Immunofluorescent analysis of the MET/PI3K/AKT activation and GRP78 expression on the microfluidic chip.

<p>A549 cells were cultured in triplicate in the maintenance medium alone, mixed with the CAF matrix in the presence or absence of anti-HGF or containing 40 ng/ml of human HGF in the 3D chambers for 48h. The cells were stained with the indicated FITC-conjugated antibodies, and examined under a fluorescent microscope. Furthermore, the cells were cultured in the mixture of maintenance medium and CAF matrix in the presence or absence of an inhibitor for c-Met, PI3K or GRP78 for 48h and stained as described above. Data are representative images (magnification x 200) from three separate experiments. (A)The CAF matrix or HGF enhances the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells. (B)The effect of an inhibitor of c-Met, PI3K or GRP78 on the CAF-enhanced c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.</p

The design and validation of a 3D culture microfluidic chip.

<p>(a)The schematic design of the microfluidic chip with CGG and downstream cell chambers (the upper panel) and the fabricated chip with pumping machine (the lower panel). (b)The diffused Rh-123 in the 3D chamber within 30 min and >95% cells were viable (green). Magnification ×100. (c) The morphological features of A549 cells in the 3D chamber without or with CAF matrix. The white arrows indicate apoptotic cells. (d)The α-SMA immunofluorescence assay of HFL1 cells. HFL1 cells induced by A549 medium showed a positive α-SMA staining (right) compared to the untreated HFL1 (left). Magnification ×400. (e) Immunohistochemistry assay for lung cancer tissues. The expression of α-SMA protein in the lung cancer tissues is higher than that in adjacent tissues. Magnification ×200.</p

The validation in in vivo xenograft model.

<p>(A) Representative images of tumor size in different groups of nude mice on the 60th day after treatment. (B) Growth curves in different groups of nude mice. *<i>P</i><0.05 vs control group, ^#<i>P</i><0.05 vs HGF group.</p

Western blot analysis of the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.

<p>A549 cells were cultured in the condition as described above and the relative levels of phosphorylated Met, PI3Kp85, AKT and GRP78 expression in the different groups of cells were characterized by Western blot assays and quantified. Data are representative images and expressed as the means ± SD of each protein in individual groups of cells from three separate experiments. *<i>P</i><0.05; **<i>P</i><0.01 vs. the controls.</p

Illustration of medium flow direction in the microfluidic device.

<p>The blue, red and green indicators represented control group, EGF group, GM6001/EGF group respectively. These three indicators were perfused into microchannels from inlet A, B, C simultaneously and separately, while these indicators could spread out to cell chambers of both sides via oval microchannels uniformly and in parallel without crossing.</p

A microfluidic chip designed for the study of cancer cells invasion in 3D matrix.

<p>(A) Schematic representation of the microfluidic platform. Layout of the integrated microfluidic device is composed of three units sharing a common outlet, each of which contains an inlet, three parallel main channels, three cell culture chambers and an outlet. (B) A magnified illustration of one cell culture chamber. (C) Photograph of the microfluidic system.</p

Invadopodia formation assay and quantification analysis with confocal system in A549 cells.

<p>The cells in control group (A), EGF group (B), and GM6001/EGF group (C), were cultured on a 3D microfluidic device for 12 h. The cells were stained green represented combined with cortactin, stained red represented combined with F-actin, and arrowheads in merge pictures indicated cells displaying invadopodia. (D) The percent of the cells with invadopodia formation. (E) The average number of invadopodia per cell. Error bars represented the SD of three different determinations. *Statistically significant between control group and EGF group; **statistically significant between EGF group and GM6001/EGF group, p<0.05. Magnification: ×1200.</p

Actual invadopodia formation of A549 cells in control group (A), EGF group (B), and GM6001/EGF group (C) in 3D extracellular matrix in the microfluidic device with confocal system.

<p>Invadopodia could be obviously induced by EGF in (B), <b>while</b> this induction could be inhibited by GM6001 in (C). White arrowheads represented invadopodia. Magnification: ×1200.</p

Fluoresent analysis of apoptotic in A549 cells.

<p>Cells were cultured on the 3D microfluidic device for 96 h and then stained by Hoechst and PI. Live cells were stained blue and dead cells were stained red. Magnification: ×200.</p