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While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.

Three-dimensional (3D) optical imaging of whole biological organs with microscopic resolution has remained a challenge. Most versions of such imaging techniques require special preparation of the tissue specimen. Here we demonstrate microtomy-assisted photoacoustic microscopy (mPAM) of mouse brains and other organs, which automatically acquires serial distortion-free and registration-free images with endogenous absorption contrasts. Without tissue staining or clearing, mPAM generates micrometer-resolution 3D images of paraffin- or agarose-embedded whole organs with high fidelity, achieved by label-free simultaneous sensing of DNA/RNA, hemoglobins, and lipids. mPAM provides histology-like imaging of cell nuclei, blood vessels, axons, and other anatomical structures, enabling the application of histopathological interpretation at the organelle level to analyze a whole organ. Its deep tissue imaging capability leads to less sectioning, resulting in negligible sectioning artifact. mPAM offers a new way to better understand complex biological organs. The state-of-the-art three-dimensional biomedical imaging often requires specific tissue preparation that may alter the physical properties of the specimen causing loss of information. Here Wong et al. develop a microtomy-assisted photoacoustic microscopy that allows imaging of biological samples without labelling agents and with reduced sectioning.

Ultrasound imaging is a highly valuable tool in imaging human tissues due to its non-invasive and easily accessible nature. Despite advances in the field of ultrasound research, conventional transducers with frequencies lower than 20 MHz face limitations in resolution for cellular applications. To address this challenge, we employed ultrahigh frequency (UHF) transducers and demonstrated their potential applications in the field of biomedical engineering, specifically for cell imaging and acoustic tweezers. The lateral resolution achieved with a 110 MHz UHF transducer was 20 μm, and 6.5 μm with a 410 MHz transducer, which is capable of imaging single cells. The results of our experiments demonstrated the successful imaging of a single PC-3 cell and a 15 μm bead using an acoustic scanning microscope equipped with UHF transducers. Additionally, the dual-mode multifunctional UHF transducer was used to trap and manipulate single cells and beads, highlighting its potential for single-cell studies in areas such as cell deformability and mechanotransduction.

Efficient intracellular delivery of biologically active macromolecules has been a challenging but important process for manipulating live cells for research and therapeutic purposes. There have been limited transfection techniques that can deliver multiple types of active molecules simultaneously into single-cells as well as different types of molecules into physically connected individual neighboring cells separately with high precision and low cytotoxicity. Here, a high frequency ultrasound-based remote intracellular delivery technique capable of delivery of multiple DNA plasmids, messenger RNAs, and recombinant proteins is developed to allow high spatiotemporal visualization and analysis of gene and protein expressions as well as single-cell gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9), a method called acoustic-transfection. Acoustic-transfection has advantages over typical sonoporation because acoustic-transfection utilizing ultra-high frequency ultrasound over 150 MHz can directly deliver gene and proteins into cytoplasm without microbubbles, which enables controlled and local intracellular delivery to acoustic-transfection technique. Acoustic-transfection was further demonstrated to deliver CRISPR-Cas9 systems to successfully modify and reprogram the genome of single live cells, providing the evidence of the acoustic-transfection technique for precise genome editing using CRISPR-Cas9.

Single-cell analysis is essential to understand the physical and functional characteristics of cells. The basic knowledge of these characteristics is important to elucidate the unique features of various cells and causative factors of diseases and determine the most effective treatments for diseases. Recently, acoustic tweezers based on tightly focused ultrasound microbeam have attracted considerable attention owing to their capability to grab and separate a single cell from a heterogeneous cell sample and to measure its physical cell properties. However, the measurement cannot be performed while trapping the target cell, because the current method uses long ultrasound pulses for grabbing one cell and short pulses for interrogating the target cell. In this paper, we demonstrate that short ultrasound pulses can be used for generating acoustic trapping force comparable to that with long pulses by adjusting the pulse repetition frequency (PRF). This enables us to capture a single cell and measure its physical properties simultaneously. Furthermore, it is shown that short ultrasound pulses at a PRF of 167 kHz can trap and separate either one red blood cell or one prostate cancer cell and facilitate the simultaneous measurement of its integrated backscattering coefficient related to the cell size and mechanical properties.

High-resolution quantitative imaging of cerebral oxygen metabolism in mice is crucial for understanding brain functions and formulating new strategies to treat neurological disorders, but remains a challenge. Here, we report on our newly developed ultrasound-aided multi-parametric photoacoustic microscopy (PAM), which enables simultaneous quantification of the total concentration of hemoglobin (C Hb ), the oxygen saturation of hemoglobin (sO 2 ) and cerebral blood flow (CBF) at the microscopic level and through the intact mouse skull. The three-dimensional skull and vascular anatomies delineated by the dual-contrast (i.e., ultrasonic and photoacoustic) system provide important guidance for dynamically focused contour scan and vessel orientation-dependent correction of CBF, respectively. Moreover, bi-directional raster scan allows determining the direction of blood flow in individual vessels. Capable of imaging all three hemodynamic parameters at the same spatiotemporal scale, our ultrasound-aided PAM fills a critical gap in preclinical neuroimaging and lays the foundation for high-resolution mapping of the cerebral metabolic rate of oxygen (CMRO 2 )—a quantitative index of cerebral oxygen metabolism. This technical innovation is expected to shed new light on the mechanism and treatment of a broad spectrum of neurological disorders, including Alzheimer’s disease and ischemic stroke.

