The transcriptome of lung tumor-infiltrating dendritic cells reveals a tumor-supporting phenotype and a microRNA signature with negative impact on clinical outcome

OncoImmunology

Volumn 6, Issue 1, 2017, Pages

  Pyfferoen, Lotte ^a,b   Brabants, Elisabeth ^a   Everaert, Celine ^a   De Cabooter, Nancy ^a   Heyns, Kelly ^a   Deswarte, Kim ^b   Vanheerswynghels, Manon ^b   De Prijck, Sofie ^b   Waegemans, Glenn ^c   Dullaers, Melissa ^a,b   Hammad, Hamida ^b   De Wever, Olivier ^c   Mestdagh, Pieter ^a   Vandesompele, Jo ^a   Lambrecht, Bart N ^b,d   Vermaelen, Karim Y ^a  

^a GHENT UNIVERSITY HOSPITAL   (Belgium)

^b DEPARTMENT OF PLANT SYSTEMS BIOLOGY   (Belgium)

^c GHENT UNIVERSITY   (Belgium)

^d ERASMUS MEDICAL CENTER   (Netherlands)

Author keywords

Dendritic cell; Hypoxia; Lung cancer; MiR 31; PD L1

Indexed keywords

CD103 ANTIGEN; CD11B ANTIGEN; CD64 ANTIGEN; MICRORNA; MICRORNA 31; TRANSCRIPTOME;

ANIMAL CELL; ARTICLE; CELL INFILTRATION; CELL INVASION ASSAY; CLINICAL OUTCOME; CONTROLLED STUDY; DENDRITIC CELL; FLOW CYTOMETRY; FLUORESCENCE ACTIVATED CELL SORTING; GENE OVEREXPRESSION; GENETIC TRANSFECTION; HISTOLOGY; HYPOXIA; IMMUNOCOMPETENT CELL; IMMUNOFLUORESCENCE; IMMUNOSUPPRESSIVE TREATMENT; LUNG PARENCHYMA; LUNG TUMOR; MICROARRAY ANALYSIS; MOUSE; NONHUMAN; OVERALL SURVIVAL; PROTEIN EXPRESSION; PROTEOMICS; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; TUMOR ASSOCIATED LEUKOCYTE; TUMOR IMMUNOGENICITY; TUMOR MICROENVIRONMENT; UPREGULATION;

EID: 85007408216     PISSN: 21624011     EISSN: 2162402X     Source Type: Journal    
DOI: 10.1080/2162402X.2016.1253655     Document Type: Article

References (63)

