Abstract
Multiple in vivo animal models for uterine leiomyoma do not adequately represent human disease based on etiology, molecular phenotype, or limited fixed life span. Our objective was to develop a xenograft model with sustained growth, by transplanting a well-established actively growing three-dimensional (3D) cell culture of human leiomyoma and myometrium in NOD/SCID ovariectomized female mice. We demonstrated continued growth to at least 12 weeks and the overexpression of extracellular matrix (ECM). Further, we confirmed maintenance of hormonal response that is comparable to human disease in situ. Leiomyoma xenografts under hormonal treatment demonstrated 8 to12-fold increase of volume over the xenografts not treated with hormones. Estradiol-treated xenografts were more cellular as compared to progesterone or combination milieu, at the end of 8-week time frame. There was also a non-statistically significant 2–4 mm3 increase in volume between 8-week and 12-week xenografts with higher matrix to cell ratio in 12-week xenografts compared to the 8-week and placebo xenografts. Increased expression of ECM proteins, fibronectin, versican, and collagens, indicated an actively growing cell matrix formation in the xenografts. In conclusion, we have developed and validated a xenograft in vivo model for uterine leiomyoma that shares the genomic and proteomic characteristics with the human surgical specimens of origin and recapitulates the most important features of the human tumors, the aberrant ECM expression that defines the leiomyoma phenotype and gonadal hormone regulation. Using this model, we demonstrated that combination of estradiol and progesterone resulted in increased cellularity and ECM production leading to growth of the xenograft tumors.
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References
Stewart EA, Laughlin-Tommaso SK, Catherino WH, Lalitkumar S, Gupta D, Vollenhoven B. Uterine fibroids. Nat Rev Dis Primers. 2016;2:16043.
Stewart EA, Cookson CL, Gandolfo RA, Schulze-Rath R. Epidemiology of uterine fibroids: a systematic review. BJOG. 2017;124(10):1501–12.
Marsh EE, Al-Hendy A, Kappus D, Galitsky A, Stewart EA, Kerolous M. Burden, prevalence, and treatment of uterine fibroids: A survey of U.S. women. J Women's Health (Larchmt). 2018;27:1359–67.
McWilliams MM, Chennathukuzhi VM. Recent advances in uterine fibroid etiology. Semin Reprod Med. 2017;35:181–9.
Malik M, Norian J, McCarthy-Keith D, Britten J, Catherino WH. Why leiomyomas are called fibroids: the central role of extracellular matrix in symptomatic women. Semin Reprod Med. 2010;28:169–79.
Commandeur AE, Styer AK, Teixeira JM. Epidemiological and genetic clues for molecular mechanisms involved in uterine leiomyoma development and growth. Hum Reprod Update. 2015;21:593–615.
Moravek MB, Bulun SE. Endocrinology of uterine fibroids: steroid hormones, stem cells, and genetic contribution. Curr Opin Obstet Gynecol. 2015;27:276–83.
Styer AK, Rueda BR. The epidemiology and genetics of uterine leiomyoma. Best Pract Res Clin Obstet Gynaecol. 2016;34:3–12.
Mehine M, Kaasinen E, Heinonen HR, et al. Integrated data analysis reveals uterine leiomyoma subtypes with distinct driver pathways and biomarkers. Proc Natl Acad Sci U S A. 2016;113:1315–20.
Fritton K, Borahay MA. New and emerging therapies for uterine fibroids. Semin Reprod Med. 2017;35:549–59.
El Andaloussi A, Chaudhry Z, Al-Hendy A, Ismail N. Uterine fibroids: bridging genomic defects and chronic inflammation. Semin Reprod Med. 2017;35:494–8.
Rafique S, Segars JH, Leppert PC. Mechanical signaling and extracellular matrix in uterine fibroids. Semin Reprod Med. 2017;35:487–93.
Elkafas H, Qiwei Y, Al-Hendy A. Origin of uterine fibroids: conversion of myometrial stem cells to tumor-initiating cells. Semin Reprod Med. 2017;35:481–6.
Dvorská D, Braný D, Danková Z, Halašová E, Višňovský J. Molecular and clinical attributes of uterine leiomyomas. Tumor Biol. 2017;39. https://doi.org/10.1177/1010428317710226.
Pavone D, Clemenza S, Sorbi F, Fambrini M, Petraglia F. Epidemiology and risk factors of uterine fibroids. Best Pract Res Clin Obstet Gynaecol. 2018;46:3–11.
Malik M, Catherino WH. Novel method to characterize primary cultures of leiomyoma and myometrium with the use of confirmatory biomarker gene arrays. Fertil Steril. 2007;87:1166–72.
