analysis. Flowjo was used to generate the dot plots.
Lentiviral infection. Lentivirus containing short hairpin RNAs (shRNAs) expressed in a lentiviral vector (pLKO.1-puro) were generated in 293T cells as previously described48
7. Plasmids pLKO-Luciferase-i, pLKO-KLF4-i-3, pLKO-KLF4-i-934, pLKO-GFP, and packaging plasmid pCMVΔR8.91 were obtained from S.C. Teng (National Taiwan University, Taiwan). Plasmids 2 and pLKO-BMI1-i-3 were provided by National RNAi Core Facility of Academia Sinica, Taipei, Taiwan.
For lentivirus production, 293T cells were transfected with 15 μg pLKO.1-puro lentiviral vectors expressing different shRNAs along with 1.5 μg of envelope plasmid pMD.G and 15 μg of packaging plasmid pCMVΔR8.91. Virus was collected 48 h after transfection. To prepare KLF4 or Bmi1 knockdown cells, OECM-1-Twist1 cells were infected with lentivirus for 24 h, and stable clones were generated by selection with appropriate antibiotics. The sequences for the lentivirus were shown in Supplementary Table S4.
2.5 Dimensional (2.5D) tube formation assay49
8. For experiments in which cells were cultured on top of the Matrigel, 50 l growth factor reduced Matrigel were plated in 8-well chamber slides. The chambers were then incubated at 37°C for 30 min to allow Matrigel to polymerize. 1 × 104 cells were added to the top of the
Matrigel in each well. After incubation for 5 days, the slides were examined for endothelial tubes using light microscope. Five random images per well were quantified by the MetaMorph Imaging System, an image analysis program a digital imaging software to analyze biological images. The Angiogenesis Tube Formation Module was used to analyze tube formation activity. The image was run through the software to identify tubes or nodes. A pixel is a single point in a graphic image as a
unit of measurement.
Three dimensional (3D) tube formation assay. For 3D culture, tumor cells were resuspended in 5 mg/ml-1
growth factor reduced Matrigel (BD Biosciences) on ice. 1
× 104 cells each well were seeded in 8-well chamber slides and allowed to polymerize at 37°C for 30 min followed by adding 10% serum-containing MCDB131 culture medium to the top of the wells. The chambers were then incubated at 37°C. After incubation for 5 days, the slides were used for DiI-AcLDL uptake analysis and
immunofluorescence assay.
DiI-AcLDL uptake analysis. The functional assay for endothelial cells was performed by incubation of cells with 10 g/ml of DiI-labeled acetylated low density lipoproteins (DiI-AcLDL)(Molecular Probes, Invitrogen) for 4 h followed by observation by fluorescence microscope. A picture with 3D tumor formation assay was usually accompanied with the LDL uptake picture to indicate the presence of cell
clones tested.
In vivo sSubcutaneous implantation and, hemoglobin quantification., and drug
treatment of xenografted mice. For in vivo monitoring of tumor-associated angiogenesis, 1 x 106 cells of different cell lines in 0.1 ml Matrigel were injected under the abdominal skin of nude 6 week old female Balb/c nude mice. After tumor formation (4 to 6 weeks after injection), animals were sacrificed and matrigel implants were collected, weighed, and fixed in 4% paraformaldehyde for IHC analysis or in PBS for hemoglobin quantification. Hemoglobin contents of matrigel implants were measured using a Drabkin’s reagent kit (Sigma) as described previously49
8. The tissue was transferred into the Eppendorf containing 0.5 ml PBS buffer under overnight incubation at 37℃ to lyse RBC and release hemoglobin. The tissue samples were homogenized and spun down to collect the clear supernatant.
Samples (50 l) of tissue supernatant were added to 1 ml Drabkin’s reagent and then incubated for 15 minutes at room temperature. Absorbance of each sample was measured at 540 nm. The results are divided by tissue weight and expressed as hemoglobin concentration (mgmg-1
).
Drug treatment of xenografted mice. In the xenotransplanted tumor model, the OECM1-Twist1 cells (2x106) in 0.2 mL PBS were injected into the right lateral flank of 6 week old female Balb/c Nude mice. Treatment was started when the tumors grew
to certain size. Mice were treated once a week by intraperitoneal injections (i.p.) of 10 (Selleck Chemicals., Houston, USA) or both together. The tumor size of each group were monitored and measured every week. Tumor volume (mm3) was estimated
according to the formula of length x (width) 2 x 0.5 in mm3. Tumor sphere formation24
. The procedure was performed as described24. Cells (1
×103) were seeded and incubated with serum-free medium composed of DMEM/F-12 (Thermo Fisher Scientific Inc. United States), N2 supplement (Thermo Fisher Scientific Inc. United States), 10 ng/ml-1
human recombinant bFGF (R&D Systems, Inc. United States) and 10 ng/ml ml-1
EGF (R&D Systems, Inc. United States) at 24 well plate. The spheroids were resuspended to form secondary and tertiary spheroids.