1. Kastenmuller W, Kastenmuller K, Kurts C, Seder RA. Dendritic cell-targeted vaccines—hope or hype? Nat Rev Immunol 2014; 14:705-11; PMID:25190285; http://dx.doi.org/10.1038/nri3727
2. Palucka K, Banchereau J. Cancer immunotherapy via dendritic cells. Nat Rev Cancer 2012; 12:265-77; PMID:22437871; http://dx.doi.org/10.1038/nrc3258
3. Koebel CM, Vermi W, Swann JB, Zerafa N, Rodig SJ, Old LJ, Smyth MJ, Schreiber RD. Adaptive immunity maintains occult cancer in an equilibrium state. Nature 2007; 450:903-7; PMID:18026089; http://dx.doi.org/10.1038/nature06309
4. Remark R, Becker C, Gomez JE, Damotte D, Dieu-Nosjean MC, Sautes-Fridman C, Fridman WH, Powell CA, Altorki NK, Merad M et al. The non-small cell lung cancer immune contexture. A major determinant of tumor characteristics and patient outcome. Am J Respir Critical Care Med 2015; 191:377-90; PMID:25369536; http://dx.doi.org/1242963810.1164/rccm.201409-1671PP
5. Inoshima N, Nakanishi Y, Minami T, Izumi M, Takayama K, Yoshino I, Hara N. The influence of dendritic cell infiltration and vascular endothelial growth factor expression on the prognosis of non-small cell lung cancer. Clin Cancer Res 2002; 8:3480-6; PMID:12429638
6. Goc J, Germain C, Vo-Bourgais TK, Lupo A, Klein C, Knockaert S, de Chaisemartin L, Ouakrim H, Becht E, Alifano M et al. Dendritic cells in tumor-associated tertiary lymphoid structures signal a Th1 cytotoxic immune contexture and license the positive prognostic value of infiltrating CD8+ T cells. Cancer Res 2014; 74:705-15; PMID:24366885; http://dx.doi.org/10.1158/0008-5472.CAN-13-1342
7. Zong J, Keskinov AA, Shurin GV, Shurin MR. Tumor-derived factors modulating dendritic cell function. Cancer Immunol Immunother 2016; 65:821-33; PMID:26984847; http://dx.doi.org/10.1007/s00262-016-1820-y
8. Perrot I, Blanchard D, Freymond N, Isaac S, Guibert B, Pacheco Y, Lebecque S. Dendritic cells infiltrating human non-small cell lung cancer are blocked at immature stage. J Immunol 2007; 178:2763-9; PMID:17312119; http://dx.doi.org/1929318610.4049/jimmunol.178.5.2763
9. Norian LA, Rodriguez PC, O'Mara LA, Zabaleta J, Ochoa AC, Cella M, Allen PM. Tumor-infiltrating regulatory dendritic cells inhibit CD8+ T cell function via L-arginine metabolism. Cancer Res 2009; 69:3086-94; PMID:19293186; http://dx.doi.org/10.1158/0008-5472.CAN-08-2826
10. Zheng X, Koropatnick J, Chen D, Velenosi T, Ling H, Zhang X, Jiang N, Navarro B, Ichim TE, Urquhart B et al. Silencing IDO in dendritic cells: a novel approach to enhance cancer immunotherapy in a murine breast cancer model. Int J Cancer 2013; 132:967-77; PMID:22870862; http://dx.doi.org/10.1002/ijc.27710
11. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol 2012; 12:253-68; PMID:22437938; http://dx.doi.org/10.1038/nri3175
12. Plantinga M, Guilliams M, Vanheerswynghels M, Deswarte K, Branco-Madeira F, Toussaint W, Vanhoutte L, Neyt K, Killeen N, Malissen B et al. Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen. Immunity 2013; 38:322-35; PMID:23352232; http://dx.doi.org/10.1016/j.immuni.2012.10.016
13. Shi L, Reid LH, Jones WD, Shippy R, Warrington JA, Baker SC, Collins PJ, de Longueville F, Kawasaki ES, Lee KY et al. The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol 2006; 24:1151-61; PMID:16964229; http://dx.doi.org/10.1038/nbt1239
14. Shen XZ, Okwan-Duodu D, Blackwell WL, Ong FS, Janjulia T, Bernstein EA, Fuchs S, Alkan S, Bernstein KE. Myeloid expression of angiotensin-converting enzyme facilitates myeloid maturation and inhibits the development of myeloid-derived suppressor cells. Lab Invest 2014; 94:536-44; PMID:24614194; http://dx.doi.org/10.1038/labinvest.2014.41
15. Deng J, Fujimoto J, Ye XF, Men TY, Van Pelt CS, Chen YL, Lin XF, Kadara H, Tao Q, Lotan D et al. Knockout of the tumor suppressor gene Gprc5a in mice leads to NF-kappaB activation in airway epithelium and promotes lung inflammation and tumorigenesis. Cancer Prevent Res 2010; 3:424-37; PMID:20354164; http://dx.doi.org/10.1158/1940-6207.CAPR-10-0032
16. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005; 102:15545-50; PMID:16199517; http://dx.doi.org/10.1073/pnas.0506580102
17. Balwierz PJ, Pachkov M, Arnold P, Gruber AJ, Zavolan M, van Nimwegen E. ISMARA: automated modeling of genomic signals as a democracy of regulatory motifs. Genome Res 2014; 24:869-84; PMID:24515121; http://dx.doi.org/10.1101/gr.169508.113
18. Belladonna ML, Renauld JC, Bianchi R, Vacca C, Fallarino F, Orabona C, Fioretti MC, Grohmann U, Puccetti P. IL-23 and IL-12 have overlapping, but distinct, effects on murine dendritic cells. J Immunol 2002; 168:5448-54; PMID:12023338; http://dx.doi.org/2205310610.4049/jimmunol.168.11.5448
19. Fukao T, Frucht DM, Yap G, Gadina M, O'Shea JJ, Koyasu S. Inducible expression of Stat4 in dendritic cells and macrophages and its critical role in innate and adaptive immune responses. J Immunol 2001; 166:4446-55; PMID:11254700; http://dx.doi.org/2205310610.4049/jimmunol.166.7.4446
20. Wu L, Yan C, Czader M, Foreman O, Blum JS, Kapur R, Du H. Inhibition of PPARgamma in myeloid-lineage cells induces systemic inflammation, immunosuppression, and tumorigenesis. Blood 2012; 119:115-26; PMID:22053106; http://dx.doi.org/10.1182/blood-2011-06-363093
21. Xu Y, Zhan Y, Lew AM, Naik SH, Kershaw MH. Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking. J Immunol 2007; 179:7577-84; PMID:18025203; http://dx.doi.org/2608402910.4049/jimmunol.179.11.7577
22. Helft J, Bottcher J, Chakravarty P, Zelenay S, Huotari J, Schraml BU, Goubau D, Reis e Sousa C. GM-CSF mouse bone marrow cultures comprise a heterogeneous population of CD11c(+)MHCII(+) macrophages and dendritic cells. Immunity 2015; 42:1197-211; PMID:26084029; http://dx.doi.org/10.1016/j.immuni.2015.05.018
23. Shibue T, Brooks MW, Inan MF, Reinhardt F, Weinberg RA. The outgrowth of micrometastases is enabled by the formation of filopodium-like protrusions. Cancer Disco 2012; 2:706-21; PMID:22609699; http://dx.doi.org/10.1158/2159-8290.CD-11-0239
24. Franklin RA, Liao W, Sarkar A, Kim MV, Bivona MR, Liu K, Pamer EG, Li MO. The cellular and molecular origin of tumor-associated macrophages. Science 2014; 344:921-5; PMID:24812208; http://dx.doi.org/10.1126/science.1252510
25. Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erle DJ, Barczak A, Rosenblum MD, Daud A, Barber DL et al. Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell 2014; 26:638-52; PMID:25446897; http://dx.doi.org/10.1016/j.ccell.2014.09.007
26. Lohela M, Casbon AJ, Olow A, Bonham L, Branstetter D, Weng N, Smith J, Werb Z. Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations. Proc Natl Acad Sci USA 2014; 111:E5086-95; PMID:25385645; http://dx.doi.org/10.1073/pnas.1419899111
27. Diao J, Mikhailova A, Tang M, Gu H, Zhao J, Cattral MS. Immunostimulatory conventional dendritic cells evolve into regulatory macrophage-like cells. Blood 2012; 119:4919-27; PMID:22490680; http://dx.doi.org/10.1182/blood-2011-11-392894
28. Peng Y, Latchman Y, Elkon KB. Ly6C(low) monocytes differentiate into dendritic cells and cross-tolerize T cells through PDL-1. J Immunol 2009; 182:2777-85; PMID:19234172; http://dx.doi.org/2709632110.4049/jimmunol.0803172
29. Salmon H, Idoyaga J, Rahman A, Leboeuf M, Remark R, Jordan S, Casanova-Acebes M, Khudoynazarova M, Agudo J, Tung N et al. Expansion and activation of CD103(+) dendritic cell progenitors at the tumor site enhances tumor responses to therapeutic PD-L1 and BRAF inhibition. Immunity 2016; 44:924-38; PMID:27096321; http://dx.doi.org/10.1016/j.immuni.2016.03.012
30. Fehrenbacher L, Spira A, Ballinger M, Kowanetz M, Vansteenkiste J, Mazieres J, Park K, Smith D, Artal-Cortes A, Lewanski C et al. Atezolizumab versus docetaxel for patients with previously treated non-small-cell lung cancer (POPLAR): a multicentre, open-label, phase 2 randomised controlled trial. Lancet 2016; 387:1837-46; PMID:26970723; http://dx.doi.org/10.1016/S0140-6736(16)00587-0
31. Liu Q, Zhang C, Sun A, Zheng Y, Wang L, Cao X. Tumor-educated CD11bhighIalow regulatory dendritic cells suppress T cell response through arginase I. J Immunol 2009; 182:6207-16; PMID:19414774; http://dx.doi.org/2062285910.4049/jimmunol.0803926
