Ultrasound Molecular Imaging and Visualization of Immune Biomarkers: A New Paradigm for Tumor Immunotherapy Response...

Cancer immunotherapy has revolutionized the treatment landscape, yet its clinical efficacy is limited by modest objective response rates and the emergence of atypical response patterns such as pseudoprogression and hyperprogression. Conventional RECIST criteria relying on anatomical size changes and invasive tissue biopsies suffer from prominent sampling bias and cannot dynamically reflect the heterogeneous tumor immune microenvironment (TIME), creating an urgent demand for non-invasive, real-time functional imaging tools to longitudinally profile intra-tumoral immune landscapes. Ultrasound molecular imaging (USMI) stands out as a distinctive imaging modality complementary to PET-CT and MRI, featuring radiation-free operation, low cost, superior spatiotemporal resolution and repeatable whole-tumor visualization-advantages that overcome the limitations of ionizing radiation, high expense and static single-spot sampling inherent to mainstream molecular imaging modalities. This review systematically elaborates state-of-the-art advances in USMI for visualizing tumor immune biomarkers, with in-depth dissection of core acoustic imaging mechanisms, rational design and multi-functional optimization strategies of immune-targeted microbubble/nanobubble probes and comprehensive collation of landmark pre-clinical investigations across melanoma, hepatocellular carcinoma, non-small cell lung cancer, colorectal and breast cancers. We thoroughly correlate USMI signal readouts with pathological immunohistochemistry, transcriptomic profiles and longitudinal immunotherapy outcomes, and elaborate on its core translational applications: dynamic tracking of immune cell infiltration and spatial distribution, quantitative mapping of global immune checkpoint expression, early prediction of therapeutic efficacy and differential diagnosis of pseudoprogression, hyperprogression and true tumor progression. We further highlight the inherent uniqueness of USMI for TIME surveillance and its complementary value relative to PET/MRI and objectively dissect critical translational bottlenecks, including probe off-target binding, insufficient standardized quantitative pipelines and deep-tissue ultrasound attenuation. Rather than overstating preliminary exploratory work, we rationally discuss the synergistic integration of USMI with multi-omics and artificial intelligence radiomics as a forward-looking developmental direction and propose theranostic probe engineering and standardized multi-center validation frameworks to accelerate clinical translation. This review constructs a complete theoretical and technical framework positioning USMI as a novel functional assessment paradigm for tumor immunotherapy, clarifies its irreplaceable strengths in immune molecular imaging and provides targeted insights to advance precision tumor immunotherapy evaluation.