Only the tumor spheres with a size greater than 100m were counted. The number of
spheroids was counted after 14 days.
Tissue microarray construction50
. A high-density tissue microarray (TMA) was constructed using formalin-fixed, paraffin-embedded specimens of HNSCC patient samples as previously described11. H&E-stained sections were made from each paraffin block to define representative tumor regions. Tissue cylinders with a diameter of 0.6 mm were punctured from these regions in the paraffin block and arrayed into a recipient block using the tissue chip microarrayer (Beecher Instruments, Silver
Spring, MD). The recipient block was serially cut into 5-μm sections on silanized slides (Dako, Glostrup, Denmark). Informed consent was obtained from all subjects
and Tthe protocol was approved by the IRB of the Taipei Veterans General Hospital.
In situ hybridization. The paraffin-embedded tissue specimens were de-paraffinized in xylene, rehydrated through 100%, 90%, 70%, 50% ethanol, and rinsed in PBS buffer. Antigen retrieval processes were then performed twice by pressure cooker at 1.2 atm for 15 min with target retrieval solution (#S1700, Dako Corporation, Denmark) and then blocked with 0.3% hydrogen peroxide for 30 minutes. 20 uL of the biotinylated probes for general screening (HPV 6, 11, 16, 18, 30, 31, 33, 45, 51, 52) (#Y1404, DAKO) were applied on separate specimen, and were covered with coverslips. Denaturation of DNA was performed in 92° C for 5 minutes in an oven.
Hybridization was performed in a humid chamber at 37° C overnight. After washing, the GenPoint Kit (#K0620; DAKO) was used according to the manufacturer's
instruction.
Immunohistochemstry (IHC), scoring, and validation of antibodies11,24,47. The sample processing and IHC procedure for determining the immunoreactivity of Twist1, Jagged1, and KLF4 were described as the following.11,24,46. Briefly, The paraffin-embedded tissue specimens were de-paraffinized in xylene, rehydrated through 100%, 90%, 70%, 50% ethanol, and rinsed in PBS buffer. Antigen retrieval
processes were then performed by pressure cooker at 1.2 atm for 15 min with 1 mM sodium citrate buffer (pH 6.0) and then blocked with 3% hydrogen peroxide for 30 minutes. The specimens were incubated with primary antibodies in 5% BSA buffer in a humidity chamber overnight at 40C. Detection of the staining was used by the Super Sensitive Non-Biotin Polymer HRP Detection System according to the manufacturer’s instructions (BioGenex, San Ramon, CA). The immunoreactivity of Twist1, Jagged1 and KLF4 was graded from 0 to 3+ (0, no staining; 1+, 1 25%; 2+, 26 50%; 3+, >50% nuclear staining) according to nuclear expression, and only 3+
(>50% nuclear staining) was considered as a positive immunohistochemistry result.
All IHC staining was independently scored by two experienced pathologists. If there was discordance with IHC scoring, a pathologic peer review will be performed to consolidate the result into a final score. The pathologists scoring the IHC were blinded to the clinical data. The interpretation was performed in five high power views for each slide, and 100 cells per for each view were counted for analysis.
Validation of Twist1 antibody was described11. Validation of antibodies against Jagged1 and KLF4 was performed from various overexpression and knockdown experiments (e.g. Jagged1 from Supplementary Figs. 1611a,b, 1712b,c; KLF4 from Fig. 46a, Supplementary Fig. S3121b) as well as from the manufacturer’s protocols.
Statistical analysis. The independent Student’s t-test was used to compare the
continuous variables between two groups, and the 2 test was applied for comparison of dichotomous variables in tables. The control groups of all the statistical analyses were specified in the figure legends. The level of statistical significance was set at
0.05 for all tests unless specified.
Deposition of microarray data. The raw data of microarray (H1299 control vs.
H1299-Twist1-si) were deposited to GenBank in MIAME format and the accession number is GSE57756.
References
1. Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell
144, 646-674 (2011).
2. Le Bras, A. et al. Molecular mechanism of endothelial differentiation. Vasc. Med.
15, 321 (2010).