32. Herber DL, Cao W, Nefedova Y, Novitskiy SV, Nagaraj S, Tyurin VA, Corzo A, Cho HI, Celis E, Lennox B et al. Lipid accumulation and dendritic cell dysfunction in cancer. Nature Med 2010; 16:880-6; PMID:20622859; http://dx.doi.org/10.1038/nm.2172
33. Blengio F, Raggi F, Pierobon D, Cappello P, Eva A, Giovarelli M, Varesio L, Bosco MC. The hypoxic environment reprograms the cytokine/chemokine expression profile of human mature dendritic cells. Immunobiology 2013; 218:76-89; PMID:22465745; http://dx.doi.org/10.1016/j.imbio.2012.02.002
34. Bosco MC, Pierobon D, Blengio F, Raggi F, Vanni C, Gattorno M, Eva A, Novelli F, Cappello P, Giovarelli M et al. Hypoxia modulates the gene expression profile of immunoregulatory receptors in human mature dendritic cells: identification of TREM-1 as a novel hypoxic marker in vitro and in vivo. Blood 2011; 117:2625-39; PMID:21148811; http://dx.doi.org/10.1182/blood-2010-06-292136
35. Pierobon D, Bosco MC, Blengio F, Raggi F, Eva A, Filippi M, Musso T, Novelli F, Cappello P, Varesio L et al. Chronic hypoxia reprograms human immature dendritic cells by inducing a proinflammatory phenotype and TREM-1 expression. Eur J Immunol 2013; 43:949-66; PMID:23436478; http://dx.doi.org/10.1002/eji.201242709
36. Chapoval SP, Lee CG, Tang C, Keegan AD, Cohn L, Bottomly K, Elias JA. Lung vascular endothelial growth factor expression induces local myeloid dendritic cell activation. Clinical Immunol 2009; 132:371-84; PMID:19553159; http://dx.doi.org/10.1016/j.clim.2009.05.016
37. Hiratsuka S, Nakamura K, Iwai S, Murakami M, Itoh T, Kijima H, Shipley JM, Senior RM, Shibuya M. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell 2002; 2:289-300; PMID:12398893; http://dx.doi.org/10.1016/S1535-6108(02)00153-8
38. Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK, Hansen KR, Thompson LF, Colgan SP. Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest 2002; 110:993-1002; PMID:12370277; http://dx.doi.org/10.1172/JCI0215337
39. Challier J, Bruniquel D, Sewell AK, Laugel B. Adenosine and cAMP signalling skew human dendritic cell differentiation towards a tolerogenic phenotype with defective CD8(+) T-cell priming capacity. Immunology 2013; 138:402-10; PMID:23278551; http://dx.doi.org/10.1111/imm.12053
40. Curiel TJ, Wei S, Dong H, Alvarez X, Cheng P, Mottram P, Krzysiek R, Knutson KL, Daniel B, Zimmermann MC et al. Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity. Nat Med 2003; 9:562-7; PMID:12704383; http://dx.doi.org/10.1038/nm863
41. Noman MZ, Desantis G, Janji B, Hasmim M, Karray S, Dessen P, Bronte V, Chouaib S. PD-L1 is a novel direct target of HIF-1alpha, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med 2014; 211:781-90; PMID:24778419; http://dx.doi.org/10.1084/jem.20131916
42. Chen X, Iliopoulos D, Zhang Q, Tang Q, Greenblatt MB, Hatziapostolou M, Lim E, Tam WL, Ni M, Chen Y et al. XBP1 promotes triple-negative breast cancer by controlling the HIF1alpha pathway. Nature 2014; 508:103-7; PMID:24670641; http://dx.doi.org/10.1038/nature13119
43. Cubillos-Ruiz JR, Silberman PC, Rutkowski MR, Chopra S, Perales-Puchalt A, Song M, Zhang S, Bettigole SE, Gupta D, Holcomb K et al. ER stress sensor XBP1 controls anti-tumor immunity by disrupting dendritic cell homeostasis. Cell 2015; 161:1527-38; PMID:26073941; http://dx.doi.org/10.1016/j.cell.2015.05.025
44. Pyfferoen L, Mestdagh P, Vergote K, De Cabooter N, Vandesompele J, Lambrecht BN, Vermaelen KY. Lung tumours reprogram pulmonary dendritic cell immunogenicity at the microRNA level. Int J Cancer 2014; 135:2868-77; PMID:24789737; http://dx.doi.org/10.1002/ijc.28945
45. Wang S, Hu J, Zhang D, Li J, Fei Q, Sun Y. Prognostic role of microRNA-31 in various cancers: a meta-analysis. Tumour Biol 2014; 35:11639-45; PMID:25139099; http://dx.doi.org/10.1007/s13277-014-2492-x
46. Meng W, Ye Z, Cui R, Perry J, Dedousi-Huebner V, Huebner A, Wang Y, Li B, Volinia S, Nakanishi H et al. MicroRNA-31 predicts the presence of lymph node metastases and survival in patients with lung adenocarcinoma. Clin Cancer Res 2013; 19:5423-33; PMID:23946296; http://dx.doi.org/10.1158/1078-0432.CCR-13-0320
