Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.

Engineering Heterogeneous Tumor Models for Biomedical Applications.

Tumor tissue engineering holds great promise for replicating the physiological and behavioral characteristics of tumors in vitro. Advances in this field have led to new opportunities for studying the tumor microenvironment and exploring potential anti-cancer therapeutics. However, the main obstacle to the widespread adoption of tumor models is the poor understanding and insufficient reconstruction of tumor heterogeneity. In this review, the current progress of engineering heterogeneous tumor models is discussed. First, the major components of tumor heterogeneity are summarized, which encompasses various signaling pathways, cell proliferations, and spatial configurations. Then, contemporary approaches are elucidated in tumor engineering that are guided by fundamental principles of tumor biology, and the potential of a bottom-up approach in tumor engineering is highlighted. Additionally, the characterization approaches and biomedical applications of tumor models are discussed, emphasizing the significant role of engineered tumor models in scientific research and clinical trials. Lastly, the challenges of heterogeneous tumor models in promoting oncology research and tumor therapy are described and key directions for future research are provided.

Acoustofluidic assembly of primary tumor-derived organotypic cell clusters for rapid evaluation of cancer immunotherapy.

Cancer immunotherapy shows promising potential for treating breast cancer. While patients may have heterogeneous treatment responses for adjuvant therapy, it is challenging to predict an individual patient's response to cancer immunotherapy. Here, we report primary tumor-derived organotypic cell clusters (POCCs) for rapid and reliable evaluation of cancer immunotherapy. By using a label-free, contactless, and highly biocompatible acoustofluidic method, hundreds of cell clusters could be assembled from patient primary breast tumor dissociation within 2 min. Through the incorporation of time-lapse living cell imaging, the POCCs could faithfully recapitulate the cancer-immune interaction dynamics as well as their response to checkpoint inhibitors. Superior to current tumor organoids that usually take more than two weeks to develop, the POCCs can be established and used for evaluation of cancer immunotherapy within 12 h. The POCCs can preserve the cell components from the primary tumor due to the short culture time. Moreover, the POCCs can be assembled with uniform fabricate size and cell composition and served as an open platform for manipulating cell composition and ratio under controlled treatment conditions with a short turnaround time. Thus, we provide a new method to identify potentially immunogenic breast tumors and test immunotherapy, promoting personalized cancer therapy.

Activation of MAP2K signaling by genetic engineering or HF-rTMS promotes corticospinal axon sprouting and functional regeneration.

Facilitating axon regeneration in the injured central nervous system remains a challenging task. RAF-MAP2K signaling plays a key role in axon elongation during nervous system development. Here, we show that conditional expression of a constitutively kinase-activated BRAF in mature corticospinal neurons elicited the expression of a set of transcription factors previously implicated in the regeneration of zebrafish retinal ganglion cell axons and promoted regeneration and sprouting of corticospinal tract (CST) axons after spinal cord injury in mice. Newly sprouting axon collaterals formed synaptic connections with spinal interneurons, resulting in improved recovery of motor function. Noninvasive suprathreshold high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) activated the BRAF canonical downstream effectors MAP2K1/2 and modulated the expression of a set of regeneration-related transcription factors in a pattern consistent with that induced by BRAF activation. HF-rTMS enabled CST axon regeneration and sprouting, which was abolished in MAP2K1/2 conditional null mice. These data collectively demonstrate a central role of MAP2K signaling in augmenting the growth capacity of mature corticospinal neurons and suggest that HF-rTMS might have potential for treating spinal cord injury by modulating MAP2K signaling.

Microfluidics guided by deep learning for cancer immunotherapy screening.

Immunocyte infiltration and cytotoxicity play critical roles in both inflammation and immunotherapy. However, current cancer immunotherapy screening methods overlook the capacity of the T cells to penetrate the tumor stroma, thereby significantly limiting the development of effective treatments for solid tumors. Here, we present an automated high-throughput microfluidic platform for simultaneous tracking of the dynamics of T cell infiltration and cytotoxicity within the 3D tumor cultures with a tunable stromal makeup. By recourse to a clinical tumor-infiltrating lymphocyte (TIL) score analyzer, which is based on a clinical data-driven deep learning method, our platform can evaluate the efficacy of each treatment based on the scoring of T cell infiltration patterns. By screening a drug library using this technology, we identified an epigenetic drug (lysine-specific histone demethylase 1 inhibitor, LSD1i) that effectively promoted T cell tumor infiltration and enhanced treatment efficacy in combination with an immune checkpoint inhibitor (anti-PD1) in vivo. We demonstrated an automated system and strategy for screening immunocyte-solid tumor interactions, enabling the discovery of immuno- and combination therapies.