3. Weis, S.M. & Cheresh, D.A. Tumor angiogenesis: molecular pathways and
therapeutic targets. Nature Med. 17, 1359-1370 (2011).
4. Pezzolo, A. et al. Tumor origin of endothelial cells in human neuroblastoma. J.
Clin. Oncol. 25, 376-383 (2007).
5. Streubel, B. et al. Lymphoma-specific genetic aberrations in microvascular
endothelial cells in B-cell lymphoma. N. Engl. J. Med. 351, 250-259 (2004).
6. Ricci-Vitiani, L. et al. Tumor vascularization via endothelial differentiation of
glioblastoma stem-like cells. Nature 468, 824-828 (2010).
7. Soda, Y. et al. Transdifferentiation of glioblastoma cells into vascular endothelial
cells. Proc. Natl. Acad. Sci. USA 108, 4274-4280 (2011).
8. Wang, R. et al. Glioblastoma stem-like cells give rise to tumor endothelium.
Nature 468, 829-833 (2010).
9. Furlong, E.E. et al. Patterns of gene expression during Drosophila mesoderm development. Science 293, 1629-1633 (2001).
10. Thiery, J.P. et al. Epithelial-mesenchymal transitions in development and disease.
Cell 139, 871-890 (2009).
11. Yang, M.H. et al. Direct regulation of TWIST by HIF-1promotes metastasis.
Nat. Cell Biol. 10, 295-305 (2008).
12. Yang, M.H. & Wu, K.J. TWIST activation by hypoxia inducible factor-1 (HIF-1):
implications in metastasis and development. Cell Cycle 7, 2090-2096 (2008).
13. Dufraine, J. et al. Notch signaling regulates tumor angiogenesis by diverse
mechanisms. Oncogene 27, 5132-5137 (2008).
14. Bridges, E. et al. Notch regulation of tumor angiogenesis. Future Oncol. 7,
569-588 (2011).
15. Zeng, Q. et al. Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling. Cancer Cell 8, 13-23
(2005).
16. Yamanaka, S. & Blau, H.M. Nuclear reprogramming to a pluripotent state by
three approaches. Nature 465, 704-712 (2010).
17. Suzuki, T. et al. Vascular implications of the Kruppel-like family of transcription
factors. Arterioscler. Thromb. Vasc. Biol. 25, 1135-1141 (2005).
18. Yet, S.F. et al. Human EZF, a Kruppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains. J. Biol. Chem. 273, 1026-1031 (1998).
19. Teo, A.K. et al. Pluripotency factors regulate definitive endoderm specification
through eomesodermin. Genes Dev. 25, 238-250 (2011).
20. Lawson, C. & Wolf, S. ICAM-1 signaling in endothelial cells. Pharmacol. Rep.
61, 22-32 (2009).
21. Panwar, A. et al. Human papilloma virus positive oropharyngeal squamous cell
carcinomas: a growing epidemic. Cancer Treat. Rev. 40, 215-219 (2014).
22. Adams, R.H. Molecular control of arterial-venous blood vessel identity. J. Anat.
202, 105-121 (2003).
23. You, L.R. et al. Suppression of Notch signaling by the COUP-TFII transcription
factor regulates vein identity. Nature 435, 98-104 (2005).
24. Yang, M.H. et al. Bmi1 is essential in Twist1-induced epithelial-mesenchymal
transition. Nat. Cell Biol. 12, 982-992 (2010).
25. Suzuki, T. et al. Vascular implications of the Kruppel-like family of transcription
factors. Arterioscler. Thromb. Vasc. Biol. 25, 1135-1141 (2005).
26. Lu, J. et al. Endothelial cells promote the colorectal cancer stem cell phenotype
through a soluble form of Jagged-1. Cancer Cell. 23,171-185 (2013).
27. Lee, J. et al. Genetic reconstruction of mouse spermatogonial stem cell
self-renewal in vitro by Ras-cyclin D2 activation. Cell Stem Cell 5, 76-86 (2009).
28. Yang, D.H. et al. Wnt5a is required for endothelial differentiation of embryonic stem cells and vascularization via pathways involving both Wnt/-catenin and protein kinase C. Circ. Res. 104, 372-379 (2009).
29. Yeh, J.R. et al. Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse
spermatogonial stem cells. J Cell Sci. 124, 2357-2366 (2011).
30. Wu, C.Y. et al. Epigenetic reprogramming and post-transcriptional regulation of
the epithelial-mesenchymal transition. Trends Genet 28, 454-463 (2012).
