{"paper_id":"5714a150-310d-4f8e-93fb-dbaf9a9f9225","body_text":"1\nVol.:(0123456789)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports\nCollagen I triggers directional \nmigration, invasion and matrix \nremodeling of stroma cells in a 3D \nspheroid model of endometriosis\nAnna Stejskalová1*, Victoria Fincke1, Melissa Nowak1,3, Yvonne Schmidt1, Katrin Borrmann2, \nMarie‑Kristin von Wahlde1, Sebastian D. Schäfer1, Ludwig Kiesel1, Burkhard Greve2 & \nMartin Götte1*\nEndometriosis is a painful gynecological condition characterized by ectopic growth of endometrial \ncells. Little is known about its pathogenesis, which is partially due to a lack of suitable experimental \nmodels. Here, we use endometrial stromal (St‑T1b), primary endometriotic stromal, epithelial \nendometriotic (12Z) and co‑culture (1:1 St‑T1b:12Z) spheroids to mimic the architecture of \nendometrium, and either collagen I or Matrigel to model ectopic locations. Stromal spheroids, but not \nsingle cells, assumed coordinated directional migration followed by matrix remodeling of collagen \nI on day 5 or 7, resembling ectopic lesions. While generally a higher area fold increase of spheroids \noccurred on collagen I compared to Matrigel, directional migration was not observed in co‑culture or in \n12Z cells. The fold increase in area on collagen I was significantly reduced by MMP inhibition in stromal \nbut not 12Z cells. Inhibiting ROCK signalling responsible for actomyosin contraction increased the \nfold increase of area and metabolic activity compared to untreated controls on Matrigel. The number \nof protrusions emanating from 12Z spheroids on Matrigel was decreased by microRNA miR ‑200b and \nincreased by miR‑145. This study demonstrates that spheroid assay is a promising pre‑clinical tool that \ncan be used to evaluate small molecule drugs and microRNA‑based therapeutics for endometriosis.\nEndometriosis is a common gynaecological disease in which the uterine lining, the endometrium, grows at \nectopic locations such as the ovaries and peritoneal  cavity1. This disease is currently treated using hormonal \ntherapy and excision surgery. Unfortunately, these treatments are not curative and have high associated side \neffects and remission  rates1,2. While targeted therapies are urgently needed, their development has been hindered \nby the  heterogeneity3,4 and limited mechanistic understanding of the disease.\nBased on the widely accepted Sampson’s theory, endometriosis arises when tissue fragments shed during \nmenstruation implant in the surrounding  tissue5 (Fig. 1). To implant, endometrial fragments have to first pen-\netrate either through intact barriers consisting of epithelial cells, basement membranes and collagen or directly \nthrough a damaged tissue (e.g. due to  microtrauma6 or previous surgical  procedure7–9) and then  spread10. In \nthis regard, endometriosis shares many similarities with metastatic  cancer11. However, while cancer researchers \nhave devoted considerable attention to dissecting the invasive processes involved in cancer  metastases12, little is \nknown about invasive processes in endometriosis.\nOne significant hurdle in studying how endometrial cells invade ectopic tissues has been a lack of suitable \nexperimental  models13. To address this, in vitro models of endometriosis consisting of endometrial  explants14, \n organoids3 or single cells combined with chorioallantoic  membrane15, amniotic  membrane16, peritoneal meso-\nthelial cell  monolayers17, peritoneal  explants18 and  hydrogels19 have been developed. Nevertheless, each of these \napproaches has some inherent limitations. 2D cell culture is the gold standard, but the invasive and migratory \nstrategies in 2D are markedly different from the coordinated multicellular collective invasion through the extra-\ncellular matrix (ECM) that has been observed in vivo 20,21. Organoid models typically focus predominantly on \nepithelial cells and lack the stromal  component22. Explants suffer from high heterogeneity, mixed cell population \nOPEN\n1Department of Gynecology and Obstetrics, Münster University Hospital, Albert -Schweitzer Campus 1, \nD11, 48149 Münster, Germany. 