Establishment of endometriotic models: the past and future

Gu ZY, Zhi-Yue Gu, Zhiyue Gu, Shuang-Zheng Jia, Shuangzheng Jia, Leng JH, Jin-Hua Leng, Jinhua Leng
Chinese medical journal · 2020 · vol. 133(14) , pp. 1703–1710 · doi:10.1097/cm9.0000000000000885 · PMID:32568880 · PMC7401770 · W3036610361
review OA: gold CC0 ⤵ 11 in-corpus citations
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This review summarizes the past and future of endometriotic models, including animal models, cell lines, and emerging organoids, which are crucial for studying endometriosis etiology and developing new therapies.

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This review describes how endometriotic experimental models—including endometriosis-derived cell lines and emerging organoid cultures—have been developed and used to study disease mechanisms, with a literature search of PubMed, Embase, and Web of Science up to December 2019. It highlights key findings from specific immortalized epithelial and stromal cell lines (often SV40 T-antigen–based) that show invasive behavior and characteristic marker patterns (e.g., E-cadherin negative/N-cadherin positive), while noting limitations such as limited primary-cell lifespan and senescence barriers in ovarian epithelial models. The review further discusses hTERT/TERT-immortalized lines such as EEC16-TERT and EMOsis-CC/TERT1/ER, which retain estrogen/progestin responsiveness or other features relevant to endometriosis-associated ovarian cancer, though some lines show issues like reduced marker expression over passages or contamination. This paper is centrally about endometriosis — it summarizes and compares established endometriotic cell line models and related future modeling approaches (including organoids) for endometriosis research.

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Abstract

Endometriosis is a prevalent chronic disease that affects approximately 6% to 10% of reproductive-aged women. Although numerous researchers have endeavored to explore the etiology of endometriosis over a century, its etiology still remains an enigma. The exploration of pathophysiologic mechanism and novel therapy for endometriosis depends on ideal endometriotic models. In the previous decade, various endometriotic models have been established; therefore, we made a conclusion for available information on these models. This review summarized the common experimental models used in endometriotic studies, including their origins, characteristics, applications, and limitations. Endometriotic models played an important role in studying etiologies and novel treatments of endometriosis during the last decades. Among them, animal models and endometriotic cell lines were viewed as most common studying tools to explore the intrinsic entities of endometriosis. In addition, endometrial organoid also emerged and was regarded as an ideal studying tool for endometriosis research. Different research models collectively complement each other to advance the endometriosis research. The successful establishment of endometrial organoids means that organoids are expected to become an ideal model for studying endometriosis in the future.
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Intro

As a prevalent disease, endometriosis affects approximately 6% to 10% of all women during their reproductive age. [ 1 ] The incidence of endometriosis ranges within 40% to 60% in women with dysmenorrhea and within 20% to 30% in women with subfertility. [ 2 , 3 ] Endometriosis is characterized by the presence of endometrioid epithelial and stromal cells outside the uterus. Endometriosis is mostly found in the pelvic cavity, and mainly consists of three categories: peritoneal, ovarian, and deep infiltrating endometriosis (DIE). [ 4 , 5 ] Patients often experience pelvic pain, dysmenorrhea, deep dyspareunia, ovary cyst and infertility, and even face a higher risk of epithelial ovarian cancer. [ 4 , 6 , 7 ] Present treatments contain pain medication, hormonal intervention, and surgery. [ 7 ] Despite these numerous studies, the etiologies of endometriosis remain indistinct, which depends on ideal studying models. Among these, animal models and endometriotic cell lines are viewed as the most common tools to support the research of endometriosis. In addition, the basis on organoid cultures that concern the liver and gut, endometrial organoid (EO) has also emerged, and has been regarded as a reliable model for endometriosis research. Therefore, the present review summarizes the experimental models that have been used in endometriotic studies, and it was considered that organoid culture could be an ideal model complementary to others for endometriosis study in the future. Published literatures were searched from the PubMed, Embase, and Web of Science databases with the following relative terms: “endometriosis cell line∗,” “(endometriosis epithelial cell line∗[Title/Abstract]) OR endometriosis stromal cell line∗[Title/Abstract]),” “(endometriosis) AND (stem cell),” “organoid culture,” “(endometrium) AND (organoid culture),” and “(endometriosis) AND (animal models).” The search was performed until December 2019. In addition, relevant reviews and the reference lists of all the included articles were analyzed to search for related articles.

Funding

This study was supported by a grant from the National Key Research and Development Program of China (No. 2017YFC1001200).