31. Gerber, D.E. & Choy, H. Cetuximab in combination therapy: from bench to clinic.
Cancer Metastasis Rev. 29, 171-180 (2010).
32. Vokes, E.E. Induction chemotherapy for head and neck cancer: recent data.
Oncologist 15 (Suppl 3), 3-7 (2010).
33. Sjolund, J. et al. Suppression of renal cell carcinoma growth by inhibition of
Notch signaling in vitro and in vivo. J. Clin. Invest. 118, 217-228 (2008).
34. Kirschmann, D.A. et al. Molecular pathways: vasculogenic mimicry in tumor cells: diagnostic and therapeutic implications. Clin. Cancer Res. 18, 2726-2732
(2012).
35. Sun, T. et al. Expression and functional significance of Twist1 in hepatocellular
carcinoma: its role in vasculogenic mimicry. Hepatology 51, 545-556 (2010).
36. Chen, Z.F. & Behringer, R.R. twist is required in head mesenchyme for cranial
neural tube morphogenesis. Genes Dev. 9, 686-699 (1995).
37. Yen, H.Y. et al. Jagged1 functions downstream of Twist1 in the specification of the coronal suture and the formation of a boundary between osteogenic and non-osteogenic cells. Dev. Biol. 347, 258-270 (2010).
38. Ghaleb, A.M. et al. Notch inhibits expression of the Kruppel-like factor 4 tumor
suppressor in the intestinal epithelium. Mol. Cancer Res. 6, 1920-1927 (2008).
39. Miyamoto, S. et al. Suppression of colon carcinogenesis by targeting Notch
signaling. Carcinogenesis 34, 2415-2423 (2013).
40. Zhao, W. et al. Identification of Kruppel-like factor 4 as a potential tumor
suppressor gene in colorectal cancer. Oncogene 23, 395-402 (2004).
41.Wei, D. et al. Kruppel-like factor 4 induced p27Kip1 expression in and suppresses the growth and metastasis of human pancreatic cancer cells. Cancer Res. 68,
4631-4639 (2008)
42.Zammarchi, F. et al. KLF4 is a novel candidate tumor suppressor gene in pancreatic ductal carcinoma. Am. J. Pathol. 178, 361-372 (2011).
43. Rowland, B.D. et al. The KLF4 tumor suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nat. Cell Biol. 7, 1074-1082
(2005).
44. Yu, F. et al. Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion. Oncogene 30, 2161-2172
(2011).
Wei, D. et al. KLF4 up-regulation promotes cell cycle progression and reduces survival time of patients with pancreatic cancer. Gastroenterology 139, 2135-2145 (2010).
45. Tai, S.K. et al. Persistent Kruppel-like factor 4 expression predicts progression
and poor prognosis of head and neck squamous cell carcinoma. Cancer Sci. 102,
895-902 (2011).
46.Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25,
402-408 (2001).
47.Wu, M.Z. et al. Interplay between HDAC3 and WDR5 is essential for
hypoxia-induced epithelial-mesenchymal transition. Molecular Cell 43, 811-822 (2011).
48.Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied
to an arrayed viral high-content screen. Cell 124, 1283-1298 (2006).
49.Passaniti, A. et al. A simple, quantitative method for assessing angiogenesis and
antiangiogenic agents using reconstituted basement membrane, heparin, and
fibroblast growth factor. Lab. Invest. 67, 519-528 (1992).
50.Freier, K. et al. Tissue microarray analysis reveals site-specific prevalence of oncogene amplifications in head and neck squamous cell carcinoma. Cancer Res.
63, 1179-1182 (2003).
Acknowledgments
We appreciate Dr. L.R. You for critical comments on the manuscript. We thank Dr. H.W. Wang, Dr. T.Y. Chou, C.Y. Hsieh and Yang-Ming Genome Research Center for technical assistance. This work was supported in part to K.J.W.
by National Science Council Frontier grant (NSC101-2321-B-010-002, NSC102-2321-B-010-001), Keelung Chang-Gang Memorial Hospital (CMRPG2D0031), National Research Program for Biopharmaceuticals (NSC102-2325-B-010-004), a grant from Ministry of Education, Aim for the Top University Plan (102AC-TC13, 103AC-T301), center of excellence for cancer research at Taipei Veterans General Hospital (DOH102-TD-C-111-007, MOHW103-TD-B-111-02), Taichung Veterans General Hospital (TCVGH-YM1000301, TCVGH-YM1010301), and National Health Research Institutes (NHRI-EX102-10230SI, NHRI-EX103-10230SI).