Tracking adoptive natural killer cells via ultrasound imaging assisted with nanobubbles

The recent years has witnessed an exponential growth in the field of natural killer (NK) cell-based immunotherapy for cancer treatment. As a prerequisite to precise evaluations and on-demand interventions, the noninvasive tracking of adoptive NK cells plays a crucial role not only in post-treatment monitoring, but also in offering opportunities for preclinical studies on therapy optimizations. Here, we describe an NK cell tracking strategy for cancer immunotherapy based on ultrasound imaging modality. Nanosized ultrasound contrast agents, gas vesicles (GVs), were surface-functionalized to label NK cells. Unlike traditional microbubble contrast agents, nanosized GVs with their unique thermodynamical stability enable the detection of labeled NK cells under nonlinear contrast-enhanced ultrasound (nCEUS), without a noticeable impact on cellular viability or migration. By such labeling, we were able to monitor the trafficking of systematically infused NK cells to a subcutaneous tumor model. Upon co-treatment with interleukin (IL)-2, we observed a rapid enhancement in NK cell trafficking at the tumor site as early as 3 h post-infusion. Altogether, we show that the proposed ultrasound-based tracking strategy is able to capture the dynamical changes of cell trafficking in NK cell-based immunotherapy, providing referencing information for early-phase monotherapy evaluation, as well as understanding the effects of modulatory co-treatment. Statement of significance: In cellular immunotherapies, the post-infusion monitoring of the living therapeutics has been challenging. Several popular imaging modalities have been explored the monitoring of the adoptive immune cells, evaluating their trafficking and accumulation in the tumor. Here we demonstrated, for the first time, the ultrasound imaging-based immune cell tracking strategy. We showed that the acoustic labeling of adoptive immune cells was feasible with nanosized ultrasound contrast agents, overcoming the size and stability limitations of traditional microbubbles, enabling dynamical tracking of adoptive natural killer cells in both monotherapy and synergic treatment with cytokines. This article introduced the cost-effective and ubiquitous ultrasound imaging modality into the field of cellular immunotherapies, with broad prospectives in early assessment and on-demand image-guided interventions.