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 3h 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. This article, for the first time, demonstrates an ultrasound imaging-based cell tracking strategy for tracking systematically infused adoptive natural killer cells. The proposed strategy enabled the dynamical monitoring of tumor recruitment of adoptive NK cells, as well as evaluating the boosting effects when immune adjuvant co-treatment was applied. As a radiation-free and cost-effective imaging modality, the ultrasound is an appealing candidate for tracking adoptive immune cells with its extensive use in clinical practices including real-time image-guided administration of therapeutic cells. This article has shown the capability of ultrasound imaging in tracking adoptive immune cells in cellular immunotherapy, introducing the versatile modality into the field of immunotherapy with broad prospective in early assessment and on-demand image-guided interventions.