2Department of Radiotherapy-Radiooncology, Münster University Hospital, \n48149 Münster, Germany. 3Present address: Institut für Molekulare Medizin III, Heinrich-Heine-Universität \nDüsseldorf, 40225 Düsseldorf, Germany. *email: anna.stejskalova@gmail.com; mgotte@uni-muenster.de\n\n2\nVol:.(1234567890)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nand low  throughput23. The promising and integrative tissue engineering approach has so far recreated models of \ndecidual eutopic endometrium rather than lesions or menstrual stage  endometrium19.\nMenstrual stage endometrium is characterized by stromal reorganization into tightly packed cellular con-\ndensates sometimes referred to as ‘blue balls’ , collapsed glands and blood  debris24. We hypothesized that such \ncollapsed architecture could be modelled using the spheroid culture in vitro. Spheroid culture is a well-established \ntechnique that commonly used to study  malignancies25. Indeed, endometrial epithelial spheroids generated from \n12Z, an endometriotic lesion derived epithelial cell  line26 and endometriotic stromal  cells27 have already been \nshown to share histological similarities to lesions better than 2D culture. However, it has not been investigated \nhow endometrial spheroids interact with the ECM.\nIn this study, we show that endometrial spheroids create structures resembling lesions on collagen I and \nMatrigel in vitro within 5–7 days. We demonstrate that this assay can dissect the effect of the cell and ECM type \nas well as of small molecule- and RNA- drugs.\nResults\nAn endometrial stromal cell line (St‑T1b), primary endometriotic stromal cells and the endo ‑\nmetriotic epithelial cell line (12Z) self‑organize into spheroids in hanging drop culture. To \ncapture the heterogeneity of endometrial cells found in lesions, the cells we employed in this study were an \nimmortalized eutopic stromal cell line St-T1b 28, primary endometriotic stromal cells (ESCs) and the ectopic \nlight red peritoneal lesion derived epithelial 12Z cell line that was previously shown to be invasive in a Matrigel \ninvasion  assay29.\nFirst, we validated that the cells retained their stromal and epithelial morphology in culture. Figure 2A shows \nthat while the St-T1b and ESCs cells have an elongated, fibroblast-like stromal morphology, 12Z cells have a \nmostly polygonal shape and grow in clusters. Furthermore, on tissue culture (TC) plastic, the stromal cells exhibit \nmore defined actin fibers compared to the 12Z cells. Quantitative analysis (Fig. 2B) confirmed that 12Z cells are \nsignificantly smaller (p < 0.0001) than St-T1b and ESCs, where the average area for St-T1b, 12Z and ESCs cells \non TC plastic were 2086 ± 904.1 µm2 (n = 29), 787.7 ± 380.9 µm2 (n = 32) and 1989 ± 889.5 µm2 (n = 30).\nRecent studies suggested that spheroid culture offers several advantages over 2D culture and confirmed \nthat 12Z  cells26 and endometriotic stromal  cells27 can assemble into spheroids using the U-bottom 96 well \n plates27. However, it has not been investigated whether also the hanging drop method can be used to fabricate \nendometrial spheroids and whether there are any differences between spheroids fabricated from epithelial and \nstromal endometrial cells alone or their co-culture. We, therefore, evaluated the hanging-drop method, each \ndrop containing 20,000 of stromal or epithelial cells or their co-culture in 20 µL of standard media and selected \nday 4 as the harvesting day.