Understanding Immune-Driven Brain Aging by Human Brain Organoid Microphysiological Analysis Platform.

The aging of the immune system drives systemic aging and the pathogenesis of age-related diseases. However, a significant knowledge gap remains in understanding immune-driven aging, especially in brain aging, due to the limited current in vitro models of neuroimmune interaction. Here, the authors report the development of a human brain organoid microphysiological analysis platform (MAP) to discover the dynamic process of immune-driven brain aging. The organoid MAP is created by 3D printing that confines organoid growth and facilitates cell and nutrition perfusion, promoting organoid maturation and their committment to forebrain identity. Dynamic rocking flow is incorporated into the platform that allows to perfuse primary monocytes from young (20 to 30-year-old) and aged (>60-year-old) donors and culture human cortical organoids to model neuroimmune interaction. The authors find that the aged monocytes increase infiltration and promote the expression of aging-related markers (e.g., higher expression of p16) within the human cortical organoids, indicating that aged monocytes may drive brain aging. The authors believe that the organoid MAP may provide promising solutions for basic research and translational applications in aging, neural immunological diseases, autoimmune disorders, and cancer.

Evaluation of cancer immunotherapy using mini-tumor chips.

Rationale: Predicting tumor responses to adjuvant therapies can potentially help guide treatment decisions and improve patient survival. Currently, tumor pathology, histology, and molecular profiles are being integrated into personalized profiles to guide therapeutic decisions. However, it remains a grand challenge to evaluate tumor responses to immunotherapy for personalized medicine. Methods: We present a microfluidics-based mini-tumor chip approach to predict tumor responses to cancer immunotherapy in a preclinical model. By uniformly infusing dissociated tumor cells into isolated microfluidic well-arrays, 960 mini-tumors could be uniformly generated on-chip, with each well representing the ex vivo tumor niche that preserves the original tumor cell composition and dynamic cell-cell interactions and autocrine/paracrine cytokines. Results: By incorporating time-lapse live-cell imaging, our mini-tumor chip allows the investigation of dynamic immune-tumor interactions as well as their responses to cancer immunotherapy (e.g., anti-PD1 treatment) in parallel within 36 hours. Additionally, by establishing orthotopic breast tumor models with constitutive differential PD-L1 expression levels, we showed that the on-chip interrogation of the primary tumor's responses to anti-PD1 as early as 10 days post tumor inoculation could predict the in vivo tumors' responses to anti-PD1 at the endpoint of day 24. We also demonstrated the application of this mini-tumor chip to interrogate on-chip responses of primary tumor cells isolated from primary human breast and renal tumor tissues. Conclusions: Our approach provides a simple, quick-turnaround solution to measure tumor responses to cancer immunotherapy.

Rapid Profiling of Tumor-Immune Interaction Using Acoustically Assembled Patient-Derived Cell Clusters.

Tumor microenvironment crosstalk, in particular interactions between cancer cells, T cells, and myeloid-derived suppressor cells (MDSCs), mediates tumor initiation, progression, and response to treatment. However, current patient-derived models such as tumor organoids and 2D cultures lack some essential niche cell types (e.g., MDSCs) and fail to model complex tumor-immune interactions. Here, the authors present the novel acoustically assembled patient-derived cell clusters (APCCs) that can preserve original tumor/immune cell compositions, model their interactions in 3D microenvironments, and test the treatment responses of primary tumors in a rapid, scalable, and user-friendly manner. By incorporating a large array of 3D acoustic trappings within the extracellular matrix, hundreds of APCCs can be assembled within a petri dish within 2 min. Moreover, the APCCs can preserve sensitive and short-lived (≈1 to 2-day lifespan in vivo) tumor-induced MDSCs and model their dynamic suppression of T cell tumor toxicity for up to 24 h. Finally, using the APCCs, the authors succesully model the combinational therapeutic effect of a multi-kinase inhibitor targeting MDSCs (cabozantinib) and an anti-PD-1 immune checkpoint inhibitor (pembrolizumab). The novel APCCs may hold promising potential in predicting treatment response for personalized cancer adjuvant therapy as well as screening novel cancer immunotherapy and combinational therapy.

