Epithelial–mesenchymal transition (EMT) plays a critical role in the early stages of dissemination of carcinoma leading to metastatic tumors, which are responsible for over 90% of all cancer-related deaths. Current therapeutic regimens, however, have been ineffective in the cure of metastatic cancer, thus an urgent need exists to revisit existing protocols and to improve the efficacy of newly developed therapeutics. Strategies based on preventing EMT could potentially contribute to improving the outcome of advanced stage cancers. To achieve this goal new assays are needed to identify targeted drugs capable of interfering with EMT or to revert the mesenchymal-like phenotype of carcinoma to an epithelial-like state. Current assays are limited to examining the dispersion of carcinoma cells in isolation in conventional 2-dimensional (2D) microwell systems, an approach that fails to account for the 3-dimensional (3D) environment of the tumor or the essential interactions that occur with other nearby cell types in the tumor microenvironment. Here we present a microfluidic system that integrates tumor cell spheroids in a 3D hydrogel scaffold, in close co-culture with an endothelial monolayer. Drug candidates inhibiting receptor activation or signal transduction pathways implicated in EMT have been tested using dispersion of A549 lung adenocarcinoma cell spheroids as a metric of effectiveness. We demonstrate significant differences in response to drugs between 2D and 3D, and between monoculture and co-culture.

Microwell technology has revolutionized many aspects of in vitro cellular studies from 2-dimensional (2D) traditional cultures to 3-dimensional (3D) in vivo-like functional assays. However, existing lithography-based approaches are often costly and time-consuming. This study presents a rapid, low-cost prototyping method of CO2 laser ablation of a conventional untreated culture dish to create concave microwells used for generating multicellular aggregates, which can be readily available for general laboratories. Polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), and polystyrene (PS) microwells were investigated, and each produced distinctive microwell features. Among these three materials, PS cell culture dishes produced the optimal surface smoothness and roundness. A549 lung cancer cells were grown to form cancer aggregates of controllable size from ~40 to ~80 μm in PS microwells. Functional assays of spheroids were performed to study migration on 2D substrates and in 3D hydrogel conditions as a step towards recapitulating the dissemination of cancer cells. Preclinical anti-cancer drug screening was investigated and revealed considerable differences between 2D and 3D conditions, indicating the importance of assay type as well as the utility of the present approach.

Tumor-associated macrophages (TAMs) can constitute up to 50% of the tumor mass and have strong implications in tumor progression and metastasis. Macrophages are plastic and can polarize to various subtypes that differ in terms of surface receptor expression as well as cytokine and chemokine production and effector function. Conventionally, macrophages are grouped into two major subtypes: the classically activated M1 macrophages and the alternatively activated M2 macrophages. M1 macrophages are pro-inflammatory, promote T helper (Th) 1 responses, and show tumoricidal activity, whereas M2 macrophages contribute to tissue repair and promote Th2 responses. Herein, we present a microfluidic system integrating tumor cell aggregates and subtypes of human monocyte-derived macrophages in a three-dimensional hydrogel scaffold, in close co-culture with an endothelial monolayer to create an in vitro tumor microenvironment. This platform was utilized to study the role of individual subtypes of macrophages (M0, M1, M2a, M2b and M2c) in human lung adenocarcinoma (A549) aggregate dispersion, as a representation of epithelial-mesenchymal transition (EMT). A significant difference was observed when M2a macrophages were in direct contact with or separated from A549 aggregates, suggesting a possible mechanism for proximity-induced, contact-dependent dissemination via ICAM-1 and integrin β2 interactions. Indeed, M2a macrophages tended to infiltrate and release cells from carcinoma cell aggregates. These findings may help in the development of immunotherapies based on enhancing the tumor-suppressive properties of TAMs.

Morella rubra, red bayberry, is an economically important fruit tree in south China. Here, we assembled the first high-quality genome for both a female and a male individual of red bayberry. The genome size was 313-Mb, and 90% sequences were assembled into eight pseudo chromosome molecules, with 32 493 predicted genes. By whole-genome comparison between the female and male and association analysis with sequences of bulked and individual DNA samples from female and male, a 59-Kb region determining female was identified and located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, four of them are related to sex floral development. This 59-Kb female-specific region was likely to be derived from duplication and rearrangement of paralogous genes and retained non-recombinant in the female-specific region. Sex-specific molecular markers developed from candidate genes co-segregated with sex in a genetically diverse female and male germplasm. We propose sex determination follow the ZW model of female heterogamety. The genome sequence of red bayberry provides a valuable resource for plant sex chromosome evolution and also provides important insights for molecular biology, genetics and modern breeding in Myricaceae family.