\nBright-field images (Fig. 2C) show that all the studied cell types self-organized into spheroids. Interestingly, \nthe morphology of the spheroids varied across cell types. St-T1b and ESCs cells assembled into compact, round-\nspheroids, while the 12Z spheroids were larger and sometimes exhibited slightly branching morphology. We \nalso generated co-culture spheroids from the epithelial 12Z and stromal St-T1b cell lines combined at 1:1 ratio \n(Fig. 2D). Cell Tracker staining and confocal imaging suggest the two cell populations were homogeneously \ndistributed throughout the spheroid on day 4. Interestingly, while the size of individual 12Z cells in 2D is sig-\nnificantly smaller compared to the ESCs and St-T1b cells, 12Z spheroids were significantly larger compared to \nSt-T1b and ESCs (n = 14, p < 0.0001 and p < 0.001) (Fig. 2E). To exclude that this is due to a cell-counting error, \nthe spheroid size was measured on spheroids prepared three independent times. The co-culture spheroids were \nalso significantly larger compared to St-T1b spheroids (n = 11) and had a higher metabolic activity that is indica-\ntive of higher cell count and proliferation over the spheroid formation period (Fig. 2F ,G).\nNext, we evaluated whether the condensation into spheroids induces changes in gene expression. We analysed \na subset of genes related to ectopic tissue invasion. Gene expression analysis revealed that while organisation into \nFigure 1.  Endometriosis modelling in vitro. Left. Sampson’s theory of retrograde menstruation. Menstrual \ntissue contains stromal condensates (dark blue) and collapsed epithelial glands (pink). Ectopic lesions are \nfrequently described to have a ‘bullet-like appearance’ Right. Spheroids generated using the hanging drop \nmethod as a model of collapsed endometrium architecture are placed on either Matrigel or collagen I on day 4 \nand their phenotype on the hydrogels and the effect of pharmacological intervention is evaluated.\n\n3\nVol.:(0123456789)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nspheroids alters the gene expression of several markers in St-T1b cells, none of these markers was significantly \naltered in 12Z spheroids compared to monoculture across three independent preparations (Fig. 2H,I).\nFigure 2.  Spheroid formation by endometrial cells. (A) F-Actin (red) and nuclei (blue) stained St-T1b, 12Z and \nESCs. (B) The projected area in 2D of St-T1b and ESCs is significantly larger than of 12Z cells (n = 29, 32 and \n30 cells, Kruskal–Wallis with Dunn’s multiple comparisons post hoc test. Data show mean ± s.d.). (C) Bright-\nfield images of fixed spheroids that formed after 4-days using the hanging drop method. Scale bars 250 µm. \n(D) Co-Culture 1:1 St-T1b:12Z spheroids on day 4 stained by Cell Tracker. Red are St-T1b and green 12Z cells. \nScale bar 200 µm. (E) The 12Z spheroids were significantly larger compared to St-T1b and ESCs spheroids \n(n = 14 prepared across three different preparations, Kruskal–Wallis with Dunn’s multiple comparisons post hoc \ntest), the area was measured manually on bright-field images, 10 × magnification. (F) Metabolic-based assay \nsuggests 12Z and Co-Culture spheroids on day 4 consist of a higher number of cells than St-T1b spheroids (n = 3 \nindependent wells and one preparation, one-way ANOV A with Tukey’s multiple comparisons test). (G) Spheroid \nprojected area is also significantly larger in 12Z cells and co-culture groups than in the St-T1b group (n = 10–15 \nindependent wells from two different spheroid preparations, Kruskal–Wallis with Dunn’s multiple comparisons \npost hoc test). (H) qPCR analysis comparing gene expression in 2D and 3D spheroids on day 4 of the hanging \ndrop culture in St-T1b cells and (I) qPCR analysis comparing gene expression in 2D and 3D spheroids on day 4 \nof the hanging drop culture in 12Z cells (n = 3 independent preparations on the same cell lines, multiple t tests). \nFor all figures in the panel *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001 and not significant (n.s.) p > 0.05; \nData shown as mean ± standard deviation (s.d.) or as mean + s.d.\n\n4\nVol:.(1234567890)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nFirst, we examined the expression of Ras-related C3 botulinum toxin substrate 1(RAC1 /Rac1), a small sig -\nnalling G protein that directs actin-driven cellular protrusion, microtubule prolongation and the formation of \n lamellipodia30 both in single cells and at the leading edge during collective  migration31. The expression of RAC1 \nwas significantly downregulated in 3D compared to 2D St-T1b (p < 0.01, n = 3) (Fig. 2H).