Modeling and validating three dimensional human normal cervix and cervical cancer tissues in vitro

Abstract

The use of three dimensional in vitro systems in cancer research is a promising path for developing effective anticancer therapies. The aim of this study was to engineer a functional 3-Din vitro model of normal and cancerous cervical tissue.Normal epithelial and immortalized cervical epithelial carcinoma cell lines were used to construct 3-D artificial normal cervical and cervical cancerous tissues. De-epidermised dermis (DED) was used as a scaffold for both models. Morphological analyses were conducted by using hematoxylin and eosin staining and characteristics of the models were studied by analyzing the expression of different structural cytokeratins and differential protein marker Mad1 using immunohistochemical technique.Haematoxylin and eosin staining results showed that normal cervical tissue had multi epithelial layers while cancerous cervical tissue showed dysplastic changes. Immunohistochemistry staining results revealed that for normal cervix model cytokeratin 10 was expressed in the upper stratified layer of epithelium while cytokeratin 5 was expressed mainly in the middle and basal layer. Cytokeratin 19 was weakly expressed in a few basal cells. Cervical cancer model showed cytokeratin 19 expression in different epithelial layers and weak or no expression for cytokeratin 5 and cytokeratin 10. Mad1 expression was detected in some suprabasal cells.The 3-Din vitro models showed stratified epithelial layers and expressed the same types and patterns of differentiation marker proteins as seen in correspondingin vivo tissue in either normal cervical or cervical cancerous tissue. Findings imply that they can serve as functional normal and cervical cancer models.

Keywords

cervical cancer / MAX dimerisation protein 1 / cytokeratins / three dimensional in vitro models

References

[1] Kim JB. Three-dimensional tissue culture models in cancer biology[J]. Semin Cancer Biol, 2005, 15(5): 365–377

[2] Ellingsen C, Natvig  I, Gaustad JV , Human cervical carcinoma xenograft models for studies of the physiological microenvironment of tumors[J]. J Cancer Res ClinOncol, 2009, 135(9): 1177–1184

[3] Padrón JM, van der Wilt CL, Smid  K, The multilayered postconfluent cell culture as a model for drug screening[J]. Crit Rev Oncol Hematol, 2000, 36(2-3): 141–157

[4] Sun T, Jackson  S, Haycock JW , Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents[J]. J Biotechnol, 2006, 122(3): 372–381

[5] Benam KH, Dauth  S, Hassell B , Engineered in vitro disease models[J]. Annu Rev Pathol, 2015, 10(10): 195–262

[6] Burdick AD, Bility  MT, Girroir EE , Ligand activation of peroxisome proliferator-activated receptor-beta/delta(PPARbeta/delta) inhibits cell growth of human N/TERT-1 keratinocytes[J]. Cell Signal, 2007, 19(6): 1163–1171

[7] York M, Griffiths  HA, Whittle E , Evaluation of a human patch test for the identification and classification of skin irritation potential[J]. Contact Dermatitis, 1996, 34(3): 204–212

[8] Horning JL, Sahoo  SK, Vijayaraghavalu S , 3-D tumor model for in vitro evaluation of anticancer drugs[J]. Mol Pharm, 2008, 5(5): 849–862

[9] Roskelley CD, Bissell  MJ. Dynamic reciprocity revisited: a continuous, bidirectional flow of information between cells and the extracellular matrix regulates mammary epithelial cell function[J]. Biochem Cell Biol, 1995, 73(7-8): 391–397

[10] Fischbach C, Chen  R, Matsumoto T , Engineering tumors with 3D scaffolds[J]. Nat Methods, 2007, 4(10): 855–860

[11] Szot CS, Buchanan  CF, Freeman JW , 3D in vitro bioengineered tumors based on collagen I hydrogels[J]. Biomaterials, 2011, 32(31): 7905–7912

[12] Hsiao AY, Torisawa  YS, Tung YC , Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids[J]. Biomaterials, 2009, 30(16): 3020–3027

[13] Xu F, Celli  J, Rizvi I , A three-dimensional in vitro ovarian cancer coculture model using a high-throughput cell patterning platform[J]. Biotechnol J, 2011, 6(2): 204–212

