animal experiment confirmed that anti-PD-L1 mAb combined with CXCR4-targeted MBs effectively induced a robust immune response in liver cancer. These findings establish a solid foundation for future molecular CEUS imaging applications and the development of sensitization strategies for liver cancer therapy. STATEMENT OF SIGNIFICANCE: Ultrasound molecular imaging plays a pivotal role in advancing precision medicine by optimizing tumor diagnosis and treatment. This study pioneers ultrasound molecular imaging in liver tumor therapy using CXCR4-targeted microbubbles (MBs) conjugated with LFC131 peptides. Achieving 99.77 % ligand binding efficiency, the CXCR4-targeted MBs group suppressed tumor migration and enabled precise molecular imaging validated by immunohistochemistry. Moreover, the integration of CXCR4-targeted MBs with anti-PD-L1 immunotherapy resulted in a remarkable tumor inhibition rate of 94.6 %, accompanied by increased CD8+ T cells and decreased FOXP3+ regulatory T cells. These findings underscore the dual role of CXCR4-targeted MBs in both imaging and enhancing chemotherapy/immunotherapy, establishing a foundational framework for the future advancement of molecular imaging-guided liver cancer treatment.