Organotypic culture as a research and preclinical model to study uterine leiomyomas

Abstract

Organotypic cultures of tissue slices have been successfully established in lung, prostate, colon, gastric and breast cancer among other malignancies, but until now an ex vivo model based on tissue slices has not been established for uterine leiomyoma. In the present study, we describe a method for culturing tumour slides onto an alginate scaffold. Morphological integrity of tissue slices was maintained for up to 7 days of culture, with cells expressing desmin, estrogen and progesterone receptors. Driver mutations were present in the ex vivo slices at all-time points analyzed. cultivated tumour slices responded to ovarian hormones stimulation upregulating the expression of genes involved in leiomyoma pathogenesis. This tissue model preserves extracellular matrix, cellular diversity and genetic background simulating more in-vivo-like situations. As a novelty, this platform allows encapsulation of microspheres containing drugs that can be tested on the ex vivo tumour slices. After optimizing drug release rates, microspheres would then be directly tested in animal models through local injection. Uterine leiomyoma (UL), also called fibroid or myoma, is the most commonly diagnosed tumour of the female genital tract with an incidence of 40% at the age of 35 and nearly 70-80% around the age of 50 1,2. Severe symptoms develop in 15-30% of patients being irregular, prolonged or heavy vaginal bleeding the most common manifestations frequently associated with moderate to severe anemia 1. In addition, UL may interfere with embryo implantation, complications during pregnancy and childbirth, decreasing reproductive success 3. Fibroids are the leading cause of hysterectomy with an estimated US healthcare cost of ~$21 billion annually 4. Although the etiology remains elusive, some progress has been made about the molecular mechanisms involved in tumour initiation and growth including (1) steroid hormone-dependency 2 , (2) excessive accumulation of extracellular matrix (ECM), a reservoir for growth factors, cytokines, chemokines, angiogenic and inflammatory response mediators and proteases that contribute to cell growth, differentiation and ECM turnover 5,6 and (3) alterations in driver genes such as HMGA2 (high mobility group AT-hook 2) and MED12 (mediator complex subunit 12) occurring in most of the tumours 7,8. Different cell populations are found in leiomyomas, including smooth muscle (SMC), fibroblast and stem cells 9-12 , all of them embedded in the abundant ECM. Communication between these cells seems to be critical for tumour proliferation and survival 9-11. Thus, it has been proposed that leiomyoma stem-progenitor cells devoid of sex hormonal receptors, respond to paracrine signals sent by surrounding tumour cells upon ovarian steroid stimulation, inducing self-renewal and tumour maintenance and growth 13. Furthermore, UL derived fibroblast stimulate the proliferation of leiomyoma cells and collagen type I production 6,11,14. Most studies published to date have used traditional 2D culture models of primary or immortalize...

Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering

“…147 In-situ hybridisation using labelled probes for specific DNA sequences, molecular methods such as real time quantitative PCR and western blot have also been used to identify cell types, gene expression and protein production in other ex vivo tissue models. 46,158,159…”

Potential Effects of Melatonin on the Development of Uterine Fibroids in Women of Reproductive Age (Review of Literature)

“…Асоціюється з 2-3% всіх випадків безпліддя та 1,4-2% патологічним перебігом вагітності [3]. Отже, ЛМ -найпоширеніша гормонзалежна доброякісна пухлина, що виникає з фенотипово зміненої гладком᾽язової тканини матки (моноклональний проліферат) [4,5]. Від 20-50% жінок виявляють скарги, однак значна частина припадає на безсимптомний перебіг.…”

Development of Primary Monolayer Cell Model and Organotypic Model of Uterine Leiomyoma

“…UL is a benign tumor consisting of various cell populations (fibroblasts, smooth muscle, and stem cells) embedded in the extracellular matrix, interacting with each other, thereby ensuring proliferation. Organotypic cultures are used to study the pathogenesis and various therapeutic approaches for a number of malignant and benign tumors [10]. They retain a histological and three-dimensional structure with inter-and extracellular interactions, cellular matrix components, and intact metabolic activity.…”