Background
Methods
Cell line and culture conditions
Immunocytochemistry
Immunohistochemistry
Obtaining red-channel images
Sulforhodamine B cell proliferation assay
Creation of modified cell lines
Cell line | Clone ID | Sequence |
---|---|---|
468-shCDH1-A | V2LHS_14834 | CTGTTGGTGTCTTTATTAT |
468-shCDH1-B | V2LHS_14838 | GTCGTAATCACCACACTGA |
468-shCDH1-C | V2LHS_14837 | CCAACTGGCTGGAGATTAA |
468-shCDH1-D | V2LHS_ 243170 | GAGAGAGTTTCCCTACGTA |
Western blotting and quantitative real-time polymerase chain reaction experiments
Species | Source | Catalogue number | |
---|---|---|---|
Primary antibody | |||
Human E-cadherin | Rabbit | Abcam, Cambridge, UK | Ab15148 |
Human E-cadherin | Mouse | BD Biosciences, San Jose, CA, USA | 610404 |
Human vimentin | Mouse | Dako, Glostrup, Denmark | M0725 |
α-Pan-actin | Mouse | Abcam, Cambridge, UK | Ab14128 |
Human mitochondrial | Mouse | Thermo Fisher Scientific, Waltham, MA, USA | MA5-12017 |
Mouse CD31 | Rat | BD Pharmingen, San Diego, CA, USA | 550274 |
Human HIF-1α | Mouse | BD Biosciences, San Jose, CA, USA | 610958 |
Secondary antibody | |||
Antimouse biotin | Rabbit | Dako, Campbellfield, Australia | E0354 |
Antirabbit biotin | Swine | Dako, Campbellfield, Australia | E0431 |
Antirat biotin | Goat | Vector Laboratories, Burlingame, CA, USA | BA-9401 |
Antirabbit Alexa Fluor® 594 | Donkey | Molecular Probes, Eugene, OR, USA | R37119 |
Antimouse Alexa Fluor® 488 | Goat | Molecular Probes, Eugene, OR, USA | A11001 |
Tertiary label | |||
Streptavidin/HRP | Dako, Campbellfield, Australia | P0397 |
Primer name | Nucleotide sequence | |
---|---|---|
L32 | RT sequence | CAGAAAACGTGCACATGAGCTGC |
Forward sequence (outer nested) | CAGGGTTCGTAGAAGATTCAAGGG | |
Reverse sequence (outer nested) | CTTGGAGGAAACATTGTGAGCGATC | |
Forward sequence (inner nested) | GATCTTGATGCCCAACATTGGTTATG | |
Reverse sequence (inner nested) | GCACTTCCAGCTCCTTGACG | |
Carbonic Anhydrase 9 (for hypoxia validation) | Random priming used | |
Forward | CCTCAAGAACCCCAGAATAATGC | |
Reverse | CCTCCATAGCGCCAATGACT | |
E-cadherin | RT sequence | GTCAGCCAGCTTCTTGAAGCGATT |
Forward sequence | GCCCTGCCAATCCCGATGAAA | |
Reverse sequence | GGGGTCAGTATCAGCCGCT | |
Vimentin | RT sequence | CCGTGAGGTCAGGCTTGGAAA |
Forward sequence | GCTTCAGAGAGAGGAAGCCGAAAA | |
Reverse sequence | CCGTGAGGTCAGGCTTGGAAA | |
Endogenous E-cadherin (CDH1-3′UTR) | RT sequence | GCACTTGGGGATTCTGGGCTTT |
Forward sequence | GTGCCTAAAGTGCTGCAGCCAAA | |
Reverse sequence | GTACAAACCACGGATCTTGTGTCAGAAA | |
Exogenous E-cadherin (for 468-CDH1 construct) | RT sequence | GAAAGGACAGTGGGAGTGGCACTTT |
Forward sequence | CCTGAACTCCTCAGAGTCAGACAAA | |
Reverse sequence | GTGGCACCTTCCAGGGTCAAGGAA | |
E-cadherin shRNA (for 468-shCDH1 constructs) | RT sequence | CCAGCTCAGCCCGAGTGGAAAT |
Forward sequence | CCTCCCATCAGCTGCCCAGAAAA | |
Reverse sequence | CTCTGTCACCTTCAGCCATCCTGTTT | |
Snail 1 | RT sequence | CGCAGACAGGCCAGCTCAGGAAT |
Forward sequence | CACATCCTTCTCACTGCCATGGAATT | |
Reverse sequence | GCTGCCCTCCCTCCACAGAAAT | |
Estrogen Receptor 1 | RT sequence | CCAGGGCCACGCTGGGAAATGAA |
Forward sequence | GTTCCAGTGGGCACTGTACTTGGATCTT | |
Reverse sequence | CAGCTCCATGCCCCAGGGCTAAAT |
Transwell chemotaxis migration assay
Matrigel outgrowth assay (nubbin assay)
Inoculation of severe combined immunodeficiency mice with E-cadherin-modified cell lines
Determining tumor/necrosis ratio
Analysis of human clinical data
Statistical analyses
Results
MDA-MB-468 tumors exhibit regions of EMP associated with hypoxia
Metastases of MDA-MB-468 xenograft primary tumors exhibit evidence of epithelial differentiation, consistent with MET
Group | Number observed | With lung metastasis | Percentage of metastasis |
---|---|---|---|
468-shSCR | 10 | 7 | 70 |
468-shCDH1-B | 10 | 3 | 30 |
468-shCDH1-D | 10 | 5 | 50 |
468-pcDNA3 | 9 | 5 | 55.6 |
468-CDH1 | 10 | 7 | 70 |
Creation and validation of E-cadherin-modified MDA-MB-468 cell lines
Forced E-cadherin expression blocked cellular invasion, whereas its knockdown stimulated invasion and reduced proliferative growth
E-cadherin-manipulated tumors grew slower than their respective controls
Group | Number of mice inoculated | Tumors | Died early | Tissue harvest |
---|---|---|---|---|
468-WT | 13 | 13 | 0 | 13 |
468-shSCR | 13 | 13 | 0 | 13 |
468-shCDH1-B | 13 | 13 | 0 | 13 |
468-shCDH1-D | 13 | 13 | 01 | 12 |
468-pcDNA3 | 12 | 09 | 0 | 12 |
468-CDH1 | 12 | 12 | 0 | 12 |
E-cadherin-knockdown tumors are less necrotic
Tumor microvasculature is more developed in 468-shCDH1-B xenografts
EMT in xenograft tumors from E-cadherin-manipulated MDA-MB-468 cells
E-cadherin-knockdown cells displayed EMT in vivo
E-cadherin was expressed in tumor emboli located within local vasculature, regardless of E-cadherin manipulation status
Group | Number of mice inoculated | With tumors | With local invasion | Percentage of invasion |
---|---|---|---|---|
468-shSCR | 13 | 13 | 8 | 61.5 |
468-shCDH1-B | 13 | 13 | 8 | 61.5 |
468-shCDH1-D | 12 | 12 | 11 | 91.7 |
468-pcDNA3 | 12 | 9 | 5 | 41.7 |
468-CDH1 | 12 | 12 | 11 | 91.7 |