[14] Andrei G, van den Oord  J, Fiten P , Organotypic epithelial raft cultures as a model for evaluating compounds against alphaherpesviruses[J]. Antimicrob Agents Chemother, 2005, 49(11): 4671–4680

[15] Anacker D, Moody  C. Generation of organotypic raft cultures from primary human keratinocytes[J]. J Vis Exp, 2012, 60(60): 3–7

[16] Delvenne P, Hubert  P, Jacobs N ,  Giannini SL ,  Havard L ,  Renard I ,  Saboulard D  and  Boniver J . The organotypic culture of HPV-transformed keratinocytes: an effective in vitro model for the development of new immunotherapeutic approaches for mucosal (pre)neoplastic lesions[J]. Vaccine, 200, 19 (17–19): 2557–2564.

[17] Gibbs S, Vicanová  J, Bouwstra J , Culture of reconstructed epidermis in a defined medium at 33 degrees C shows a delayed epidermal maturation, prolonged lifespan and improved stratum corneum[J]. Arch Dermatol Res, 1997, 289(10): 585–595

[18] Regnier M and Darmon  M.25-Dihydroxyvitamin D3 stimulates specifically the last steps of epidermal differentiation of cultured human keratinocytes[J]. Differentiation; research in biological diversity, 1991, 47 (3): 173–188.

[19] Breiden B, Gallala  H, Doering T , Optimization of submerged keratinocyte cultures for the synthesis of barrier ceramides[J]. Eur J Cell Biol, 2007, 86(11-12): 657–673

[20] Oudhoff MJ, Kroeze  KL, Nazmi K , Structure-activity analysis of histatin, a potent wound healing peptide from human saliva: cyclization of histatin potentiates molar activity 1,000-fold[J]. FASEB J, 2009, 23(11): 3928–3935

[21] Chakrabarty KH, Dawson  RA, Harris P , Development of autologous human dermal-epidermal composites based on sterilized human allodermis for clinical use[J]. Br J Dermatol, 1999, 141(5): 811–823

[22] Xie Y, Rizzi  SC, Dawson R , Development of a three-dimensional human skin equivalent wound model for investigating novel wound healing therapies[J]. Tissue Eng Part C Methods, 2010, 16(5): 1111–1123

[23] Smedts F, Ramaekers  F, Troyanovsky S , Keratin expression in cervical cancer[J]. Am J Pathol, 1992, 141(2): 497–511

[24] Kim CJ, Um  SJ, Kim TY , Regulation of cell growth and HPV genes by exogenous estrogen in cervical cancer cells[J]. Int J Gynecol Cancer, 2000, 10(2): 157–164

[25] Abe H, Oikawa  T. Effects of estradiol and progesterone on the cytodifferentiation of epithelial cells in the oviduct of the newborn golden hamster[J]. Anat Rec, 1993, 235(3): 390–398

[26] Comer MT, Leese  HJ, Southgate J . Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium. Hum Reprod, 1998, 13(11): 3114–3120

[27] Wang Q, Li  X, Wang L , Antiapoptotic effects of estrogen in normal and cancer human cervical epithelial cells[J]. Endocrinology, 2004, 145(12): 5568–5579

[28] Wang H, Yuang  F, Huang X ,  Zhang H ,  Yang S and Shi B.Effects of Estrogen and Progestogen on the Growth and Apoptosis of Human Cervical Cancer Cells[J]. U.S Chinese Journal of Lymphology and Urology, 2006, 5(2): 65–70.

[29] Arbeit JM, Howley  PM, Hanahan D . Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus type 16 transgenic mice[J]. Proc Natl AcadSci U S A, 1996, 93(7): 2930–2935

[30] Elson DA, Riley  RR, Lacey A , Sensitivity of the cervical transformation zone to estrogen-induced squamous carcinogenesis[J]. Cancer Res, 2000, 60(5): 1267–1275

[31] Park JS, Rhyu  JW, Kim CJ , Neoplastic change of squamo-columnar junction in uterine cervix and vaginal epithelium by exogenous estrogen in hpv-18 URR E6/E7 transgenic mice[J]. GynecolOncol, 2003, 89(3): 360–368

