Figure 1.
Establishment of anoikis-resistant sublines of ACC cells.
A, Schematic of establishing anoikis-resistant sublines of ACC by cycles of culture under suspension and adhesion. B, The cell viabilities of ACC and ACCAr in suspension were measured using TBE assay (Mean ± SE, **p<0.01 versus ACC-2, ##p<0.01 versus ACC-M). C, Hoechst 33258 staining was performed to detect the apoptosis of ACC and ACCAr cells that cultured in suspension. D, DNA fragments of ACC and ACCAr in suspension were detected using Cell Death Detection ELISAPLUS (Mean ± SE, **p<0.01 versus ACC-2, ##p<0.01 versus ACC-M). E, Anchorage-independent growth of ACC and ACCAr cells was analyzed using soft agar assay. F, Expression levels of cleaved PARP, cleaved caspase-3, cleaved caspase-9, Bax, and Bcl-2 were determined using Western blot analysis (**p<0.01 versus ACC-2, ##p<0.01 versus ACC-M).
Figure 2.
Enhanced migration and invasion of ACCAr cells.
A and B, The migration of ACC and ACCAr was analyzed using wound healing and transwell Boyden system assays. C, The invasion of ACC and ACCAr was measured using a transwell Boyden system coated with Matrigel. D, Quantification of migrated and invaded cells in wound healing assays and transwell migration/invasion assays. The results were represented as relative ratio to ACC-2 (Mean ± SE, **p<0.01 versus ACC-2, ##p<0.01 versus ACC-M). E, The relative mRNA expression levels of VEGF, MMP9, and SDF-1α were analyzed by real-time RT-PCR. The results were represented as relative ratio to ACC-2 (Mean ± SE, **p<0.01 versus ACC-2, ##p<0.01 versus ACC-M).
Figure 3.
EMT-like transformation in ACCAr cells.
A, A mesenchymal morphology as well as epithelial (E-cadherin) and mesenchymal (vimentin) markers were detected in ACC-2Ar and ACC-MAr. B, Protein expression levels of EMT-related genes in ACCAr cells were measured using Western blot analysis. C, The mRNA expression levels of E-cadherin, N-cadherin, and vimentin in ACCAr cells were determined using qRT-PCR. D, The mRNA expression levels of Snail, Slug, and Twist in ACCAr cells were determined using qRT-PCR. E, The mRNA expression levels of bone morphogenetic protein 4 (BMP4) and connective tissue growth factor (CTGF) in ACCAr cells were determined using qRT-PCR. The results of qRT-PCR were represented as relative ratio to ACC-2 cells (Mean ± SE, **p<0.01 versus ACC-2, ##p<0.01 versus ACC-M).
Figure 4.
Involvement of EGFR/PI3K/Akt pathway in acquisition of anoikis resistance and EMT-like transformation in ACCAr cells.
A, The mRNA expression levels of EGF in ACCAr cells were measured using qRT-PCR, and results were expressed as relative ratio to ACC-2 cells (Mean ± SEM, **p<0.01 versus ACC-2, ##p<0.01 versus ACC-M). B, The protein expression levels of EGFR, PI3K, Akt, and their phosphorylated forms in ACC and ACCAr cells were determined using Western blot analysis. C, The transformed ACC-2 cells (both ACCAr and EGF-treated cells) were treated with gefitinib (1 µM) and LY294002 (10 µM) for 12 h before detection. The parental ACC-2 cells were used as Control. The expression levels of the phosphorylated form of EGFR, Akt, and E-cadherin, vimentin, and Slug were subjected to Western blot analysis. D, A mesenchymal morphology as well as epithelial (E-cadherin) and mesenchymal (vimentin) markers were detected in ACC cells with indicated treatment as describe above. E, The apoptosis rate in suspension was analyzed using Cell Death Detection ELISAPLUS (Mean ± SE, *p<0.05, **p<0.01 versus ACC-2; ##p<0.01 versus ACC-2Ar). F, The migration of ACC cells with indicated treatment was analyzed using transwell Boyden system.
Figure 5.
Prevention of ACCAr and EGF-induced metastasis in vivo by gefitinib.