\nSpheroid St-T1b culture exhibited higher proteolytic gene expression compared to 2D (Fig.  2H). qPCR \nanalysis revealed that the spheroids exhibit higher expression of the secreted MMP2 (p < 0.0001, n = 3) and the \nmembrane-type metalloproteinase MMP14 (p < 0.05, n = 3) than cells grown in 2D.\nAs the epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) have \nbeen implicated in the progression of the disease, we further investigated the expression of mesenchymal mark-\ners vimentin (VIM) and cadherin-2 (CDH2) and the epithelial marker cadherin-1 (CDH1) (Fig. 2H). Vimentin \nexpression remained unchanged in both cell lines (p > 0.05, n = 3). The expression of CDH2, a cadherin known to \npromote invasion in many cell  types32, was downregulated in St-T1b spheroids (p < 0.01, n = 3) while the expres-\nsion of CDH1 was upregulated in St-T1b spheroids (< 0.05 = n = 3) compared to the 2D control.\nMatrigel and collagen I trigger distinct phenotypes in single cells and spheroids where stromal \ncondensates create defects on collagen I. Having confirmed that endometrial stromal and epithelial \nendometriotic cell line as well as their co-culture were able to form spheroids, we evaluated their invasive behav-\niour on two different ECM-derived hydrogels: Matrigel and collagen I using confocal imaging.\nSingle cells of all studied cells on Matrigel formed cellular aggregates by day 3(Fig.  3A). While these aggre-\ngates remained mostly rounded in St-T1b and ESCs groups, the 12Z cell line aggregates consistently developed \nmultiple multicellular protrusions across several preparations. Cells seeded on collagen I were invading collagen \nI as single cells (Fig. 3A).\nWe next evaluated the spheroid behaviour on Matrigel and collagen I. On the basement membrane (BM) \nmimic Matrigel, the stromal St-T1b spheroids remained rounded with ESCs exhibiting few protrusions and only \nthe 12Z spheroids consistently developed multiple multicellular protrusions across several preparations. Confo-\ncal imaging on day 7(Fig.  3B) revealed that the 12Z protrusive edges consisted of tightly packed cells (DNA in \nblue) with scant cytoplasm (actin staining in red).\nThe response of all studied cell types to collagen I as spheroids was markedly different compared to single cells \n(Fig. 3B). St-T1b and ESC spheroids on collagen I developed into invasive lesion-like structures (Fig. 3B). More \nspecifically, the St-T1b and ESC spheroids gradually invaded collagen I, leaving behind a circularly remodeled \nmatrix with a ring of tightly adhering cells at its margins (Fig. 3B,C). These rings appeared to stabilize the defect \nand to limit further random cellular spreading outside of the defect in many but not all spheroids.\nFigure 3.  Lesion-like structures on collagen I and Matrigel. (A) Confocal images of a suspension of \nendometrial cells after 3 days on Matrigel (top row). Stromal St-T1b and ESCs cellular aggregates consisted of \nonly a few cells and were highly circular and 12Z aggregates were larger and showed protrusions. All cell types \ninvaded collagen I (bottom row) as single cells (maximal intensity projection, scale bar 200 µm, f-actin red, \nnuclei blue). (B) Confocal images of spheroids after 7 days on Matrigel and collagen I. 12Z exhibited the highest \nnumber of protrusions on Matrigel. St-T1b and ESCs created circular defects in collagen I surrounded by cells, \nwhereas epithelial 12Z cells migrated as a sheet and confocal imaging revealed no invasion (maximal intensity \nprojection, scale bar, 200 µm, actin cytoskeleton red, nuclei blue). (C) Detail of three different imaging planes of \nthe edge of the circular defect in St-T1b spheroid in collagen I group. F-actin in red and DNA in blue. Scale bar \n100 µm. (D) S-T1b: 12Z co-culture after 7 days on Matrigel (left) and collagen I (right). Scale bar 200 µm.\n\n5\nVol.:(0123456789)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nInterestingly, no matrix defect or directional spreading was detected in co-culture St-T1b:12Z spheroids \non collagen I (Fig.  3D). Co-culture spheroids on Matrigel developed protrusive edges similar to the 12Z-only \nspheroids.