PROJECT SUMMARY Dynamic cell-cell interactions are crucial for healthy cell behavior and proper intercellular communication. Impaired intercellular communication has been implicated in the pathologies of various diseases including cancer, neurodegenerative diseases, bacterial and viral infections, autoimmune diseases, and cardiovascular diseases. As a result, probing cell-cell interactions is essential to many areas of biomedical research. It can lead to a more detailed understanding of various diseases and the development of novel therapeutic strategies, such as personalized immuno-oncology. However, current single-cell analysis techniques are slow and require potentially harmful physical contact with cells of interest, hindering the progress in elucidating these phenomena. Recently, we invented Harmonic Acoustics for Neighboring cell Dynamic studies (HANDs), an acoustic- based, automated, contact-free, cell-cell-pairing technology, which overcomes the key obstacles associated with the existing technologies. In this R01 project, we will develop and validate the HANDs platform with the following features: (1) Contactless nature and high biocompatibility: Instead of requiring direct contact with solid substrates or beads, the proposed HANDs technology is a contactless method. In addition, rather than exposure to large shear forces, strong pressures, or powerful optics, which can cause physiological damage, the cells in our setup are manipulated gently with low-power acoustic waves. The proposed HANDs platform allows long- term (>24 hours) cell-cell interaction studies. This feat cannot easily be achieved using existing state-of-the-art technologies such as atomic force spectroscopy. (2) High-throughput reversible cell-cell interactions and precise quantitative analysis at the single-cell level: The multi-trapping nature of the HANDs technology enables the simultaneous and parallel study of numerous (>20,000) cell pairs with single-cell precision. Existing single-cell techniques are either limited to studying a single pair of cells at any given time or lack the precision needed to control cell pairing and separation, and precise quantitative analysis. (3) Automated operation: Unlike existing cell pairing technologies which require complicated procedures and tools to achieve operation, the proposed technology automatically aligns cell-cell pairs using acoustic traps. Additionally, once the control signal is specified, cells can be brought into contact and separated in whatever automated and prescribed contact pattern is desired for testing. (4) High resolution (~100 nm): Using single-phase unidirectional transducers and harmonic frequency modulation, we will improve the spatial resolution of our HANDs technology from ~1 μm to ~100 nm. We will validate the performance of our HANDs platform across two well-established models: interactions between T cells and cancer cells, and interactions between stem cells and macrophages. In this regard, we aim to d...