Malik M, Webb J, Catherino WH. Retinoic acid treatment of human leiomyoma cells transformed the cell phenotype to one strongly resembling myometrial cells. Clin Endocrinol. 2008;69:462–70.
Malik M, Catherino WH. Development and validation of a three-dimensional in vitro model for uterine leiomyoma and patient-matched myometrium. Fertil Steril. 2012;97:1287–93.
Markowski DN, Holzmann C, Bullerdiek J. Genetic alterations in uterine fibroids - a new direction for pharmacological intervention? Expert Opin Ther Targets. 2015;19:1485–94.
Al-Hendy A, Laknaur A, Diamond MP, Ismail N, Boyer TG, Halder SK. Silencing Med12 gene reduces proliferation of human leiomyoma cells mediated via Wnt/β-catenin signaling pathway. Endocrinology. 2017;158:592–603.
Yeung RS, Xiao GH, Everitt JI, Jin F, Walker CL. Allelic loss at the tuberous sclerosis 2 locus in spontaneous tumors in the Eker rat. Mol Carcinog. 1995;14:28–36.
Everitt JI, Wolf DC, Howe SR, Goldsworthy TL, Walker C. Rodent model of reproductive tract leiomyomata. Clinical and pathological features. Am J Pathol. 1995;146:1556–67.
Howe SR, Gottardis MM, Everitt JI, Goldsworthy TL, Wolf DC, Walker C. Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. Am J Pathol. 1995;146:1568–79.
Walker CL, Hunter D, Everitt JI. Uterine leiomyoma in the Eker rat: a unique model for important diseases of women. Genes Chromosom Cancer. 2003;38:349–56.
Howe SR, Gottardis MM, Everitt JI, Walker C. Estrogen stimulation and tamoxifen inhibition of leiomyoma cell growth in vitro and in vivo. Endocrinology. 1995;136:4996–5003.
Cook JD, Walker CL. The Eker rat: establishing a genetic paradigm linking renal cell carcinoma and uterine leiomyoma. Curr Mol Med. 2004;4:813–24.
Miyake A, Takeda T, Isobe A, Wakabayashi A, Nishimoto F, Morishige K, et al. Repressive effect of the phytoestrogen genistein on estradiol-induced uterine leiomyoma cell proliferation. Gynecol Endocrinol. 2009;25:403–9.
Halder SK, Sharan C, Al-Hendy A. 1,25-dihydroxyvitamin D3 treatment shrinks uterine leiomyoma tumors in the Eker rat model. Biol Reprod. 2012;86:116.
Goldberg AA, Joung KB, Mansuri A, Kang Y, Echavarria R, Nikolajev L, et al. Oncogenic effects of urotensin-II in cells lacking tuberous sclerosis complex-2. Oncotarget. 2016;7:61152–65.
Chen HY, Huang TC, Lin LC, Shieh TM, Wu CH, Wang KL, et al. Fucoidan inhibits the proliferation of leiomyoma cells and decreases extracellular matrix-associated protein expression. Cell Physiol Biochem. 2018;49:1970–86.
Prizant H, Sen A, Light A, Cho SN, DeMayo F, Lydon JP, et al. Uterine-specific loss of Tsc2 leads to myometrial tumors in both the uterus and lungs. Mol Endocrinol. 2013;27:1403–14.
Mäkinen N, Vahteristo P, Kämpjärvi K, Arola J, Bützow R, Aaltonen LA. MED12 exon 2 mutations in histopathological uterine leiomyoma variants. Eur J Hum Genet. 2013;21:1300–3.
Mäkinen N, Vahteristo P, Bützow R, Sjöberg J, Aaltonen LA. Exomic landscape of MED12 mutation-negative and -positive uterine leiomyomas. Int J Cancer. 2014;134:1008–12.
Halder SK, Laknaur A, Miller J, Layman LC, Diamond M, Al-Hendy A. Novel MED12 gene somatic mutations in women from the southern United States with symptomatic uterine fibroids. Mol Gen Genomics. 2015;290:505–11.
Lee M, Cheon K, Chae B, et al. Analysis of MED12 mutation in multiple uterine leiomyomas in south Korean patients. Int J Med Sci. 2018;15:124–8.
Mittal P, Shin YH, Yatsenko SA, Castro CA, Surti U, Rajkovic A. Med12 gain-of-function mutation causes leiomyomas and genomic instability. J Clin Invest. 2015;125:3280–4.