\nDirectional invasion followed by the formation of a circular defect occurs in St‑T1b and ESCs \nspheroids but not in St‑T1b:12Z co‑culture. Next, we quantified the invasive and migratory patterns \non Matrigel and collagen I using bright-field imaging and a parameter that we termed ‘Fold change in the area’ \nthat we defined as the overall projected area, including matrix defects on the day of interest divided by the area \non the day 0 or 1 without any sprouts (Fig. 4A). All analysis was done manually in FIJI using the freehand selec-\ntion tool. While manual measurement has its limitation, especially when the ‘ Area’ increases and its margins \nbecome irregular, no significant difference in measured areas was observed between different assessors (Fig. 4B).\nOur data show that the ‘Fold change in area’ is significantly higher on collagen I compared to Matrigel across \nall studied cell types by day 5 (Fig. 4C,D). Confocal imaging combined with brightfield microscopy suggested the \nstromal spheroids invade (Fig. 3B,C) and migrate on the collagen I matrix directionally (Fig. 4C). To quantify this, \nwe used the parameter ‘Directionality’ that is calculated as the ratio of the distance of the centre of the spheroid \ncore from the centre of the overall migrated area b to the semi-major axis of the overall migrated area a (Fig. 4E). \nThe normalized directionality increased for St-T1b but not for 12Z or co-culture spheroids with time on collagen \nI, especially between days 3 and 5 (Fig. 4F). The directional invasion was typically followed by matrix remodeling \nresulting in a circular defect at the area with the densest stromal cell population (Fig. 4G). In our system (3 mg/\nmL, 40 µL/well) this typically occurred around day 5 or 7 with 84.6% and 53.3% of St-T1b and ESCs, respectively, \nhaving a defect on day 7 (n = 13–15 per time point) (Fig.  4G). The defects formed both on 1 mg/mL and 3 mg/\nmL collagen I hydrogels, suggesting this behavior occurs across a range of collagen I concentrations (Fig. 4H).\nSpheroid 3D culture as an effective tool to screen small molecule drug and microRNA‑based \ntherapeutics. We then evaluated the potential of the here presented endometrial spheroid in vitro assay to \nscreen the potential therapeutic effect of mechanoregulatory small molecules and micro RNAs (Supplementary \nTable ST1).\nThe broad‑spectrum MMP inhibitor NNGH limits the invasive behaviour of stromal spheroids \non collagen I. Previous studies implicated that MMP signalling plays a role in the formation of early endo-\nmetriotic  lesions15. Our study shows that the broad-spectrum MMP inhibitor 15 µM N-isobutyl-N-(4-methoxy-\nphenylsulfonyl) glycyl hydroxamic acid (NNGH) significantly reduced ‘Fold change in the area’ on collagen I \nfrom 10.4 fold to 2.3 fold (n = 6–9) and 9.2 fold to 3.3 fold (n = 6–9) in St-T1b and ESCs, respectively, but did not \nsignificantly affect the ‘Fold change in the area’ in 12Z cells (n = 6–9) (Fig.  5A, Supplementary Figure S1). Fur-\nthermore, it can be seen from Fig. 5B, that while NNGH treatment prevents the formation of the circular defect \non collagen I even after 7 days in culture, the migration of St-T1b and ESCs is not completely eliminated. The \neffect of NNGH inhibitor on the St-T1b:12Z co-culture was less pronounced and neither the control nor NNGH \ngroup formed matrix defects by day 5 (Fig. 5C).\nROCK inhibition significantly enhances spreading and invasion of all studied endometrial cell \ntypes on Matrigel. The ROCK inhibitor Y27632 significantly (p < 0.01) increased the ‘Fold change in the \narea’ of all studied cell types on Matrigel compared to DMSO (Fig.  5D). The area occupied by St-T1b, 12Z \nand ESCs was 17.3, 6.6 and 22.3 fold larger compared to day 0 (Fig.  5E). Y27632 also affected the numbers of \nmetabolically active cells, which were significantly higher compared to controls for St-T1b and 12Z cells on day \n5 on Matrigel. Moreover, Y27632 affected spheroid morphology (Fig.  5F). Y27632 on Collagen I resulted in a \ndisaggregation of the spheroid core in St-T1b and ESCs as shown in the Supplementary Figure S2. Treatment \nwith Y27632, in contrast to the MMP inhibitor NNGH, did not prevent Collagen I matrix remodeling in ESCs \n(Fig. 5G) suggesting the directional remodeling is rather due to proteolytic action than acto-myosin contraction.