The clinical application of breast ultrasound for the assessment of cancer risk and of deep learning for the classification of breast-ultrasound images has been hindered by inter-grader variability and high false positive rates and by deep-learning models that do not follow Breast Imaging Reporting and Data System (BI-RADS) standards, lack explainability features and have not been tested prospectively. Here, we show that an explainable deep-learning system trained on 10,815 multimodal breast-ultrasound images of 721 biopsy-confirmed lesions from 634 patients across two hospitals and prospectively tested on 912 additional images of 152 lesions from 141 patients predicts BI-RADS scores for breast cancer as accurately as experienced radiologists, with areas under the receiver operating curve of 0.922 (95% confidence interval (CI) = 0.868–0.959) for bimodal images and 0.955 (95% CI = 0.909–0.982) for multimodal images. Multimodal multiview breast-ultrasound images augmented with heatmaps for malignancy risk predicted via deep learning may facilitate the adoption of ultrasound imaging in screening mammography workflows. An explainable deep-learning system prospectively predicts clinical scores for breast cancer risk from multimodal breast-ultrasound images as accurately as experienced radiologists.

Prostate cancer is the second leading cause of cancer death in men. Its current treatment includes various physical and chemical approaches for the localized and advanced prostate cancer [e.g. metastatic castrate resistant prostate cancer (mCRPC)]. Although many new drugs are now available for prostate cancer, none is suitable for local treatment that can reduce adverse effects often associated with the current physical treatment. Of the drugs approved by FDA for mCRPC, the best mean improvement in overall survival is only about 4.8 months. Therefore, there is a need for improved treatment approaches for prostate cancer, especially drug-resistant cancer. Ultrasound therapy represents a useful new physical approach for the drug-resistant cancer treatment by facilitating the entry of the related chemotherapy drug into the target cancer cells. There are two versions of ultrasound: High Intensity Focused Ultrasound (HIFU) and Low Intensity Pulsed Ultrasound (LIPUS). HIFU has been a promising treatment option for prostate cancer due to its noninvasiveness and various biological effects on cancer tissue. It has been approved for the treatment of cancer and in recent years there have been numerous findings suggesting HIFU can reduce cancer cell viability and possibly reverse the spread of cancerous tumors. LIPUS is currently being studied as an alternative treatment option for prostate cancer. Preliminary studies have found LIPUS to reduce cancer cell viability without the side effects seen in HIFU. Reversible cell membrane damage caused by LIPUS could allow increased uptake of anticancer drugs, enhancing cytotoxicity and death of cancer cells. In this way, a low dose of anticancer drug is more effective toward cancer cells while there is less damage to normal cells. The combination of LIPUS with certain chemotherapeutic agents can be an exciting physical-chemical combination therapy for prostate cancer. This review will focus on this topic as well as the clinical use of HIFU to provide an understanding of their current use and future potential role for prostate cancer therapy.

Ultrasound is extensively studied for biomedical engineering applications. As the core part of the ultrasonic system, the ultrasound transducer plays a significant role. For the purpose of meeting the requirement of precision medicine, the main challenge for the development of ultrasound transducer is to further enhance its performance. In this article, an overview of recent developments in ultrasound transducer technologies that use a variety of material strategies and device designs based on both the piezoelectric and photoacoustic mechanisms is provided. Practical applications are also presented, including ultrasound imaging, ultrasound therapy, particle/cell manipulation, drug delivery, and nerve stimulation. Finally, perspectives and opportunities are also highlighted.

Cell deformability is a useful feature for diagnosing various diseases (e.g., the invasiveness of cancer cells). Existing methods commonly inflict pressure on cells and observe changes in cell areas, diameters, or thickness according to the degree of pressure. Then, the Young’s moduli (i.e., a measure of deformability) of cells are estimated based on the assumption that the degrees of the changes are inversely proportional to Young’s moduli. However, manual measurements of the physical changes in cells are labor-intensive, and the subjectivity of the operators can intervene during this step, thereby causing considerable uncertainty. Further, because the shapes of cells are nonuniform, we cannot ensure the assumption for linear correlations of physical changes in cells with their deformability. Therefore, this study aims at measuring non-linear elastic moduli of live cells (degrees of cell deformability) automatically by employing conventional neural networks (CNN) and multilayer perceptrons (MLP) while preserving (or enhancing) the accuracy of the manual methods. First, we obtain photomicrographs of cells on multiple pressure levels using single-beam acoustic tweezers, and then, we suggest an image preprocessing method for emphasizing changes in cell areas on the photomicrographs. The CNN model is trained to measure the ratios of the cell area change at each pressure level. Then, we apply the multilayer perceptron (MLP) to learn the correlations of the cell area change ratios according to the pressure levels with cell deformability. The accuracy of the CNN was evaluated using two types of breast cancer cells: MDA-MB-231 (invasive) and MCF-7 (noninvasive). The MLP was assessed using five different beads (Young’s moduli from 0.214 to 9.235 kPa), which provides standardized reference data of the non-linear elastic moduli of live cells. Finally, we validated the practicality of the proposed system by examining whether the non-linear elastic moduli estimated by the proposed system can distinguish invasive breast cancer cells from noninvasive ones.