[32] Li D, Wen  X, Ghali L , hCGβ expression by cervical squamous carcinoma--in vivo histological association with tumour invasion and apoptosis[J]. Histopathology, 2008, 53(2): 147–155

[33] Fuchs E, Weber  K. Intermediate filaments: structure, dynamics, function, and disease[J]. Annu Rev Biochem, 1994, 63: 345–382

[34] Alix-Panabières C ,  Vendrell JP ,  Slijper M , Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer[J]. Breast Cancer Res, 2009, 11(3): R39

[35] Coulombe PA, Omary  MB. ‘Hard’ and ‘soft’ principles defining the structure, function and regulation of keratin intermediate filaments[J]. CurrOpin Cell Biol, 2002, 14(1): 110–122

[36] Moll R, Franke  WW, Schiller DL , The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells[J]. Cell, 1982, 31(1): 11–24

[37] Moll R, Schiller  DL, Franke WW . Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns[J]. J Cell Biol, 1990, 111(2): 567–580

[38] Bobrow LG, Makin  CA, Law S , Expression of low molecular weight cytokeratin proteins in cervical neoplasia[J]. J Pathol, 1986, 148(2): 135–140

[39] Lane EB, Alexander  CM. Use of keratin antibodies in tumor diagnosis[J]. Semin Cancer Biol, 1990, 1(3): 165–179

[40] Carrilho C, Alberto  M, Buane L , Keratins 8, 10, 13, and 17 are useful markers in the diagnosis of human cervix carcinomas[J]. Hum Pathol, 2004, 35(5): 546–551

[41] Smedts F, Ramaekers  F, Robben H , Changing patterns of keratin expression during progression of cervical intraepithelial neoplasia[J]. Am J Pathol, 1990, 136(3): 657–668

[42] Smedts F, Ramaekers  F, Link M , Detection of keratin subtypes in routinely processed cervical tissue: implications for tumour classification and the study of cervix cancer aetiology[J]. Virchows Arch, 1994, 425(2): 145–155

[43] Maddox P, Sasieni  P, Szarewski A , Differential expression of keratins 10, 17, and 19 in normal cervical epithelium, cervical intraepithelial neoplasia, and cervical carcinoma[J]. J ClinPathol, 1999, 52(1): 41–46

[44] Heatley MK. Keratin expression in human tissues and neoplasms[J]. Histopathology, 2002, 41(4): 365–366

[45] Stasiak PC, Purkis  PE, Leigh IM , Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins[J]. J Invest Dermatol, 1989, 92(5): 707–716

[46] Michel M, Török  N, Godbout MJ , Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage[J]. J Cell Sci, 1996, 109(Pt 5): 1017–1028

[47] Bonifas JM, Bare  JW, Lynch ED , Regional assignment of the human keratin 5 (KRT5) gene to chromosome 12q near D12S14 by PCR analysis of somatic cell hybrids and multicolor in situ hybridization[J]. Genomics, 1992, 13(2): 452–454

[48] Roussel MF, Ashmun  RA, Sherr CJ , Inhibition of cell proliferation by the Mad1 transcriptional repressor[J]. Mol Cell Biol, 1996, 16(6): 2796–2801

[49] Amati B, Land  H. Myc-Max-Mad: a transcription factor network controlling cell cycle progression, differentiation and death[J]. CurrOpin Genet Dev, 1994, 4(1): 102–108

[50] Hurlin PJ, Foley  KP, Ayer DE , Regulation of Myc and Mad during epidermal differentiation and HPV-associated tumorigenesis[J]. Oncogene, 1995, 11(12): 2487–2501

[51] Zanotti S, Fisseler-Eckhoff  A, Mannherz HG . Changes in the topological expression of markers of differentiation and apoptosis in defined stages of human cervical dysplasia and carcinoma[J]. GynecolOncol, 2003, 89(3): 376–384

[52] Hurlin PJ, Quéva  C, Koskinen PJ , Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-myc dependent transformation and are expressed during neural and epidermal differentiation[J]. EMBO J, 1995, 14(22): 5646–5659

Acknowledgments

This work was supported by the Middlesex University, particularly in the award of a Postgraduate Research Studentship that provided the necessary financial support for this research.

RIGHTS & PERMISSIONS

2017 2017 by the Journal of Biomedical Research. All rights reserved.