\nThe spheroid model reveals context‑dependent roles of the mechanoregulatory microRNAs \nmiR‑200b and miR‑145 on the invasive behaviour of the endometriotic epithelial cell line 12Z \non Matrigel. We next investigated whether our in vitro model can be used as a tool to screen the functional \neffect of various microRNAs on endometrial phenotype. In particular, we selected two microRNAs, miR-200b33 \nand miR-14534, that have been previously shown to be dysregulated in  endometriosis35 and to modulate the \ninvasion and migration of 12Z cells in 2D and Transwell assays. miR-200b acts as a transcriptional repressor of \nZEB1/2 and thus downregulates EMT  transition36. The miR-145 is upregulated in endometrial lesions and has \nbeen described to modulate cytoskeletal dynamics in several cell types, including endometrial, and has many \nvalidated targets, including beta and gamma actin, cofilin, fascin, myosin light chain 9 and Rho kinase  Rock134,37. \nThe transfection was performed in monolayer culture before the fabrication of spheroids and the effect of micro-\nRNAs on spheroid spreading was assessed after 3 days on Matrigel to minimize the effect of miR dilution and \n degradation38 (Fig. 6A). It can be seen from Fig.  6B that microRNA transfection did not significantly alter the \nability of cells to form spheroids and the area of individual spheroids was not significantly different (p > 0.05) \nacross the treatment groups nor was the proliferation (Fig.  6C). We observed spheroid fragmentation of miR-\n200b transfected cells on Collagen I which resulted in a discontinuous nature of the projected area the size of \nwhich could not be reliably quantified (Fig.  6D). MiR-145 significantly reduced the spheroid area compared \nto scr. miR controls on day 3 on collagen I (Supplementary Figure S3). On Matrigel, the microRNAs, affected \nsprouting characteristics behaviour of 12Z cells as seen in the bright-field images in Fig. 6E. The miR-200b treat-\nment significantly decreased the number of sprouts per spheroid from ~ 17 to ~ 1, while miR-145 significantly \n\n6\nVol:.(1234567890)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nFigure 4.  Quantification of spheroid behaviour on Matrigel and collagen I. (A) Schematic illustrating how the \n‘Fold increase in the area’ was measured and calculated. (B) Validation of the manual measurement method \nshowed no significant difference between different assessors following the defined criteria (n matrigel = 13, n \ncollagen = 16, t test for each condition). (C) Brightfield images on day 1 and 5 of the spheroids on Matrigel (top \nrow) and collagen I (bottom row). Scale bar 500 µm. (D) Quantification of the fold change in area for individual \ncell types and St-T1b:12Z co-culture (n = 12–15 independent wells per time point and condition collated from \nthree independent spheroid preparations,  ncocultures = 4–5, one preparation. Two-way Repeated Measures (RM) \nANOV A, Šidák’s multiple comparisons tests). (E) Schematic illustrating how normalized directionality was \ncalculated. (F) Normalized directionally for St-T1b (circles), 12Z (squares) and co-cultures (diamonds) on day \n1, 3 and 5 (n = 5–10 wells per experiment collated from two independent preparations, n co-culture = 5 from one \npreparation). (G) Circular ECM defect quantification-grey colour represents the absence of macroscopic ECM \ndefect and the black colour a presence of a circular defect (n = 13–15 independent wells collated from three \nseparate spheroid preparations,  nco-culture = 5 from one preparation. Two-way Repeated Measures (RM) ANOV A, \nŠidák’s multiple comparisons test) (H) Directional matrix remodeling resulting in a circular defect occured on \nboth 1 mg/mL and 3 mg/mL collagen I hydrogels. For all figures in this panel *p < 0.05; **p < 0.01; ***p < 0.001, \n****p < 0.0001, and n.s. p > 0.05.