Acoustic Bioprinting of Patient-Derived Organoids for Predicting Cancer Therapy Responses.

Cancer models, which are biologically representative of patient tumors, can predict the treatment responses and help determine the most appropriate cancer treatment for individual patients. Here, a point-of-care testing system called acoustically bioprinted patient-derived microtissues (PDMs) that can model cancer invasion and predict treatment response in individual patients with colorectal cancer (CRC), is reported. The PDMs are composed of patient-derived colorectal tumors and healthy organoids which can be precisely arranged by acoustic bioprinting approach for recapulating primary tissue's architecture. Particularly, these tumor organoids can be efficiently generated and can apprehend histological, genomic, and phenotypical characteristics of primary tumors. Consequently, these PDMs allow physiologically relevant in vitro drug (5-fluorouracil) screens, thus predicting the paired patient's responses to chemotherapy. A correlation between organoid invasion speed and normalized spreading speed of the paired patients is further established. It provides a quantitative indicator to help doctors make better decisions on ultimate anus-preserving operation for extremely low CRC patients. Thus, by combing acoustic bioprinting and organoid cultures, this method may open an avenue to establish complex 3D tissue models for precision and personalized medicine.

Clinical application of indocyanine green fluorescence navigation technology to determine the safe margin of advanced oral squamous cell carcinoma.

Background: Advanced oral squamous cell carcinoma (OSCC) has large lesions and deep infiltration, and the control of safe surgical margins was difficult. If residual tumor remains after incomplete tumor resection, it can lead to local tumor recurrence or even distant metastasis. This study sought to investigate the clinical application of indocyanine green (ICG)-based near-infrared fluorescence (NIF) molecular imaging in the intraoperative detection of surgical margins of advanced OSCC. Methods: Twenty-nine patients with advanced OSCC treated at the First Ward of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital were divided into the ICG group and non-ICG group. In the ICG group, the tumors were removed with the assistance of ICG fluorescence navigation technology. In the non-ICG group, the tumors were removed with conventional methods, and the cutting-edge tissues of the two groups underwent frozen biopsies. The margin abnormality rates were calculated and compared. Results: Under the excitation of NIF in the ICG group, tumor fluorescence development was observable in all lesions, and the tumor boundary was clear. The abnormal rates of the incisional margin in the ICG group and non-ICG group were 0.78% and 6.25%, respectively (P<0.05). Conclusions: ICG-mediated NIF imaging technology provides a new method for observing and completely resecting tumors under direct vision during operation, and finding residual tumors at the cutting edge in time. These results will inform the treatment of advanced OSCC.

Modeling cancer metastasis using acoustically bio-printed patient-derived 3D tumor microtissues.

Cancer metastasis causes most cancer-related deaths, and modeling cancer invasion holds potential in drug discovery and companion diagnostics. Although 2D cocultures have been developed to study cancer invasion, it is challenging to recreate the 3D cancer invasion of an individual cancer patient. Here, we report an acoustic bioprinting technology that can precisely construct tumor microtissues for modeling cancer invasion in 3D. By using acoustic droplet technology, we can precisely encapsulate cancer associated fibroblasts (CAFs) derived from a colorectal cancer patient into gel droplets and print them into a 3D CAF microtissue. After depositing a tumor organoid derived from the same patient, our 3D bio-printed microtissue can be used to model cancer cell migration and invasion from the tumor organoid to the 3D CAF microtissue. We further used 3D bio-printed microtissues to investigate cancer invasion dynamics as well as their treatment response using time-lapse imaging. Thus, our acoustic 3D bioprinting technology can be widely used for establishing various microtissues for modeling cancer invasion and other diseases, highlighting its potential in personalized treatment.

Identification and Validation of PLOD2 as an Adverse Prognostic Biomarker for Oral Squamous Cell Carcinoma.

