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Extracellular vesicles (EVs) have attracted enormous attention for their diagnostic and therapeutic potential. However, it has proven challenging to achieve the sensitivity to detect individual nanoscale EVs, the specificity to distinguish EV subpopulations, and a sufficient throughput to study EVs among an enormous background. To address this fundamental challenge, we developed a droplet-based optofluidic platform to quantify specific individual EV subpopulations at high throughput. The key innovation of our platform is parallelization of droplet generation, processing, and analysis to achieve a throughput (~20 million droplets/min) more than 100× greater than typical microfluidics. We demonstrate that the improvement in throughput enables EV quantification at a limit of detection = 9EVs/μL, a >100× improvement over gold standard methods. Additionally, we demonstrate the clinical potential of this system by detecting human EVs in complex media. Building on this work, we expect this technology will allow accurate quantification of rare EV subpopulations for broad biomedical applications.

List of primer sequences used for RT-PCR analysis.

Breast tumors are stiffer and more hypoxic than nonmalignant breast tissue. Here we report that stiff and hypoxic microenvironments promote the development of breast cancer stem-like cells (CSC) through modulation of the integrin-linked kinase ILK. Depleting ILK blocked stiffness and hypoxia-dependent acquisition of CSC marker expression and behavior, whereas ectopic expression of ILK stimulated CSC development under softer or normoxic conditions. Stiff microenvironments also promoted tumor formation and metastasis in ovo, where depleting ILK significantly abrogated the tumorigenic and metastatic potential of invasive breast cancer cells. We further found that the ILK-mediated phenotypes induced by stiff and hypoxic microenvironments are regulated by PI3K/Akt. Analysis of human breast cancer specimens revealed an association between substratum stiffness, ILK, and CSC markers, insofar as ILK and CD44 were expressed in cancer cells located in tumor regions predicted to be stiff. Our results define ILK as a key mechanotransducer in modulating breast CSC development in response to tissue mechanics and oxygen tension. Cancer Res; 76(18); 5277–87. ©2016 AACR.

Stiff and hypoxic microenvironments regulate beta1-integrin, ILK, and CSC markers in human breast cancer cells. (A) Phase contrast images of MDA-MB-231 cells cultured on soft or stiff substrata under normoxia or hypoxia. Transcript levels of (B) ITGB1 and (C) ILK in MDA-MB-231 cells cultured on soft or stiff substrata under normoxia or hypoxia. (D) Immunoblotting analysis for beta1-integrin, ILK, and GAPDH in MDA-MB-231 cells cultured on soft or stiff substrata under normoxia or hypoxia. Transcript levels of (E) CD44, (F) Nanog, and (G) CD49f in MDA-MB-231 cells cultured on soft or stiff substrata under normoxia or hypoxia. (H) Immunoblotting analysis for CD44 and Nanog in MDA-MB-231 cells cultured on soft or stiff substrata under normoxia or hypoxia. Histograms of flow cytometry analysis of (I) CD44 and (J) Nanog expression in 4T1 cells cultured on soft or stiff substrata under normoxia or hypoxia. (K) Dot plots of flow cytometry analysis of ALDH activity in 4T1 cells cultured on soft or stiff substrata under normoxia or hypoxia. Scale bars, 50 µm. Shown are mean (plus minus) s.e.m. *, P

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Reciprocal epithelial-mesenchymal signaling is essential for morphogenesis, including branching of the lung. In the mouse, mesenchymal cells differentiate into airway smooth muscle that wraps around epithelial branches, but this contractile tissue is absent from the early avian lung. Here, we found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme. Before branching, the basement membrane wraps the airway epithelium as a spatially uniform sheath. After branch initiation, however, the basement membrane thins at branch tips; this remodeling requires mesenchymal expression of matrix metalloproteinase-2, which is necessary for branch extension but not branch initiation. As branches extend, tenascin-C (TNC) accumulates in the mesenchyme several cell diameters away from the epithelium. Despite its pattern of accumulation, TNC is expressed exclusively by epithelial cells. Branch extension coincides with deformation of adjacent mesenchymal cells, which correlates with an increase in mesenchymal fluidity at branch tips that may transport TNC away from the epithelium. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis.

Cell-generated forces drive an array of biological processes ranging from wound healing to tumor metastasis. Whereas experimental techniques such as traction force microscopy are capable of quantifying traction forces in multidimensional systems, the physical mechanisms by which these forces induce changes in tissue form remain to be elucidated. Understanding these mechanisms will ultimately require techniques that are capable of quantifying traction forces with high precision and accuracy in vivo or in systems that recapitulate in vivo conditions, such as microfabricated tissues and engineered substrata. To that end, here we review the fundamentals of traction forces, their quantification, and the use of microfabricated tissues designed to study these forces during cell migration and tissue morphogenesis. We emphasize the differences between traction forces in two- and three-dimensional systems, and highlight recently developed techniques for quantifying traction forces.

Breast tumors are stiffer and more hypoxic than nonmalignant breast tissue. Here we report that stiff and hypoxic microenvironments promote the development of breast cancer stem-like cells (CSC) through modulation of the integrin-linked kinase ILK. Depleting ILK blocked stiffness and hypoxia-dependent acquisition of CSC marker expression and behavior, whereas ectopic expression of ILK stimulated CSC development under softer or normoxic conditions. Stiff microenvironments also promoted tumor formation and metastasis in ovo, where depleting ILK significantly abrogated the tumorigenic and metastatic potential of invasive breast cancer cells. We further found that the ILK-mediated phenotypes induced by stiff and hypoxic microenvironments are regulated by PI3K/Akt. Analysis of human breast cancer specimens revealed an association between substratum stiffness, ILK, and CSC markers, insofar as ILK and CD44 were expressed in cancer cells located in tumor regions predicted to be stiff. Our results define ILK as a key mechanotransducer in modulating breast CSC development in response to tissue mechanics and oxygen tension. Cancer Res; 76(18); 5277–87. ©2016 AACR.