Section

Compared with in vitro models, in vivo models have extraordinary advantages in exploring etiology, novel therapies, and the influence of endometriosis on patients, such as fertility or pain, which require behavior analysis. The most common animal models included non-human primates (NHPs) and rodent animals. Each category has its own merits and limitations [Table 3 ]. The merits and weaknesses of animal models. Spontaneous endometriosis only occurs in humans and NHPs, such as rhesus monkeys and baboons, which have nature menstrual cycles. [ 57 ] Endometriosis in NHPs resemble the human condition, in terms of phylogenetics, reproductive anatomy and physiology, laparoscopic and microscopic aspects. [ 58 ] Furthermore, NHPs are only species with spontaneous or induced endometriosis similar to the disease in women. [ 59 ] In the wild, endometriosis infrequently and slowly develops, which result in the establishment of an induced model via the injection of autologous menstrual effluent into the pelvic cavities of baboons. [ 60 ] In general, the presence or absence of endometriosis was checked by laparoscopy. After endometrium was injected into the abdominal cavity, laparoscopic procedures were performed at different time points to observe the endometriotic lesions and development of the disease. [ 61 , 62 ] The reasons why baboons were the most frequent models include noninvasive cycle monitoring based on perineal changes, continuous breeding, suitable size and strength, spontaneous peritoneal fluid, cross-reactivity between baboons and humans, vaginal transcervical uterine access, spontaneous retrograde menstruation, and human-like minimal to severe endometriosis. [ 59 ] In a classic design, D’Hooghe et al found that compared with retroperitoneal injection, intra-peritoneal implantation using menstrual endometrium, rather than luteal endometrium, could more successfully induce endometriosis. [ 62 ] Frequently, baboons have been used to explore the possible pathophysiology and potential therapies. [ 63 , 64 ] There is also a saying that new drugs that exhibit effective potential in the rodent model needs to be further tested, in terms of the general and reproductive side effect in NHPs. [ 59 ] Although there are a lot of advantages in the NHP model, the limitation of price and ethical considerations make its frequent use difficult. On the contrary, rodent models are relatively more general with respect to economics, and easy to maneuver and perform genetic manipulation, such as the knock-out mice and transgenic mice. The rat autologous model was developed by Vernon and Wilson in 1985, [ 65 ] and subsequently modified by Berkley et al , [ 66 ] who sutured small pieces of uterus not only to the mesenteric cascade, but also onto the abdomen and ovary, to further resemble the distribution of lesions in women. In addition, Prodromidou et al attempted to establish the DIE model in rats by suturing the resectied uterine horn to the rectum of rats, and confirmed this macroscopically and microscopically. [ 67 ] Except for verifying the reduced fertility, [ 65 ] the rat model was also commonly used to explore the association between endometriosis and increase in pelvic nociception, such as the vaginal hyperalgesia and muscle hyperalgesia induced by a ureteral calculosis amid behavior analysis, and this might be partly explained via the “viscero-visceral referred hyperalgesia” and central sensitization. [ 66 , 68 ] At the same time, some studies explored relevant therapies for “viscero-visceral hyperalgesia” and central sensitization using rat models, such as ketoprofen. [ 68 ] Uterine fragments have also once been grafted onto the sciatic nerve to imitate neuropathic pain in endometriosis. [ 69 ] In addition to exploring the pain mechanism, a rat model was used to study novel therapies and relevant pathophysiology. For instance, cisplatin and letrozole have been tested for the treatment of endometriosis on a rat model. [ 70 ] Furthermore, the rat model was used to analyze the gene expression profiles of ectopic tissues deposited in rats, and explore the association between lesions and inflammatory response, angiogenesis, extracellular environment, and so on. [ 71 – 73 ] However, the establishment of rat models were mainly surgically induced by suturing fragments of uterine tissue to the peritoneum and omentum from the same or syngeneic donor. [ 74 ] The injection method did not work in rats, because the fragments failed to attach and invade the peritoneal cavity. [ 65 ] In contrast, due to the involvement of knockout and transgenic mouse, the mouse model was more various. Apart from the autologous model, there was also the patient-derived xenograft (PDX), which indicated the humanized mouse model of endometriosis by grafting intact human tissue or human endometriosis cell lines. [ 75 ] In addition, even in the syngeneic mouse models, the experimental methods were different, ranging from the suturing tissue to the peritoneal lining, and the injection of whole uterine fragments to the injection of “menstrual” material, which was described by Greaves et al in detail. [ 75 ] Among these models, the use of steroid-induced menstruation as the source of syngeneic mouse menstrual endometrium and the introduction of this into the peritoneum of immunocompetent mice [ 76 , 77 ] further simulated the human disease process. These different mice models were complementary to each other. For example, although human tissues could be manipulated before xenografting in heterologous models, but these cannot be used to study the immune system due to immunodeficiency. In contrast, the immunocompetent mouse model could be used to study the effect of immune-modulating drugs and anti-inflammatory agents. [ 75 ] The mouse model has also been involved in genetic manipulation, which was applied to certain target genes to investigate alterations in ERβ activity during endometriosis progression. [ 78 ]