Experiments were performed in a modified microfluidic platform recapitulating part of the in vivo tumor microenvironment by co-culturing carcinoma cell aggregates embedded in a three-dimensional (3D) collagen scaffold with human umbilical vein endothelial cells (HUVECs). HUVECs were seeded in one channel of the device to initiate vessel-like structures in vitro prior to introducing the aggregates. The lung adenocarcinoma cell line A549 and the bladder carcinoma cell line T24 were tested. Dose-response assays of four drugs known to interfere with Epithelial Mesenchymal Transition (EMT) signaling pathways were quantified using relative dispersion as a metric of EMT progression. The presence of HUVECs in one channel induces cell dispersal in A549 which then can be inhibited by each of the four drugs. Complete inhibition of T24 aggregate dispersal, however, is not achieved with any single agent, although partial inhibition was observed with 10 μM of the Src inhibitor, AZD-0530. Almost complete inhibition of T24 dispersal in monoculture was achieved only when the four drugs were added in combination, each at 10 μM concentration. Coculture of T24 with HUVECs forfeits the almost-complete inhibition. The enhanced dispersal observed in the presence of HUVECs is a consequence of secretion of growth factors, including HGF and FGF-2, by endothelial cells. This 3D microfluidic co-culture platform provides an in vivo-like surrogate for anti-invasive and anti-metastatic drug screening. It will be particularly useful for defining combination therapies for aggressive tumors such as invasive bladder carcinoma.

Planar patch-clamp has revolutionized ion-channel measurement by eliminating laborious manipulation from the traditional micropipette approach and enabling high throughput. However, low yield in gigaseal formation and/or relatively high cost due to microfabricated processes are two main drawbacks. This paper presents patch clamping on glass substrate-an economical solution without sacrificing gigaseal yield rate. Two-stage CO(2) laser drilling methodology was used to generate an hourglass, funnel-like aperture of a specified diameter with smooth and debris-free surfaces on 150 microm borosilicate cover glass. For 1-3 microm apertures as patch-clamp chips, seal resistance was tested on human embryonic kidney, Chinese hamster ovary, and Jurkat T lymphoma cells with a gigaseal success rate of 62.5%, 43.6% and 66.7% respectively. Results also demonstrated both whole-cell and single channel recording on endogenously expressed ion channels to confirm the capability of different patch configurations.

This paper presents in vitro microvascular network formation within 3D gel scaffolds made from different concentrations of type-I collagen, fibrin, or a mixture of collagen and fibrin, using a simple microfluidic platform. Initially, microvascular network formation of human umbilical vein endothelial cells was examined using live time-lapse confocal microscopy every 90 min from 3 h to 12 h after seeding within three different concentrations of collagen gel scaffolds. Among the three conditions of collagen gel scaffolds (2.0 mg/ml, 2.5 mg/ml, and 3.0 mg/ml), the number of skeleton within collagen gel scaffolds was consistently the highest (3.0 mg/ml), followed by those of collagen gel scaffolds (2.5 mg/ml and 2.0 mg/ml). Results demonstrated that concentration of collagen gel scaffolds, which influences matrix stiffness and ligand density, may affect microvascular network formation during the early stages of vasculogenesis. In addition, the maturation of microvascular networks in monoculture under different gel compositions within gel scaffolds (2.5 mg/ml) was examined for 7 d using live confocal microscopy. It was confirmed that pure fibrin gel scaffolds are preferable to collagen gel or collagen/fibrin combinations, significantly reducing matrix retractions during maturation of microvascular networks for 7 d. Finally, early steps in the maturation process of microvascular networks for 14 d were characterized by demonstrating sequential steps of branching, expanding, remodeling, pruning, and clear delineation of lumens within fibrin gel scaffolds. Our findings demonstrate an in vitro model for generating mature microvascular networks within 3D microfluidic fibrin gel scaffolds (2.5 mg/ml), and furthermore suggest the importance of gel concentration and composition in promoting the maturation of microvascular networks.

This paper presents a magnetically driven valve via a permanent magnet pressing a spacer against deformable polydimethylsiloxane (PDMS) to fully close a microchannel. Its ability for electrical isolation, time response, and resistance to backpressure are interrogated. Simulation of the valve closing process was commenced along with experimental verification. Effects of PDMS thickness, and dimension and aspect ratio of microchannels were characterized. Up to 10 GΩ electrical isolation was demonstrated, as well as 50-70 ms valve response and ∼200 kPa resistible pressure. On-demand actuation for arbitrary flow patterns further quantifies its utility. With advantages of simple fabrication, flexible valving location, and no external power requirement, the on/off valve could be leveraged for proof-of-concept microfluidic devices and other applications.