Mechanotransduction is often described in the context of force-induced changes in molecular conformation, but molecular-scale mechanical stimuli arise in vivo in the context of complex, multicellular tissue structures. For this reason, we highlight and review experimental methods for investigating mechanotransduction across multiple length scales. We begin by discussing techniques that probe the response of individual molecules to applied force. We then move up in length scale to highlight techniques aimed at uncovering how cells transduce mechanical stimuli into biochemical activity. Finally, we discuss approaches for determining how these stimuli arise in multicellular structures. We expect that future work will combine techniques across these length scales to provide a more comprehensive understanding of mechanotransduction.

Cells are complex machines whose behaviors arise from their internal collection of dynamically interacting organelles, supramolecular complexes, and cytoplasmic chemicals. The current understanding of the nature by which subcellular biology produces cell-level behaviors is limited by the technological hurdle of measuring the large number (>103 ) of small-sized (

Demanding line rules and property requirements for electrochemically-grown materials used in fabricated and templated systems motivates a need for quantitative analytical methods that are compatible with fabrication systems. We demonstrate a nanoelectrode array-based analysis method analogous to the Levich–Koutecky equation to enable rapid and quantitative evaluation of surface kinetics and mass transport parameters. The only data needed for the analysis is the radius of individual deposits on the array. We apply this new method to electrodeposition and electroless deposition (ELD) of copper from quiescent electrolytes. The results yield a room temperature cupric ion diffusivity of D=5.9×10−10 m2/s in acidic copper sulfate electrodeposition bath and an ELD activation energy of EA=58 kJ/mol in a copper ELD bath with the reducing agent DMAB. Keywords: Electrodeposition, Electroless deposition, Mass transfer, Kinetics

Forces arising from contractile actomyosin filaments help shape tissue form during morphogenesis. Developmental events that result from actomyosin contractility include tissue elongation, bending, budding, and collective migration. Here, we highlight recent insights into these morphogenetic processes from the perspective of actomyosin contractility as a key regulator. Emphasis is placed on a range of results obtained through live imaging, culture, and computational methods. Combining these approaches in the future has the potential to generate a robust, quantitative understanding of tissue morphodynamics.

The mechanical and chemical properties of the cellular microenvironment can influence the phenotype of cancer cells. The hypoxic conditions within solid breast tumors can contribute to invasiveness and the formation of breast cancer stem cells (CSCs). Integrin-linked kinase (ILK) is a critical mediator for mechanotransduction, a process that converts exogenous mechanical stimuli into biochemical signals. Yet, it is unclear how the physical properties of the tumor microenvironment, including matrix stiffness and hypoxia, integrate to direct the formation of the breast CSC niche. Using an innovative engineered culture model, we found that stiff and hypoxic microenvironments activate integrin signaling and a stem-like gene signature in breast cancer cells, suggesting that integrin signaling, matrix mechanics and oxygen tension cooperatively promote the formation of breast CSCs. Stiff and hypoxic microenvironments promote integrin signaling and the expression of CSC markers including CD44 and Nanog. Knocking down ILK reverts the CSC phenotype of invasive breast cancer cells on stiff matrix, and abrogates their ability to form mammospheres and colonies in soft agar. In contrast, ectopic expression of ILK enhances CSC properties. ILK promotes integrin signaling and the CSC phenotype through the PI3K/Akt pathway. Stiff microenvironments promote tumor formation and metastasis in ovo. ILK depletion significantly abrogates the tumorigenenic and metastatic potential of invasive breast cancer cells in ovo. Importantly, we found that breast cancer cells expressing ILK and the CSC marker CD44 were only present in the regions of tumors predicted to be stiff in breast cancer patient samples. Our data suggest that ILK act as an essential mechanosensor that regulates breast CSC phenotype in response to matrix mechanics and oxygen tension to regulate the formation of breast CSCs. Citation Format: Mei-Fong Pang, Melody Stallings-Mann, Michael J. Siedlik, Victor D. Varner, Siyang Han, Derek C. Radisky, Celeste M. Nelson. Tissue stiffness and hypoxia regulate breast cancer stem cells through ILK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4395.

Orchestrated structure evolution is an alternative nanomanufacturing approach that combines the advantages of top-down patterning and bottom-up self-organizing growth. It relies upon tool-directed patterning to create 'seed' locations on a surface from which a subsequent deposition process produces the final, merged film. Despite its demonstrated ability to reduce patterning time by orders of magnitude, our prior reliance on mass transfer limited deposition and square seed arrays resulted in extraneous film growth along pattern edges, thereby limiting the pattern quality of the final film. Here, quality improvements are demonstrated by modeling and tuning the growth mechanism of the deposition step to include charge transfer effects. In addition, a seed positioning optimization technique derived from simulated annealing is introduced as a method for relocating the seeds to minimize film overgrowth at the pattern edges. These improvements enable OSE to maintain geometric quality while substantially reducing the time and cost compared to traditional direct-write manufacturing methods.

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