Conclusion

As a debilitating, chronic and recurrent disease, endometriosis affects around 6% to 10% of women in their reproductive age, and this substantially affects the quality of life of women, and imposes costs on the society, which is similar to other chronic conditions, such as type-2 diabetes mellitus, rheumatoid arthritis and Crohn disease. [ 7 ] To better understand this enigmatic disease, the establishment of reliable endometriotic models for further research are indispensable. Traditional endometriotic models include cell lines and animal models. Primary cell lines could better represent the disease, but have a limited lifespan. For immortal cell lines, EEC12 was the most widely used, others like CRL-7566 and ESC22B have also been used in research. An ideal cell line should be able to passage and maintain the original phenotype and genotype in the long-term. However, SV40 T antigen transfected cell lines usually have karyotype abnormalities. [ 79 ] Although hTERT transfection can maintain the normal karyotype, many formed cell lines do not express or express ER and/or PR only at the RNA level, when compared to the protein level, and these could not respond to hormonal stimulation. [ 28 , 35 ] Indeed, endometriosis is an estrogen-dependent disease. Therefore, although these cell lines are easy to culture for a long time, there are general limitations of using such cell lines, including their genetic background, potential changes occurring during transformation and culture. [ 80 ] In addition, ethical and economic reasons limit the use of NHPs. Although rodent models are valuable in research, the research outcomes are critical in view of the differences between animals and humans. For example, IFN-α-2b, which has been shown to be efficient in the treatment of rodent endometriosis, makes the endometriosis more severe in patients. [ 81 , 82 ] Organoids partly solved above mentioned problems. In oncology research, researchers have found that tumor cell lines cannot retain certain important mutations, and the barcode complexity of cell lines was also progressively lost. [ 83 ] Organoids could maintain the genetic stability of the original tissue, even for tumor significant genetic heterogeneity, which is better than tumor cell lines. [ 84 , 85 ] Turco et al and Boretto et al concluded that EOs phenotypically and genetically resemble the original characteristics, even after long-term expansion, which are important for establishing an ideal disease model. [ 53 , 54 ] In addition, the transcriptomic and genetic analyses of EOs could also reveal disease-associated traits, such as the gene expression differences associated with the signaling pathway, hormonal response, and the adhesion/invasion factors exhibited among the normal EOs, eutopic EOs, and ectopic EOs of endometriosis patients. [ 56 ] Therefore, EOs can also be used as reliable disease models for pathogenesis research and drug screening, which are similar to other organ-derived organoids. Similar to drug screening for cystic fibrosis [ 86 ] and colorectal cancer, [ 87 ] endometrial cancer EOs present with patient-specific drug responses. [ 56 ] In addition to being superior to cell lines, Schutte et al discovered that the response to various drugs between parallel organoids culture and PDX was generally consistent, [ 88 ] while PDX demanded more time and resources. Therefore, before conducting clinical trials, screening out sensitive drugs by combining organoids and PDX greatly improve the efficiency of drug screening, and saves time and costs. [ 85 ] Despite the numerous merits and potential application in clinical medicine, there are still some unresolved technical issues, such as the lack of blood vessels and immune cells in most of the present organoid protocols. [ 50 , 89 ] The communication between epithelial cells with stromal and immune cells play an essential role in the development of endometriosis, but present EOs models cannot solve this problem, which needs to be handled for endometriotic EOs in the future. Endometriosis is a heterogeneous condition, and the subtypes may differ from pathogenesis and require different treatments, and even require different markers for diagnosis and stratification. Zondervan et al once put forward that future research must focus on understanding the pathogenesis, identifying disease subtypes, developing non-invasive diagnostic methods, and targeting non-hormonal treatments appropriate for women who wished to conceive. [ 7 ] An exclusive endometriosis organoids biobank for pathophysiology research and drug screening are expected to solve these problems. Taken together, all kinds of models should corporate with others. As suggested by Bredenoord et al , organoids are complementary to, rather than in competition with, these classical research methodologies. [ 50 ]

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endometriosis

MeSH descriptors

Endometriosis Adult Animals Cell Line Disease Models, Animal Endometrium Female Humans

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