Deciphering specific estrogen signaling in cell populations of human breast and human endometrium Isabelle Dias Da Silva1, Vincent Wuidar1, Christophe Nizet2, Michelle Nisolle3, Christel Péqueux1 1 Laboratory of Biology, Tumor and Development, Giga-Cancer, University of Liege, Belgium 2 Surgery Department, CHU and University of Liege, Belgium 3 Gynecology Department, Giga-Cancer, CHU and University of Liege, Belgium i.diasdasilva@uliege.be vincent.wuidar@uliege.be Background: In western countries, it is estimated that 12 million women use a menopause hormone therapy (MHT) (1); moreover, 151 million women use a combined oral contraceptive (COC) worldwide (2). The Collaborative Group on Hormonal Factors in Breast Cancer recently published a meta-analysis1 revealing that: (i) half of the women having a breast cancer have used MHT, (ii) an excess risk of breast cancer was associated with 1-4 years of use and progressively increased with MHT duration, (iii) the excess risk was greater for estrogen receptor-positive (ER+) than ER- breast cancer, (iv) the risk was higher for estrogen-progestogen than for estrogen-only preparations. The progestogen is added to protect the endometrium against the proliferative effects of estrogens in non-hysterectomized menopausal women. The association of a possible increased breast cancer risk with the use of COCs is utterly difficult to determine. Nevertheless, several studies have reported that women using a COC have a slight increased risk of developing breast cancer (3-5). This slight risk disappears 10 years after treatment cessation, indicating that estrogens in these preparations promote preexisting breast cancer growth rather than induce breast carcinogenesis. The fear of an increased risk of breast cancer due to the use of MHT or even a COC leads an increasing number of women to avoid these treatments, especially MHT. Given the unequivocal benefits of MHT and COCs to women’s health and well-being, there is a medical need for the development of new generation estrogen-progestogen preparations presenting a better safety profile, especially regarding breast cancer risk. Endometrium and breast are estrogen-sensitive tissues and major side targets of these treatments. Since several years, efforts have been made by scientific community to better understand the proliferation and the signaling pathways induced by steroids in these tissues. Main advances in this field have been made thanks to animal models and evidenced important paracrine interactions. Especially, epithelial ER+ and ER- cell dialogue has been evidenced in the breast (6) and stromal to epithelial cell interactions has been depicted in the endometrium (7). However, these cell-to-cell communications are far from being completely understood, especially in human tissue. Therefore, a better understanding of these cell-cell communications upon estrogen or anti-estrogen treatments in these two estrogen-sensitive tissues is thus crucial to bring sufficient knowledge allowing the development of safer hormonal treatments. Methods: To carry out this research project, we have developed human endometrial patient-derived xenograft (PDX) mouse model, as well as human breast and endometrial organoid models. These models are associated to flow cytometry cell sorting strategy to isolate cell populations composing these tissues. Isolated cells can then be submitted to transcriptomic and proteomic analysis. Regarding the human mammary gland, we have developed in our laboratory a 3D culture technique called "organoids" mimicking the physiological structure of the breast in cell culture dishes. Indeed, immunohistochemical stainings of breast organoids have allowed us to highlight the presence of glandular structures with an organization showing luminal cells (ER+ or ER-) surrounded by basal cells. This arrangement of cells was validated by immunofluorescent staining targeting specific markers of these cells, namely cytokeratin 8 for luminal cells (K8+) and cytokeratin 5 for basal cells (K5+). We also highlighted that these human breast organoids express estrogen (ER) and progesterone (PR) receptors and are able to proliferate similarly to the primary tissue (Fig. 1-A). Following the validation of this model, a flow cytometry approach was also established to specifically isolate the different populations of cells composing organoids (luminal ER+/ER- and basal cells) (Fig. 1-B). Our current goal is to treat these organoids with different estrogenic and non-estrogenic treatments, to sort our cells of interest by flow cytometry and to analyze the transcriptome and the proteome of the corresponding subpopulations. Regarding the endometrium, a PDX model of endometrium in immunodeficient mice has been developed in our laboratory. For that purpose, the mice were first ovariectomized in order to mimic menopause and avoid any endogenous steroid stimulation. At least two weeks later, endometrial tissue fragments were implanted subcutaneously. After tissue engraftment, we initiated the steroid treatments to mice. To validate our model, the integrity of the pre/post engrafted tissue was assessed using specific markers for our populations of interest, 1) epithelial cells (EpCAM+) and 2) stromal cells (CD90+). We found the same features in our human endometrial PDX as the primary tissue regarding the ER, PR and Ki67 expression (Fig. 2-A). A flow cytometry sorting strategy specifically targeting these two cell types has been developed, providing the possibility to evaluate the transcriptome and proteome landscape of cells composing human endometrium under steroid treatments (Fig. 2-B). As with the human breast organoid model, we have set up a human endometrial organoid model, allowing us to study in-depth molecular mechanisms underlying the observations seen in the PDX model. The accomplishment of this research project will be a major step forward to better address breast/endometrial cancer but also develop safer hormone treatments for women's health. References 1. Beral, V.; Peto, R.; Pirie, K. Type and timing of menopausal hormone therapy and breast cancer risk: Individual participant meta-analysis of the worldwide epidemiological evidence. Lancet 2019, 394, 1159–1168. 2. Department of Economic and Social Affairs. Contraceptive Use by Method 2019: Data Booklet; United Nations: New York, NY, USA, 2019. 3. Mørch, L.S.; Skovlund, C.W.; Hannaford, P.C.; Iversen, L.; Fielding, S.; Lidegaard, Ø. Contemporary hormonal contraception and the risk of breast cancer. N. Engl. J. Med. 2017, 377, 2228–2239. 4. Calle, E.E.; Heath, C.W.; Miracle-McMahill, H.L.; Coates, R.J.; Liff, J.M.; Franceschi, S.; Talamini, R.; Chantarakul, N.; Koetsawang, S.; RachawatRachawat, D.; et al. Breast cancer and hormonal contraceptives: Collaborative reanalysis of individual data on 53 297 women with breast cancer and 100 239 women without breast cancer from 54 epidemiological studies. Lancet 1996, 347, 1713–1727. 5. Iversen, L.; Sivasubramaniam, S.; Lee, A.J.; Fielding, S.; Hannaford, P.C. Lifetime cancer risk and combined oral contraceptives: The Royal College of General Practitioners’ Oral Contraception Study. Am. J. Obstet. Gynecol. 2017, 216, 580.e1–580.e9. 6. Tanos, T.; Jimenez Rojo, L.; Echeverria, P.; Brisken, C. ER and PR signaling nodes during mammary gland development. Breast Cancer Research. 2012, 14:210. 7. Wang, Y.; Zhu, L.; Kuokkanen, S.; Pollard, J.W. Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17ß is mediated by a PKC-ERK 1/2-mTOR signaling pathway. Proc Nath Acad Sci USA. 2015, 112: E1382-91. Supplementary data - Figures: