Nanobubble-assisted ultrasound theragnostics in cellular immunotherapy for cancers

Abstract

With the unprecedented success of cellular immunotherapies in cancer treatment, there has been a growing endeavor in developing noninvasive monitoring and manipulation strategies for therapeutic immune cells, for broader applications of these potent therapeutics in well-managed manners. Popular modalities of physical energy transmission have been explored for the imaging and modulation purposes, including ionizing radiation, magnetism and optics. Ultrasound, as a versatile mechanical energy transmission modality, has been extensively used in clinical practices, covering applications from imaging to noninvasive interventions. However, the potentials of this ubiquitous modality have yet to be uncovered in cellular immunotherapies. The nanobubble (NB), gas-filled hollow nanostructures with various formulations, has recently brought substantial potentials into the field of ultrasound. With the assistance of NBs, the ultrasound modality, usually thought as macroscopic energy transmission modality, is now showing broadened potentials, especially on interacting with cellular targets with specificity. In this thesis, we describe strategies that, with the assistance of NBs, enable tracking and control of therapeutic immune cells by the ultrasound modality. We employ the novel biogenic NB, gas vesicles (GVs), as the acoustic tag and actuators for the implementation of cell tracking and mechanical switching strategies by ultrasound. To achieve noninvasive tracking of adoptive immune cells by ultrasound imaging, GVs are employed for the acoustic labeling of the cells. The GV surface is functionalized by streptavidin coating, which enables stable attachment of GVs on biotinylated cell surface via biotin-streptavidin conjugation. We show that the labeling is well-tolerated by the cells, with essential functionalities unaffected after the labeling. We demonstrate that, after ex vivo labeling with GVs, it is feasible to detect labeled cells by nonlinear contrast-enhanced ultrasound imaging both in vitro and in vivo. In mouse models of subcutaneous hepatocellular carcinoma, we further demonstrate the tracking of adoptive NK-92 cells by ultrasound imaging, showcasing a potential dynamic evaluation method for adoptive immune cells. We then demonstrate a mechanical switching strategy for the ultrasound control of genetic expression in engineered immune cells, utilizing GVs as cell-attached actuators. We show that surface-attached GVs enable induction of calcium influx in cells upon ultrasound stimulation, which further elicited transcriptional activities in calcium-centered signaling pathways, laying grounds for mechanical switching of genetic expression. We show in engineered NK-92 cells that the expression of target genes could be induced by the GV-actuated ultrasound stimulation. This demonstrates the feasibility of controlling genetic expression through ultrasound stimulation, indicating potentials of on-demand induction of pre-programmed therapeutic potencies in engineered cells in cellular therapies. Altogether, we demonstrate that, with the assistance of NBs, it is feasible to use the ultrasound modality for noninvasive tracking of adoptive immune cells, as well as for mechanical switching of genes. These establish a new role of the ultrasound modality in cellular therapies, with potentials in cost-effective post-treatment evaluation, therapy design and optimizations, as well as in implementing all-acoustic monitoring and manipulation on therapeutic cells. This also substantially expands the scope of ultrasound in theragnostic applications, paying the way for combining the ubiquitous ultrasound modality and the novel therapeutics.