BACKGROUND: Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), a key enzyme that catalyzes the hydroxylation of lysine, plays a crucial role in the progression of several solid tumors. However, its spatial expression profile and prognostic significance in oral squamous cell carcinoma (OSCC) have not been revealed. MATERIALS: Mass spectrometry was used to explore amino acid perturbations between OSCC tumor tissues and paired normal tissues of 28 patients. Then, PLOD2 mRNA and protein levels were assessed using several public databases and 18 pairs of OSCC patients' tissues. Additionally, PLOD2 spatial expression profiles were investigated in 100 OSCC patients by immunohistochemistry and its diagnostic and prognostic values were also evaluated. Lastly, gene set enrichment analysis (GSEA) was used to investigate the potential functions of PLOD2 in OSCC. RESULTS: Lysine was significantly elevated in OSCC tissues and could effectively distinguish tumor from normal tissues (AUC = 0.859, p = 0.0035). PLOD2 mRNA and protein levels were highly increased in tumor tissues of head and neck squamous cell carcinoma (HNSCC) (p < 0.001) and OSCC compared with those in nontumor tissues (p < 0.001). Histopathologically, PLOD2 was ubiquitously expressed in tumor cells (TCs) and fibroblast-like cells (FLCs) of OSCC patients but absent in tumor-infiltrating lymphocytes (TILs). Patients with highly expressed PLOD2 in TCs (PLOD2TCs) and FLCs (PLOD2FLCs) showed poor differentiation, a worse pattern of invasion (WPOI) and more lymph node metastasis (LNM), contributing to higher postoperative metastasis risk and poor survival time. However, PLOD2FLCs rather than PLOD2TCs was an independent risk factor for survival outcomes in OSCC patients. Molecularly, GSEA demonstrated highly expressed PLOD2 was mainly enriched in epithelial-mesenchymal transformation (EMT), TGF-beta signaling and hypoxia pathway, which are associated with poor clinical outcomes of OSCC patients. CONCLUSIONS: PLOD2 was a poor prognostic biomarker for OSCC patients and may affect the metastasis of OSCC through EMT pathway. These findings might shed novel sights for future research in PLOD2 targeted OSCC therapy.

For nasotracheal intubation, which nostril results in less epistaxis: right or left?: A systematic review and meta-analysis.

BACKGROUND: Nasotracheal intubation is usually required in patients undergoing oromaxillofacial, otolaryngological or plastic surgery to prevent the airway encroaching into the operating field. Epistaxis is the most common complication, but which nostril is associated with a lower incidence and severity of epistaxis is still unclear. OBJECTIVE: When both nostrils are patent, to determine the preferred nostril for nasotracheal intubation under general anaesthesia. DESIGN: A systematic review and meta-analysis of randomised controlled trials (RCTs). The primary outcome was the incidence of epistaxis and the secondary outcomes included the incidence of severe epistaxis, the time required to pass the tube through the nasal passage and total intubation time. DATA SOURCES: PubMed, Embase and the Cochrane Register of Controlled Trials were searched from database inception to 1 March 2020. ELIGIBILITY CRITERIA: The only studies included were RCTs comparing epistaxis related to nasotracheal intubation via right or left nostril, in adult surgery patients undergoing general anaesthesia. RESULTS: Ten RCTs with 1658 patients were included. Compared with the left nostril, intubation via the right nostril was associated with a significantly lower incidence of epistaxis: risk ratio (RR) and 95% confidence intervals (CI) were 0.78 (0.62 to 0.99), P = 0.04: a lower incidence of severe epistaxis (five studies, n=923), RR 0.40 (0.22 to 0.75), P = 0.004: and a shorter intubation time (three studies, n=345), mean difference -7.28 (-14.40 to -0.16) seconds, P = 0.05. In two studies (n=310), no significant difference between the right and left nostril was observed in the time to pass the tube through the nasal passages, mean difference -0.59 (-1.95 to 0.77) s, P = 0.40. CONCLUSION: On the basis of the current available evidence, when both nostrils are patent, the right nostril is more appropriate for nasotracheal intubation, with a lower incidence and severity of epistaxis and faster intubation time. TRIAL REGISTRATION: The study protocol has been registered in PROSPERO (CRD42020169949).

Acoustic Droplet Printing Tumor Organoids for Modeling Bladder Tumor Immune Microenvironment within a Week.

