Fig 1.
The design and validation of a 3D culture microfluidic chip.
(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.
Fig 2.
Immunofluorescent analysis of the MET/PI3K/AKT activation and GRP78 expression on the microfluidic chip.
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.
Fig 3.
Western blot analysis of the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.
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. *P<0.05; **P<0.01 vs. the controls.
Fig 4.
The percentages of apoptotic A549 cells.
A549 cells were cultured in maintenance medium for 24h and continually cultured in triplicate in the same medium or mixture of maintenance medium and CAF matrix in the presence or absence of the PI3K or GRP78 inhibitor on the 3D chambers for 24h. Subsequently, the cells were stained with Hochest33342 and PI, imaged and the percentages of apoptotic A549 cells were counted. Data are representative images (Fig 4A) (magnification x 200) or expressed as the means ± SD of the percentages of apoptotic cells in individual groups of cells from three separate experiments (Fig 4B). *P<0.05; **P<0.01.
Fig 5.
The validation in in vivo xenograft model.
(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. *P<0.05 vs control group, #P<0.05 vs HGF group.