br For the Transwell assay breast cancer cells were re
For the Transwell assay, breast cancer 10047-33-3 (2 × 104) were re-suspended in 200 μl of the CM and were seeded into the upper well of 8-μm-pore Boyden chambers (Millipore, GER) coated with Matrigel (Corning BioCoat, USA, 1:7.5), and 500 μl medium with 10% FBS was added into the lower well. After incubation at 37 °C and 5% CO2 for 12 h, the cells that adhered to the upper surface of the filter were re-moved with a cotton applicator. Stained with 0.5% crystal violet, the cells that invaded to the opposite side of the filter were counted under the microscope (Nikon, Japan). The data represent at least three ex-periments conducted in triplicate (mean ± standard error).
2.9. An integrin-binding assay
These assays were performed as described previously . Briefly, 96-well microtiter plates were coated with 1 μg/ml recombinant αvβ3 (R&D, USA) or α5β1 (R&D, USA). The plates were incubated with dif-ferent concentrations (0–160 ng/ml) of recombinant IL32 (R&D, USA) diluted in PBS. Bound proteins were quantified by ELISA using an HRP-conjugated anti-IL32 (R&D, USA) antibody at 450 nm. The data re-present the means of triplicate experiments. Saturation binding assays were conducted to calculate apparent dissociation constants as pre-viously described .
Cell growth was assessed by the MTT assay as previously described . Briefly, 3 × 103 cells were seeded in 96-well plates containing 200 μl of complete medium. The cells were treated with recombinant IL32 (20 ng/ml) or vehicle (PBS) after attachment. Cell culture for the specified time, MTT (5 mg/ml in PBS) was added to each well and in-cubated for 4 h. After DMSO (ThermoFisher, USA) was added into each well, the absorbance was recorded on a microplate reader (BioTek, USA) at 490 nm.
2.11. Tumour xenografts
Animal experiments were approved by the animal care ethics committees at Chongqing Medical University. Breast cancer BT549 cells (106) were mixed or not mixed with an equal number of NFs or CAFs in 200 μl PBS: Matrigel at a 1:1 ratio and were subcutaneously injected into 4-week-old female nude mice. Tumour growth was evaluated by monitoring tumour volume (V = length × width2 × 0.5) every 4 days. For the mice injected with BT549 cells alone, when the xenografts were palpable (∼3 mm in diameter), intra-tumour injection of vehicle or Cancer Letters 442 (2019) 320–332
rIL32 at 0.1 μg/kg was performed twice weekly for 5 consecutive weeks. The animals were euthanised on the 45th day after introduction of xenografts, and the tumours and mouse lungs were harvested for further research. Cryosections (4 μm) of the tissues were subjected to H &E staining for histological assessment.
2.12. Statistical analysis
This analysis was performed in SPSS standard version 19.0 software. The data are presented as mean ± SD from at least three independent experiments. Student's t-test was performed for single comparisons between two groups, and ANOVA followed by the Student–Newman–Keuls multiple-comparison test was conducted for a comparison between multiple groups. A P value < 0.05 in all cases was assumed to indicate statistical significance.
3.1. Integrins are dysregulated during the epithelial–mesenchymal transition (EMT) of mammary cells
Our previous study and other reports have revealed that EMT is critical for tumour cell invasion and metastasis [39–41]. Using a cDNA array and proteomic analyses, we identified a series of dysregulated genes of the integrin family in mammary cells during EMT (Fig. 1A). Some of these integrins (ITGB1, ITGB3, ITGB4, ITGB5, ITGAV, ITGA5, ITGA3, ITGA9, and ITGA11), which were simultaneously identified by the cDNA array analysis and proteomics, were next validated by qRT-PCR. Growing evidence suggests that Twist is an inducer of EMT [40,42], and ITGB1, ITGB3, and ITGB4 were proved here to be sig-nificantly up-regulated during EMT in mammary cells (Fig. 1B).
In line with this finding, the integrin family [integrin β1 (encoded by ITGB1), integrin β3 (encoded by ITGB3), and integrin β4 (encoded by ITGB4)] mRNA expression levels were found to be significantly elevated in breast cancer cells overexpressing Twist (e.g. BT549 and Hs578T cells) as compared with those with low expression of Twist (e.g. MCF10A and MCF7 cells; Fig. 1C). Furthermore, the loss of Twist ap-parently down-regulated ITGB1, ITGB3, and ITGB4 in BT549 cells (Fig. 1D).
It has been reported that breast CAFs, not NFs, can promote breast cancer cell migration and invasion [43,44]. To test whether CAFs can promote mammary cell invasion by means of the integrin family, MCF10A cells were transfected with ITGB1, ITGB3, or ITGB4 and co-cultured with CAFs or NFs. The potentials for cell invasion were tested. Although ITGB1, ITGB3, and ITGB4 increased the invasive capacity of MCF10A (Fig S1A), only ITGB3 strengthened the cell invasion of MCF10A cells during co-culture with CAFs (Fig. 1E). Similarly, a loss of Twist in BT549 cells attenuated the invasion ability (Fig S1B), but si-lencing of Twist in BT549 cells dramatically reduced their invasive capacity in co-culture with CAFs (Fig. 1F).