Current organoid models are limited by the incapability of rapidly fabricating organoids that can mimic the immune microenvironment for a short term. Here, an acoustic droplet-based platform is presented to facilitate the rapid formation of tumor organoids, which retains the original tumor immune microenvironment and establishes a personalized bladder cancer tumor immunotherapy model. In combination with a hydrophobic substrate, the acoustic droplet printer can yield a large number of homogeneous and highly viable bladder tumor organoids in vitro within a week. The generated organoids consist of all components of bladder tumor, including diverse immune elements and tumor cells. By coculturing tumor organoids with autologous immune cells for 2 days, tumor reactive T cells are induced in vitro. Furthermore, it is also demonstrated that these tumor-reactive T cells can also enhance the killing efficiency of matched organoids. Because of the easy operation, repeatability, and stability, the proposed acoustic droplet platform will provide a reliable approach for personalized tumor immunotherapy.

Scaffold-free generation of heterotypic cell spheroids using acoustofluidics.

3D cell cultures such as cell spheroids are widely used for tissue engineering, regenerative medicine, and translational medicine, but challenges remain in recapitulating the architectural complexity and spatiotemporal heterogeneity of tissues. Thus, we developed a scaffold-free and versatile acoustofluidic device to fabricate heterotypic cell spheroids with complexity over cell architectures and components. By varying the concentrations of cell suspension, we can precisely control the size of spheroids aggregated by a contact-free acoustic radiation force. By tuning the cell components including tumor cells, fibroblasts, and endothelial cells, heterotypic spheroids were controllably fabricated. These heterotypic spheroids can be used as a proof-of concept to model the spatial organization of tumor tissues. We demonstrated that the assembled components can self-assemble into layered structures as instructed by their cadherin expression. Finally, we demonstrated the acoustic assembly of mouse mammary gland components into spheroids and observed their maturation in culture. To conclude, we developed an acoustofluidic platform to fabricate complex spheroids with multiple components. We envision that this platform will pave the way for the high accuracy of spheroid fabrication and offer broad applications in numerous areas, such as tumor research, tissue engineering, developmental biology, and drug discovery.

Trapping cell spheroids and organoids using digital acoustofluidics.

The precise positioning and arrangement of cell spheroids and organoids are critical to reconstructing complex tissue architecture for tissue engineering and regenerative medicine. Here, we present a digital acoustofluidic method to manipulate cell spheroids and organoids with unprecedented dexterity. By introducing localized vibrations via a C-shaped integrated digital transducer (IDT), we can generate a trapping node to immobilize cell spheroids with a diameters ranging from 20µm to 300µm. Moreover, we digitally trapped multiple cell spheroids atop the C-shaped IDTs within a closed or open microfluidic chamber. By programming the trapping nodes within a 3 × 3 C-shaped IDT array, we can precisely position cell spheroids into designed patterns. We also demonstrated that our digital acoustofluidic device can accurately control the interaction of spheroid cells and organoids. Along with a simple fabrication procedure and setup, our digital acoustofluidic method can provide precisely manipulate and position various cell spheroids or organoids in a contactless, label-free, and highly biocompatible manner. We believe this technology can be widely used for tissue engineering, regenerative medicine, and fundamental cell biology research.

Profiling of immune-cancer interactions at the single-cell level using a microfluidic well array.

Cancer immunotherapy has achieved great success in hematological cancers. However, immune cells are a highly heterogeneous population and can vary highly in clonal expansion, migration and function status, making it difficult to evaluate and predict patient response to immune therapy. Conventional technologies only yield information on the average population information of the treatment, masking the heterogeneity of the individual T cell activation status, the formation of immune synapse, as well as the efficacy of tumor cell killing at the single-cell level. To fully interrogate these single-cell events in detail, herein, we present a microfluidic microwell array device that enables the massive parallel analysis of the immunocyte's heterogeneity upon its interaction pairs with tumor cells at the single-cell level. By precisely controlling the number and ratio of tumor cells and T cells, our technique can interrogate the dynamics of the CD8+ T cell and leukemia cell interaction inside 6400 microfluidic wells simultaneously. We have demonstrated that by investigating the interactions of T cell and tumor cell pairs at the single-cell level using our microfluidic chip, details hidden in bulk investigations, such as heterogeneity in T cell killing capacity, time-dependent killing dynamics, as well as drug treatment-induced dynamic shifts, can be revealed. This method opens up avenues to investigate the efficacy of cancer immunotherapy and resistance at the single-cell level and can explore our understanding of fundamental cancer immunity as well as determine cancer immunotherapy efficacy for personalized therapy.

