The role of biophysical microenvironment in endometrial epithelial cell invasion

Introduction:: Epithelial organoids derived from patient donors are valuable tools to study in vivo disease mechanisms. Modeling the donor tissue microenvironment in a defined and reproducible manner is crucial to better delineate the behavior of organoids in healthy and disease states. Matrigel, a commonly used extracellular matrix (ECM)-mimetic protein hydrogel to grow organoids, is a poorly defined matrix with limited biophysical features to recapitulate the in vivo tissue microenvironment. Here, we used fully defined new synthetic hydrogel with tunable biophysical properties that supports growth of human endometrial epithelial organoids and stromal cells to study endometriosis and adenomyosis (1)(2). Endometriosis, characterized by presence of inner lining of uterus (endometrium) at the outer pelvic regions, is a common gynecological disorder that affects around 10% of women at their reproductive age by causing debilitating pain and infertility (3). Endometriotic lesions are often found in the muscles of the diaphragm, bowel, and bladder (endometriosis) (4), which is similar to adenomyosis, the endometrial epithelia is found in the adjacent muscle tissue (myometrium) (5). One of the proposed hypotheses to explain the presence of these lesions is the growth and invasion of endometrial epithelial and stromal cells into the muscle tissue. Muscle tissue is a relatively stiffer microenvironment compared to endometrium and these lesions are often found within a fibrotic environment which contributes to tissue stiffening (6). Here, we model human lesion microenvironment to define morphology and invasion phenotypes of endometrial organoids as a function of ECM biophysical properties and specific adhesion ligands.


Materials and Methods:: Primary human endometrial epithelial glands were isolated from patient endometrial biopsies and ectopic lesions expanded as organoids in Matrigel. Organoids were dissociated and encapsulated as single cells in a droplet of poly (ethylene glycol) (PEG)-based hydrogel functionalized with pendant peptides and crosslinked with either an MMP-labile peptide (1)(2) or with thiol-functionalized hyaluronic acid (HA-SH). The concentration and stochiometric ratios of the gel components were adjusted to achieve mechanical properties similar to either the endometrium (soft) or muscle (stiff). Mechanical profiles of selected gel formulations were characterized via bulk oscillatory rheology. Organoids were cultured for 14 days and fixed either to stain for F-actin and nuclei to analyze polarization or for EdU staining to detect cell proliferation. Organoids emerged from single cells and invasion behavior was observed by time-lapse imaging with confocal microscopy.

Results, Conclusions, and Discussions:: Organoids encapsulated in soft PEG hydrogels (~ endometrial stiffness) crosslinked with MMP-labile peptides (PEG-peptide) exhibit apical-basal polarity, well-rounded morphology, and grow as hollow spheres. In contrast, organoids cultured in stiffer PEG hydrogels exhibited highly multilobular and invasive phenotype with retained polarization, similar to the ex vivo cleared and imaged adenomyotic lesions (Figure 1a, b). In stiff gels, organoids exhibit the emergence of a highly invasive front into an adjacent region of the gel. Soft and stiff PEG hydrogels crosslinked with HA-SH (PEG-HA) supported organoid growth and organoids exhibited stronger apical-basal polarity compared to soft PEG-peptide gels (Figure 1a, b). Also, organoids embedded in soft PEG-peptide or PEG-HA gels were more proliferative compared to the organoids in stiff PEG hydrogels. Interestingly, organoids derived from ectopic lesions exhibited strikingly higher growth and proliferation rate encapsulated in soft and stiff PEG-HA gels compared to PEG-peptide gels, indicating that PEG-HA gels may support the expansion of ectopic lesions. Additionally, we observed that the spatial organization of organoids in a droplet culture also impacted organoid morphology. Organoids close to the gel-media interface formed gland-like structures in both soft PEG-peptide and PEG-HA gels. Organoids that are close to the stiffer plastic sense the surface and endow migratory potential and invade toward and along it in soft PEG-peptide gels (Figure 1c, d). These results show that human endometrial organoids display a remarkable invasive phenotype in microenvironments of stiffness comparable to muscle, suggesting that the in vitro model may recapitulate crucial aspects of the in vivo lesion phenotype. Current efforts are directed at observing proliferation and invasion in gradients of stiffness and understand how biophysical and spatial cues contribute to healthy endometrium and lesion behavior.

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References (Optional): : 1. Hernandez-Gordillo V, Kassis T, Lampejo A, Choi G, Gamboa ME, Gnecco JS, et al. Fully synthetic matrices for in vitro culture of primary human intestinal enteroids and endometrial organoids. Biomaterials. 2020 Sep;254:120125.
2. Juan S. Gnecco, Alexander Brown, Kira Buttrey, Clara Ives, Brittany A. Goods, Lauren Baugh, et al. Organoid co-culture model of the cycling human endometrium in a fully-defined synthetic extracellular matrix reveals epithelial-stromal crosstalk. bioRxiv. 2022 Jan 1;2021.09.30.462577.
3. Giudice LC, Kao LC. Endometriosis. The Lancet. 2004 Nov 13;364(9447):1789–99.
4. Gordts S, Koninckx P, Brosens I. Pathogenesis of deep endometriosis. Fertil Steril. 2017 Dec 1;108(6):872-885.e1.
5. Vannuccini S, Tosti C, Carmona F, Huang SJ, Chapron C, Guo SW, et al. Pathogenesis of adenomyosis: an update on molecular mechanisms. Reprod Biomed Online. 2017 Nov 1;35(5):592–601.
6. Vigano P, Candiani M, Monno A, Giacomini E, Vercellini P, Somigliana E. Time to redefine endometriosis including its pro-fibrotic nature. Hum Reprod. 2018 Mar 1;33(3):347–52.