Wang H, Shen Q, Ye LH, Ye J. MED12 mutations in human diseases. Protein Cell. 2013;4:643–6.
Markowski DN, Huhle S, Nimzyk R, Stenman G, Löning T, Bullerdiek J. MED12 mutations occurring in benign and malignant mammalian smooth muscle tumors. Genes Chromosom Cancer. 2013;52:297–304.
Schwetye KE, Pfeifer JD, Duncavage EJ. MED12 exon 2 mutations in uterine and extrauterine smooth muscle tumors. Hum Pathol. 2014;45:65–70.
Osinovskaya NS, Malysheva OV, Shved NY, Ivashchenko TE, Sultanov IY, Efimova OA, et al. Frequency and spectrum of MED12 exon 2 mutations in multiple versus solitary uterine leiomyomas from Russian patients. Int J Gynecol Pathol. 2016;35:509–15.
Sadeghi S, Khorrami M, Amin-Beidokhti M, et al. The study of MED12 gene mutations in uterine leiomyomas from Iranian patients. Tumour Biol. 2016;37:1567–71.
Park MJ, Shen H, Kim NH, et al. Mediator kinase disruption in med12-mutant uterine fibroids from Hispanic women of South Texas. J Clin Endocrinol Metab. 2018;103:4283–92.
Porter KB, Tsibris JC, Nicosia SV, et al. Estrogen-induced guinea pig model for uterine leiomyomas: do the ovaries protect? Biol Reprod. 1995;52:824–32.
Tsibris J. The guinea pig model for uterine leiomyomata: gene-hormone interaction? Fertil Steril. 2004;82:988–9.
Mozzachio K, Linder K, Dixon D. Uterine smooth muscle tumors in potbellied pigs (Sus scrofa) resemble human fibroids: a potential animal model. Toxicol Pathol. 2004;32:402–7.
Machado SA, Bahr JM, Hales DB, Braundmeier AG, Quade BJ, Nowak RA. Validation of the aging hen (Gallus gallus domesticus) as an animal model for uterine leiomyomas. Biol Reprod. 2012;87:86.
Sousa WB, Garcia JB, Nogueira Neto J, Furtado PG, Anjos JA. Xenotransplantation of uterine leiomyoma in Wistar rats: a pilot study. Eur J Obstet Gynecol Reprod Biol. 2015;190:71–5.
Hassan MH, Eyzaguirre E, Arafa HM, Hamada FM, Salama SA, Al-Hendy A. Memy I: a novel murine model for uterine leiomyoma using adenovirus-enhanced human fibroid explants in severe combined immune deficiency mice. Am J Obstet Gynecol. 2008;199:156.e1–8.
Suo G, Sadarangani A, Lamarca B, Cowan B, Wang JY. Murine xenograft model for human uterine fibroids: an in vivo imaging approach. Reprod Sci. 2009;16:827–42.
Tsuiji K, Takeda T, Li B, Kondo A, Ito M, Yaegashi N. Establishment of a novel xenograft model for human uterine leiomyoma in immunodeficient mice. Tohoku J Exp Med. 2010;222:55–61.
Ishikawa H, Ishi K, Serna VA, Kakazu R, Bulun SE, Kurita T. Progesterone is essential for maintenance and growth of uterine leiomyoma. Endocrinology. 2010;151:2433–42.
Drosch M, Bullerdiek J, Zollner TM, Prinz F, Koch M, Schmidt N. A novel mouse model that closely mimics human uterine leiomyomas. Fertil Steril. 2013;99:927–35.
Wang G, Ishikawa H, Sone K, Kobayashi T, Kim JJ, Kurita T, et al. Nonobese diabetic/severe combined immunodeficient murine xenograft model for human uterine leiomyoma. Fertil Steril. 2014;101:1485–92.
Qiang W, Liu Z, Serna VA, Druschitz SA, Liu Y, Espona-Fiedler M, et al. Down-regulation of miR-29b is essential for pathogenesis of uterine leiomyoma. Endocrinology. 2014;155:663–9.
Fritsch M, Schmidt N, Gröticke I, et al. Application of a patient derived xenograft model for predicative study of uterine fibroid disease. PLoS One. 2015;10:e0142429.
Koohestani F, Qiang W, MacNeill AL, Druschitz SA, Serna VA, Adur M, et al. Halofuginone suppresses growth of human uterine leiomyoma cells in a mouse xenograft model. Hum Reprod. 2016;31:1540–51.
García-Pascual CM, Ferrero H, Juarez I, et al. Evaluation of the antiproliferative, proapoptotic, and antiangiogenic effects of a double-stranded RNA mimic complexed with polycations in an experimental mouse model of leiomyoma. Fertil Steril. 2016;105:529–38.
Serna VA, Kurita T. Patient-derived xenograft model for uterine leiomyoma by sub-renal capsule grafting. J Biol Methods. 2018;5. pii: e91.
Suzuki Y, Ii M, Saito T, et al. Establishment of a novel mouse xenograft model of human uterine leiomyoma. Sci Rep. 2018;8:8872.
Malik M, Britten J, Catherino WH. A 3D culture system of human immortalized myometrial cells. Bio-protocols. 2016;6:120 www.bioprotocols.org/e1970.
Malik M, Britten JL, Segars J, Catherino WH. Leiomyoma cells in 3-dimensional cultures demonstrate an attenuated response to fasudil, a rho-kinase inhibitor, as compared to 2-dimensional cultures. Reprod Sci. 2014;21:1126–38.
Malik M, Britten J, Cox J, Patel A, Catherino WH. Gonadotropin-releasing hormone analogues inhibit leiomyoma extracellular matrix despite presence of gonadal hormones. Fertil Steril. 2016;105:214–24.
Britten JL, Malik M, Lewis TD, Catherino WH. Ulipristal acetate mediates decreased proteoglycan expression through regulation of nuclear factor of activated T-cells (NFAT5). Reprod Sci. 2019;26:184–97.
Cox J, Malik M, Britten J, Lewis T, Catherino WH. Ulipristal acetate and extracellular matrix production in human leiomyomas. In vivo: a laboratory analysis of a randomized placebo controlled trial. Reprod Sci. 2018;25:198–206.
Sápi J, Kovács L, Drexler DA, Kocsis P, Gajári D, Sápi Z. Tumor volume estimation and quasi-continuous administration for most effective Bevacizumab therapy. PLoS One. 2015;10:e0142190.
Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol. 1989;24:148–54.
Reis FM, Bloise E, Ortiga-Carvalho TM. Hormones and pathogenesis of uterine fibroids. Best Pract Res Clin Obstet Gynaecol. 2016;34:13–24.
Lewis TD, Malik M, Britten J, San Pablo AM, Catherino WH. A comprehensive review of the pharmacologic management of uterine leiomyoma. Biomed Res Int. 2018;2018:2414609.
Singh P, Carraher C, Schwarzbauer JE. Assembly of fibronectin extracellular matrix. Annu Rev Cell Dev Biol. 2010;26:397–419.
Wang JP, Hielscher A. Fibronectin: how its aberrant expression in tumors may improve therapeutic targeting. J Cancer. 2017;8:674–82.
Shimomura Y, Matsuo H, Samoto T, Maruo T. Up-regulation by progesterone of proliferating cell nuclear antigen and epidermal growth factor expression in human uterine leiomyoma. J Clin Endocrinol Metab. 1998;83:2192–8.
Matsuo H, Kurachi O, Shimomura Y, Samoto T, Maruo T. Molecular bases for the actions of ovarian sex steroids in the regulation of proliferation and apoptosis of human uterine leiomyoma. Oncology. 1999;57(Suppl 2):49–58.
Maruo T, Matsuo H, Shimomura Y, Kurachi O, Gao Z, Nakago S, et al. Effects of progesterone on growth factor expression in human uterine leiomyoma. Steroids. 2003;68:817–24.
Norian JM, Malik M, Parker CY, Joseph D, Leppert PC, Segars JH, et al. Transforming growth factor beta3 regulates the versican variants in the extracellular matrix-rich uterine leiomyomas. Reprod Sci. 2009;16:1153–64.
Ohara N. A putative role of versican in uterine leiomyomas. Clin Exp Obstet Gynecol. 2009;36:74–5.
Kinsella MG, Bressler SL, Wight TN. The regulated synthesis of versican, decorin, and biglycan: extracellular matrix proteoglycans that influence cellular phenotype. Crit Rev Eukaryot Gene Expr. 2004;14:203–34.
Acknowledgments
We appreciate the expert assistance of the animal facilities (IACUC), histopathology, and imaging labs (BIC) at the Uniformed Services University. This research was supported by the Military Women’s Health Research grant at the Uniformed Services University.
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Malik, M., Britten, J. & Catherino, W.H. Development and Validation of Hormonal Impact of a Mouse Xenograft Model for Human Uterine Leiomyoma. Reprod. Sci. 27, 1304–1317 (2020). https://doi.org/10.1007/s43032-019-00123-3
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DOI: https://doi.org/10.1007/s43032-019-00123-3