A novel three-dimensional culture system of polarized epithelial cells to study endometrial carcinogenesis.
Development of three-dimensional (3D) cultures that mimic in vivo tissue organization has a pivotal role in the investigation of the involvement of cell adhesion and polarity genes in the pathogenesis of epithelial cancers. Here we describe a novel 3D culture model with primary mouse endometrial epithelial cells. In this model, isolated endometrial epithelial cells develop single-lumened, polarized glandular structures resembling those observed in endometrial tissue. Our in vitro 3D culture model of endometrial glands requires the use of serum-free defined medium with only epidermal growth factor and insulin as growth supplements and 3% Matrigel as reconstituted extracellular matrix. Under these culture conditions, glands of epithelial cells displaying typical apicobasal polarity and proper positioning of tight and adherent junctions are formed by hollowing as early as 7 to 8 days in culture. Addition of the phosphatidylinositol 3-kinase inhibitor LY294002 completely inhibits bromodeoxyuridine incorporation and cyclinD1 expression, confirming that in vitro growth of endometrial glands depends on phosphatidylinositol 3-kinase/Akt signaling. To prove that our culture method is a good model to study endometrial carcinogenesis, we knocked down E-cadherin or phosphatase and tensin homolog expression by lentivirus-delivered short hairpin RNAs. Down-regulation of E-cadherin resulted in complete loss of epithelial cell polarity and glandular formation, whereas phosphatase and tensin homolog down-regulation resulted in increased proliferation of glandular epithelial cells. These properties indicate that our 3D culture model is suitable to study the effect of growth factors, drugs, and gene alterations in endometrial carcinogenesis and to study normal endometrial biology/physiology.
Eritja
Núria
N
Oncologic Pathology Group, Hospital Universitari Arnau de Vilanova, Institut de Recerca Biomèdica de Lleida, IRBLleida, University of Lleida, Lleida, Spain.
Llobet
David
D
Domingo
Mónica
M
Santacana
Maria
M
Yeramian
Andree
A
Matias-Guiu
Xavier
X
Dolcet
Xavi
X
D000818:Animals; D015820:Cadherins / Q000378:metabolism; D018929:Cell Culture Techniques; D016764:Cell Polarity; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004338:Drug Combinations; D016889:Endometrial Neoplasms / Q000378:metabolism / Q000473:pathology*; D004717:Endometrium / Q000166:cytology / Q000473:pathology*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D007797:Laminin / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D015820:Cadherins; D003094:Collagen; D004338:Drug Combinations; D007797:Laminin; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D011509:Proteoglycans; D051057:Proto-Oncogene Proteins c-akt; C059485:matrigel; C487757:Pten protein, mouse
Nature. 1998 Mar 12;392(6672):190-3
9515965
In Vitro. 1977 May;13(5):316-28
559643
Tissue Cell. 1979;11(1):109-19
572104
Nat Rev Mol Cell Biol. 2008 Nov;9(11):887-901
18946477
Methods. 2003 Jul;30(3):256-68
12798140
Semin Cancer Biol. 2005 Oct;15(5):365-77
15975824
Curr Biol. 2008 Apr 8;18(7):507-13
18394894
Nat Genet. 1998 Aug;19(4):348-55
9697695
Breast Cancer Res Treat. 2004 Jun;85(3):281-91
15111767
Curr Opin Cell Biol. 2005 Oct;17(5):499-508
16107313
Curr Opin Cell Biol. 2008 Apr;20(2):227-34
18282696
Hum Pathol. 2001 Jun;32(6):569-77
11431710
Oncogene. 2008 Nov 24;27(55):6970-80
19029938
Annu Rev Pathol. 2008;3:313-39
18039125
Cancer Res. 1998 Aug 1;58(15):3254-8
9699651
J Clin Oncol. 2004 Jul 15;22(14):2954-63
15254063
Nat Cell Biol. 2007 Sep;9(9):1016-24
17762893
J Mammary Gland Biol Neoplasia. 2004 Oct;9(4):297-310
15838601
Reprod Sci. 2007 Dec;14(8 Suppl):11-9
18089605
Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10320-5
11504907
Cell. 2002 Oct 4;111(1):29-40
12372298
Science. 2000 Mar 3;287(5458):1606-9
10733430
J Cell Biol. 1972 Sep;54(3):626-37
4339818
Oncogene. 2008 Nov 24;27(55):6920-9
19029934
Cell. 2007 Aug 24;130(4):601-10
17719539
Gynecol Oncol. 2009 Jun;113(3):370-3
19272638
Nat Methods. 2007 Oct;4(10):855-60
17767164
Genes Dev. 2006 Jun 15;20(12):1569-74
16778075
Methods Enzymol. 2009;452:423-39
19200896
Dev Cell. 2007 Feb;12(2):221-34
17276340
Hum Pathol. 2000 Mar;31(3):312-7
10746673
Trends Cell Biol. 1993 Feb;3(2):36-9
14731716
Oncogene. 2008 Nov 24;27(55):6958-69
19029937
Am J Physiol. 1986 Nov;251(5 Pt 1):E630-6
3777165
Proc Natl Acad Sci U S A. 2004 Mar 9;101(10):3438-43
14993595
Autophagy. 2008 Apr;4(3):351-3
18196957
J Clin Pathol. 2009 Sep;62(9):777-85
18977806
Curr Biol. 2005 Oct 25;15(20):1839-46
16243031
J Pathol. 2003 Apr;199(4):471-8
12635138
Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1563-8
9990064
Am J Pathol. 2009 Jan;174(1):287-96
19056846
J Biol Chem. 2006 Aug 4;281(31):22118-22130
16735506
Proc Natl Acad Sci U S A. 1982 Aug;79(15):4672-6
6956885
Pathobiology. 2008;75(2):85-94
18544963
Nat Rev Cancer. 2005 Sep;5(9):675-88
16148884
Exp Cell Res. 1975 Aug;94(1):70-8
243
Hum Pathol. 2006 Nov;37(11):1465-72
16949921
Cell Cycle. 2008 Jan 1;7(1):57-62
18196964
Oncogene. 2008 Nov 24;27(55):6939-57
19029936
Mod Pathol. 2002 Oct;15(10):1032-7
12379748
J Cell Sci. 2000 Apr;113 ( Pt 8):1319-34
10725216
Clin Cancer Res. 2006 Oct 15;12(20 Pt 1):5932-5
17062663
J Cell Sci. 2008 Apr 15;121(Pt 8):1141-50
18388309
Curr Opin Cell Biol. 2008 Oct;20(5):583-8
18573652
Cancer Res. 1997 Sep 15;57(18):3935-40
9307275
Cancer Res. 2008 Jul 15;68(14):5619-27
18632614