47. Dong Z, Zhong Z, Yang L, Wang S, Gong Z. MicroRNA-31 inhibits cisplatin-induced apoptosis in non-small cell lung cancer cells by regulating the drug transporter ABCB9. Cancer Lett 2014; 343:249-57; PMID:24099915; http://dx.doi.org/10.1016/j.canlet.2013.09.034
48. Zhong Z, Dong Z, Yang L, Chen X, Gong Z. MicroRNA-31-5p modulates cell cycle by targeting human mutL homolog 1 in human cancer cells. Tumour Biol 2013; 34:1959-65; PMID:23539435; http://dx.doi.org/10.1007/s13277-013-0741-z
49. Gao W, Liu L, Xu J, Shao Q, Liu Y, Zeng H, Shu Y. A systematic analysis of predicted MiR-31-targets identifies a diagnostic and prognostic signature for lung cancer. Biomed Pharmacother 2014; 68:419-27; PMID:24726065; http://dx.doi.org/10.1016/j.biopha.2014.03.009
50. Liu CJ, Tsai MM, Hung PS, Kao SY, Liu TY, Wu KJ, Chiou SH, Lin SC, Chang KW. miR-31 ablates expression of the HIF regulatory factor FIH to activate the HIF pathway in head and neck carcinoma. Cancer Res 2010; 70:1635-44; PMID:20145132; http://dx.doi.org/10.1158/0008-5472.CAN-09-2291
51. Olson P, Lu J, Zhang H, Shai A, Chun MG, Wang Y, Libutti SK, Nakakura EK, Golub TR, Hanahan D. MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer. Genes Dev 2009; 23:2152-65; PMID:19759263; http://dx.doi.org/10.1101/gad.1820109
52. Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis. Nat Cell Biol 2006; 8:1369-75; PMID:17128264; http://dx.doi.org/10.1038/ncb1507
53. Hiratsuka S, Ishibashi S, Tomita T, Watanabe A, Akashi-Takamura S, Murakami M, Kijima H, Miyake K, Aburatani H, Maru Y. Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci. Nat Commun 2013; 4:1853; PMID:23673638; http://dx.doi.org/10.1038/ncomms2856
54. Hsu YL, Hung JY, Tsai EM, Wu CY, Ho YW, Jian SF, Yen MC, Chang WA, Hou MF, Kuo PL. Benzyl butyl phthalate increases the chemoresistance to doxorubicin/cyclophosphamide by increasing breast cancer-associated dendritic cell-derived CXCL1/GROalpha and S100A8/A9. Oncol Rep 2015; 34:2889-900; PMID:26397389; http://dx.doi.org/10.3892/or.2015.4307
55. Cheng P, Corzo CA, Luetteke N, Yu B, Nagaraj S, Bui MM, Ortiz M, Nacken W, Sorg C, Vogl T et al. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein. J Exp Med 2008; 205:2235-49; PMID:18809714; http://dx.doi.org/10.1084/jem.20080132
56. Feng PH, Lee KY, Chang YL, Chan YF, Kuo LW, Lin TY, Chung FT, Kuo CS, Yu CT, Lin SM et al. CD14(+)S100A9(+) monocytic myeloid-derived suppressor cells and their clinical relevance in non-small cell lung cancer. Am J Respir Critical Care Med 2012; 186:1025-36; PMID:22955317; http://dx.doi.org/10.1164/rccm.201204-0636OC
57. Xia B, Herbst RS. Immune checkpoint therapy for non-small-cell lung cancer: an update. Immunotherapy 2016; 8:279-98; PMID:26860624; http://dx.doi.org/10.2217/imt.15.123
58. Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 1999; 223:77-92; PMID:10037236; http://dx.doi.org/10.1016/S0022-1759(98)00204-X
59. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005; 102:15545-50; PMID:16199517; http://dx.doi.org/10.1073/pnas.0506580102
60. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstråle M, Laurila E et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 2003; 34:267-73; PMID:12808457; http://dx.doi.org/10.1038/ng1180
61. Mestdagh P, Feys T, Bernard N, Guenther S, Chen C, Speleman F, Vandesompele J. High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA. Nucleic Acids Res 2008; 36:e143; PMID:18940866; http://dx.doi.org/10.1093/nar/gkn725
62. Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F, Speleman F, Vandesompele J. A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol 2009; 10:R64; PMID:19531210; http://dx.doi.org/10.1186/gb-2009-10-6-r64
63. De Wever O, Hendrix A, De Boeck A, Westbroek W, Braems G, Emami S, Sabbah M, Gespach C, Bracke M. Modeling and quantification of cancer cell invasion through collagen type I matrices. Int J Dev Biol 2010; 54:887-96; PMID:19757378; http://dx.doi.org/10.1387/ijdb.092948ow