\n\n7\nVol.:(0123456789)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nincreased the number of sprouts per spheroid to ~ 34 (Fig. 6F ,G) and increased the overall sprouting area from \n56.12 ×  103 ± 21.87 ×  103 µm2 per scrambled control miR spheroid to 130.86 ×  103 ± 43.47 ×  103 µm2 per miR-145 \ntreated spheroids (p < 0.0001) (Fig. 6F ,H). In line with previous findings on EMT-marker analysis in 2D-cultured \n12Z  cells31,33, qPCR analysis of miR-200b-treated 12Z spheroids indicated strong upregulation of CDH1 expres-\nsion levels, however, the data were not significant due to high variability, since only minute amounts of RNA \ncould be isolated from the spheroids (Supplementary Figure S4).\nDiscussion\nEndometriosis is a complex multifactorial  disease1. The overall goal of this study was, therefore, to develop a \nmodular 3D in vitro model that makes it possible to study the interplay of different factors that have been pro -\nposed to contribute to the pathogenesis of endometriosis and screen potential therapeutics in vitro.\nFigure 5.  The effects of small molecule inhibitors on lesion formation. (A) The broad spectrum MMP inhibitor \nNNGH significantly reduced the in vitro lesion size in St-T1b and ESCs but not in 12Z cells that migrated on \ncollagen I surface. The spheroid size was measured manually on days 0 and 5 (n = 6–9 independent wells across \ntwo preparations, multiple t tests). (B) NNGH effectively prevented stromal cells from degrading collagen I \n(bright field channel) but did not completely prevent the cells from migrating. Confocal images were obtained \non fixed samples after 7 days in culture. Scale bar, 200 µm. (C) Co-cultures on collagen I without (top) and with \n(bottom) NNGH inhibitor on day 5 Scale bar, 200 µm. (D) The ROCK inhibitor Y27632 significantly increased \nthe spreading of endometrial cells on Matrigel after 5 days. Data were compared to the spheroid size on day \n0 using bright-field images (n = 8–10 independent wells across two different spheroid preparations, multiple t \ntests). (E) Y27632 significantly increases metabolic activity in all studied cell types on day 5 (n = 8–9, multiple \nt tests, three independent preparations). (F) Confocal images demonstrating the increase in the projected area \nof spheroids of all cell types on Matrigel upon Y27632 treatment Scale bar, 200 µm. (G) ESCs on Collagen I \non day 7 with and without Y27632. Y27632 did not prevent collagen I remodeling. Scale bar 500 µm *p < 0.05; \n**p < 0.01; ***p < 0.001 and n.s. p > 0.05; Data shown as mean ± s.d.\n\n8\nVol:.(1234567890)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nFirst, we demonstrate that the hanging drop method makes it possible to generate endometrial spheroids \nof reproducible size and thus provides a good alternative to the low-adhesion plate  method26,39. Our data show \nthat the spheroid size is consistently cell-type specific, with stromal cells generating smaller spheroids than the \nepithelial 12Z cells or their co-culture. This is likely due to proliferation of 12Z cell in spheroids as suggested \nby the cell proliferation assay on spheroids on day 4. qPCR analysis revealed that the spheroid culture affects \ngene expression. The stromal St-T1b had enhanced expression of the MMP2, MMP14 compared to 2D culture. \nRAC1, on the other hand, was downregulated in St-T1b spheroids. Spheroids in which Rac1 production was \neither inhibited or the gene was constitutively expressed had suppressed or enhanced migration in 3D matrices, \n respectively40. We speculate that it is possible that RAC1 is temporarily downregulated in stromal cells cultured \nas a suspension spheroid culture. While basal CDH1 expression was very low with a Ct value of 27, as expected \nfor a mesenchymal cell line, we observed a significantly increased expression in 3D culture. We could previously \nFigure 6.  