Intratumoral Heterogeneity Promotes Collective Cancer Invasion through NOTCH1 Variation.

Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a tumor bioengineering approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that intratumoral variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression.

A Rapid Single-Cell Antimicrobial Susceptibility Testing Workflow for Bloodstream Infections.

Bloodstream infections are a significant cause of morbidity and mortality worldwide. The rapid initiation of effective antibiotic treatment is critical for patients with bloodstream infections. However, the diagnosis of bloodborne pathogens is largely complicated by the matrix effect of blood and the lengthy blood tube culture procedure. Here we report a culture-free workflow for the rapid isolation and enrichment of bacterial pathogens from whole blood for single-cell antimicrobial susceptibility testing (AST). A dextran sedimentation step reduces the concentration of blood cells by 4 orders of magnitude in 20-30 min while maintaining the effective concentration of bacteria in the sample. Red blood cell depletion facilitates the downstream centrifugation-based enrichment step at a sepsis-relevant bacteria concentration. The workflow is compatible with common antibiotic-resistant bacteria and does not influence the minimum inhibitory concentrations. By applying a microfluidic single-cell trapping device, we demonstrate the workflow for the rapid determination of bacterial infection and antimicrobial susceptibility testing at the single-cell level. The entire workflow from blood to categorical AST result can be completed in less than two hours.

Digital electrical impedance analysis for single bacterium sensing and antimicrobial susceptibility testing.

Single-molecule and single-cell analysis techniques have opened new opportunities for characterizing and analyzing heterogeneity within biological samples. These detection methods are often referred to as digital assays because the biological sample is partitioned into many small compartments and each compartment contains a discrete number of targets (e.g. cells). Using digital assays, researchers can precisely detect and quantify individual targets, and this capability has made digital techniques the basis for many modern bioanalytical tools (including digital PCR, single cell RNA sequencing, and digital ELISA). However, digital assays are dominated by optical analysis systems that typically utilize microscopy to analyze partitioned samples. The utility of digital assays may be dramatically enhanced by implementing cost-efficient and portable electrical detection capabilities. Herein, we describe a digital electrical impedance sensing platform that enables direct multiplexed measurement of single cell bacterial cells. We outline our solutions to the challenge of multiplexing impedance sensing across many culture compartments and demonstrate the potential for rapidly differentiating antimicrobial resistant versus susceptible strains of bacteria.

Three-Dimensional Microtumors for Probing Heterogeneity of Invasive Bladder Cancer.

Bladder cancer is an increasingly common malignancy, and muscle invasive bladder cancer is associated with particularly high rates of morbidity and mortality. The morphologic and molecular diversity of bladder cancer poses significant challenges in elucidating the invasion mechanisms responsible for disease progression. Furthermore, conventional invasion assays do not provide a physiological context for studying bladder cancer invasion within 3D microenvironments and have limited ability to capture the contribution of cellular phenotypic heterogeneity to disease progression. Here, we describe the development of a 3D microtumor invasion model suitable for the analysis of cellular phenotypic heterogeneity in cell lines and primary tumor cells from bladder cancer patients. This model incorporates a self-assembly approach for recapitulating features of bladder cancer invasion in 3D microenvironments and probing the invasive cell subpopulations. The gene expression profiles of invading microtumors were analyzed by incorporating a gold nanorod-locked nucleic acid biosensor. The incorporation of the single cell biosensor and transient gene knockdown into the system revealed the formation of invasive leader cells with upregulated Delta-like ligand 4 (DLL4) expression as well as the role of NOTCH1-DLL4 signaling in collective bladder cancer invasion. The involvement of DLL4 expressing cells in bladder cancer invasion was also observed in patient samples obtained from transurethral resection. Collectively, our study demonstrates a 3D microtumor invasion model for investigating intracellular heterogeneity of bladder cancer invasion and analyzing patient derived samples toward personalized medicine applications.

Adaptable microfluidic system for single-cell pathogen classification and antimicrobial susceptibility testing.

Infectious diseases caused by bacterial pathogens remain one of the most common causes of morbidity and mortality worldwide. Rapid microbiological analysis is required for prompt treatment of bacterial infections and to facilitate antibiotic stewardship. This study reports an adaptable microfluidic system for rapid pathogen classification and antimicrobial susceptibility testing (AST) at the single-cell level. By incorporating tunable microfluidic valves along with real-time optical detection, bacteria can be trapped and classified according to their physical shape and size for pathogen classification. By monitoring their growth in the presence of antibiotics at the single-cell level, antimicrobial susceptibility of the bacteria can be determined in as little as 30 minutes compared with days required for standard procedures. The microfluidic system is able to detect bacterial pathogens in urine, blood cultures, and whole blood and can analyze polymicrobial samples. We pilot a study of 25 clinical urine samples to demonstrate the clinical applicability of the microfluidic system. The platform demonstrated a sensitivity of 100% and specificity of 83.33% for pathogen classification and achieved 100% concordance for AST.

Nanotube assisted microwave electroporation for single cell pathogen identification and antimicrobial susceptibility testing.

A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections.

Collective Cell Migration in 3D Epithelial Wound Healing.

Collective cell migration plays a pivotal role in development, wound healing, and metastasis, but little is known about the mechanisms and coordination of cell migration in 3D microenvironments. Here, we demonstrate a 3D wound healing assay by photothermal ablation for investigating collective cell migration in epithelial tissue structures. The nanoparticle-mediated photothermal technique creates local hyperthermia for selective cell ablation and induces collective cell migration of 3D tissue structures. By incorporating dynamic single cell gene expression analysis, live cell actin staining, and particle image velocimetry, we show that the wound healing response consists of 3D vortex motion moving toward the wound followed by the formation of multicellular actin bundles and leader cells with active actin-based protrusions. Inhibition of ROCK signaling disrupts the multicellular actin bundle and enhances the formation of leader cells at the leading edge. Furthermore, single cell gene expression analysis, pharmacological perturbation, and RNA interference reveal that Notch1-Dll4 signaling negatively regulates the formation of multicellular actin bundles and leader cells. Taken together, our study demonstrates a platform for investigating 3D collective cell migration and underscores the essential roles of ROCK and Notch1-Dll4 signaling in regulating 3D epithelial wound healing.