One-Stop Microfluidic Assembly of Human Brain Organoids To Model Prenatal Cannabis Exposure.

Prenatal cannabis exposure (PCE) influences human brain development, but it is challenging to model PCE using animals and current cell culture techniques. Here, we developed a one-stop microfluidic platform to assemble and culture human cerebral organoids from human embryonic stem cells (hESC) to investigate the effect of PCE on early human brain development. By incorporating perfusable culture chambers, air-liquid interface, and one-stop protocol, this microfluidic platform can simplify the fabrication procedure and produce a large number of organoids (169 organoids per 3.5 cm × 3.5 cm device area) without fusion, as compared with conventional fabrication methods. These one-stop microfluidic assembled cerebral organoids not only recapitulate early human brain structure, biology, and electrophysiology but also have minimal size variation and hypoxia. Under on-chip exposure to the psychoactive cannabinoid, Δ-9-tetrahydrocannabinol (THC), cerebral organoids exhibited reduced neuronal maturation, downregulation of cannabinoid receptor type 1 (CB1) receptors, and impaired neurite outgrowth. Moreover, transient on-chip THC treatment also decreased spontaneous firing in these organoids. This one-stop microfluidic technique enables a simple, scalable, and repeatable organoid culture method that can be used not only for human brain organoids but also for many other human organoids including liver, kidney, retina, and tumor organoids. This technology could be widely used in modeling brain and other organ development, developmental disorders, developmental pharmacology and toxicology, and drug screening.

Rapid Microfluidic Formation of Uniform Patient-Derived Breast Tumor Spheroids.

Breast cancer is a highly complex, heterogeneous, and multifactorial disease that poses challenges for rapid and efficient treatment and development of personalized therapy. Here, we describe a rapid and reliable method to generate three-dimensional (3D) tumor spheroids in vitro that recapitulate an individual patient's tumor for testing treatments. By employing droplet microfluidics and scaffold materials, tumor cells were encapsulated into a large number of Matrigel-in-oil droplets with precise control over cell numbers and components per droplet. After removal of the oil, large numbers of uniform tumor spheroids were formed within a few hours via Matrigel-supported cell self-assembly. Our microfluidic technique produces uniform-sized tumor spheroids in less than 1 day. This method was used to reproducibly and rapidly generate uniform-sized tumor spheroids derived from patients' breast tumor tissues. As a proof-of-concept application, this method was used to quickly evaluate cancer treatments. We demonstrated that our microfluidic patient-derived tumor cultures not only preserve the genetic characteristics of the original tumor tissue but also provide heterogeneous responses to targeted therapies within 2 days. We believe this method will enable a timely and reliable 3D in vitro culture model, which may be applicable to personalized treatment prediction, drug discovery, and toxicity testing.

Profiling Cell-Matrix Adhesion Using Digitalized Acoustic Streaming.

Profiling the kinetics of cell-matrix adhesion is of great importance to understand many physiological and pathological processes such as morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Here, we developed a novel digital acoustofluidic device for parallel profiling cell-matrix adhesion at single-cell level. By introduction of localized and uniform acoustic streaming into an open chamber microfluidic device, the adherent cells within the open chamber can be detached by the streaming-induced Stokes drag force. By digital regulation of pulsed acoustic power from a low level to high levels, the hundreds of adherent cells can be ruptured from the fibronectin-coated substrate accordingly, and their adhesive forces (from several pN to several nN) and kinetics can be determined by the applied power and cell incubation time. As a proof-of-concept application for studying cancer metastasis, we applied this technique to measure the adhesion strength and kinetics of human breast cancer cells to extracellular matrix such as fibronectin and compared their metastatic potentials by measuring the rupture force of cancer cells representing malignant (MCF-7 cells and MDA-MB-231 cells) and nonmalignant (MCF-10A cells) states. Our acoustofluidic device is simple, easy to operate, and capable of measuring, in parallel, hundreds of individual cells' adhesion forces with a resolution at the pN level. Thus, we expect this device could be widely used for both fundamental cell biology research as well as development of cancer diagnostics and tissue engineering technologies.