For in vivo metastasis analyses, the pulmonary metastatic models of mice were established by injected with ACC cells with indicated treatment by tail vein. The ACC-2 cells were pretreated with EGF (10 ng/ml) for 48 h, and gefitinib (1 µM) for 1 h, as well as ACC-2Ar were used in the assays. ACC-2 parental cells were also injected as Control. A, Representative hematoxylin and eosin-stained histological sections of lungs from the mice injected with ACC cells. B, Quantitative analysis of pulmonary metastasis from mice as described above. Lesions of 0.1 mm or higher were counted from 20 slides per treatment (Mean ± SE, n = 5 for each group, *p<0.05 versus ACC-2). C, Survival curve of the mice with indicated treatments. The survival analysis were performed by OriginPro (n = 5 for each group, *p<0.05 versus ACC-2). D, Survival times of the mice as indicated treatments were expressed as Mean and SD.
Figure 6.
Requirement of Slug-mediated EMT in acquisition of anoikis resistance and migration/invasion.
ACC-2Ar and ACC-2 were transfected with Slug siRNA and NC siRNA. The ACC-2-transfected cells were then stimulated with EGF (10 ng/ml). A, The protein expression levels of Slug, E-cadherin, and vimentin were determined using Western blot analysis. B, The morphology and expression of E-cadherin were analyzed by immunofluorescence. C, Apoptosis in suspension was measured using Cell Death Detection ELISAPLUS (Mean ± SE, **p<0.01 versus ACC-2Ar with NC siRNA treatment). D, Anchorage-independent growth was analyzed using soft agar assay. Quantitative analyses were performed by counting the visible clones using ImageJ. The results were expressed as relative ratio to ACC-2 with pEGFP transfection (Mean ± SE, **p<0.05 versus ACC-2 with NC siRNA transfection, ##p<0.05 versus ACC-2Ar with NC siRNA transfection). E, ACC-2 cells were transfected with NC siRNA and Slug siRNA, respectively, and then cultured in suspension. The EGFP protein was carried by the lentivirus as marker. The number of EGFP-positive cells was counted by flow cytometry and represented as relative ratio to ACC-2 cells cultured in adhesion (Mean ± SE, *p<0.05 versus ACC-2 in adhesion). F, Migration (top panel) and invasion (low panel) were determined using transwell Boyden systems coated without and with Matrigel, respectively.
Figure 7.
Prevention of apoptosis in ACC cells in suspension by EMT induction.
The ACC-2 cells were transfected with pEGFP, pEGFP-Twist, and pEGFP-Slug. A, The morphology and location of E-cadherin were analyzed by immunofluorescence. B, The protein expression levels of Slug, Twist, E-cadherin, and vimentin were determined using Western blot analysis. C, Apoptosis in suspension was measured using Cell Death Detection ELISAPLUS (Mean ± SE, *p<0.05 versus ACC-2 with pEGFP transfection, #p<0.05 versus pEGFP-Twist group). D, Anchorage-independent growth was analyzed using soft agar assay Quantitative analyses were performed by counting the visible clones using ImageJ. The results were expressed as relative ratio to ACC-2 with pEGFP transfection (Mean ± SE, **p<0.05 versus ACC-2 with pEGFP transfection, ##p<0.05 versus pEGFP-Twist group). E, Migration and invasion were determined using transwell Boyden systems coated without and with Matrigel, respectively. F, The mRNA expression levels of VEGF, MMP9, and SDF-1α were analyzed using qRT-PCR, and the results were expressed as relative ratio to ACC-2 transfected with pEGFP (*p<0.05 versus ACC-2 pEGFP group, **p<0.01 versus ACC-2 pEGFP group).
Figure 8.
Expression levels of EGFR, Slug, E-cadherin, and MMP9 in ACC tissues.
A, Representative immunohistochemical staining of EGFR, Slug, E-cadherin, and MMP9 in human adenoid cystic carcinoma and negative gland tissue (NSG). B, Spearman’s correlation were used to determine the relationship between EGFR and Slug (left panel) and that between Slug and E-cadherin (right panel) (p<0.05). Histoscore based on quantification as described in the method and statistics with OriginPro.