The effect of microRNA on 12Z sprouting on Matrigel. (A) Schematic of the workflow (B) none \nof the microRNAs affected the ability of 12Z cells to self-organize into spheroids and all groups resulted in \nspheroids with similar area (scale bar = 250 µm, n = 6 independent spheroids prepared across two preparations, \nANOV A). (C) None of the microRNA affected overall metabolic activity measured as luminescence compared \nto scr.miR treated controls. Data are normalized to controls without any microRNA (n = 3 independent wells, \nANOV A, one repeat). (D) Representative images of microRNA treated 12Z spheroids after 3 days on collagen \nI. Scale bar, 500 µm. (E) Representative images of microRNA treated 12Z spheroids after 3 days on Matrigel. \nScale bar, 250 µm. (F) A diagram showing how the number of sprouts and the sprouting area parameters were \ncalculated. (G) miR-200 significantly decreased while miR-145 significantly increased the number of sprouts per \nspheroid after 3 days on Matrigel. (n = 9–10 independent wells across two independent preparations, ANOV A, \nTukey’s multiple comparisons). (H) The overall area occupied by sprouts was significantly larger and smaller \nwhen treated with miR-145 and miR-200b, respectively, compared to scr.miR after 3 days on Matrigel (n = 8–10 \nindependent wells across two different preparations, ANOV A, Tukey’s multiple comparisons, two independent \nexperiments), *p < 0.05; **p < 0.01; ***p < 0.001 and n.s. p > 0.05; data expressed as mean ± s.d.\n\n9\nVol.:(0123456789)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nshow that CDH1 mRNA expression can be induced in St-T1b cells by external stimuli such as seminal  plasma41, \nhowever, the upregulation in a 3D environment warrants further investigation. While 12Z cells have been initially \ncharacterized as being CDH1  negative28, low expression levels have been subsequently detected by our group in \ncells authenticated by STR  analysis32,33. Our experiments suggest there is no significant difference in any of the \nanalysed markers  CDH133, RAC1, ROCK, MMP2 and MMP14 in the 12Z cell line. While another study showed \nMMP2 expression is upregulated in spheroid compared 2D culture in 12Z  cells26, this difference could be due \nto different spheroid size and culture time.\nEndometriosis is marked by the growth of endometrium at ectopic  locations1. We, therefore, investigated \nhow epithelial 12Z, ESCs and St-T1b and co-culture spheroids; and single cells interact with two ectopic ECM \nmimics Matrigel resembling the basement membrane and collagen I mimicking the exposed stroma. The ‘fold \nincrease in the area’ of the spheroids was markedly higher on collagen I than on Matrigel on day 5. Similarly, \nsingle cells seeded on top of these hydrogels preferentially invaded collagen I hydrogels. Our results are in agree-\nment with previous studies conducted on cancer cells suggesting that collagen I alone can increase the invasive \ncellular phenotype and show that this effect is significant across cell  types42–44. These data also tie well with the \npreviously reported clinical observations that tissue scarring either due to surgery or persistent microtrauma \ncould contribute to the pathogenesis of  endometriosis7–9.\nWhile the 12Z cell line was created from lesion-derived  cells29 based on their ability to penetrate through \nMatrigel coated invasion chambers, the 12Z single cells in our study only assembled into cellular aggregates with \nprocesses and 12Z spheroids developed invasive edges. Previously, Pollock and colleagues also observed only low \nlevels of basal invasion in 12Z cells on Matrigel  hydrogels45. We speculate that the limited invasive capacity of 12Z \ncell observed in this study could be due to the chemotactic gradient that is a key part of the invasion chamber \nsetup. Our group has indeed previously demonstrated that 12Z are invasive under a fetal calf serum  gradient33.\nUnexpectedly, there was a marked difference between the behaviour of stromal single-cell suspension and \nspheroids on collagen I. The St-T1b and ESC spheroids but not single cells consistently migrated on, invaded \nand remodelled collagen I in a directional manner leaving behind a circular defect in the material encircled by \nthe cells that visually resembled peritoneal endometriotic lesions. Given that this was the case for both the St-\nT1b cell line derived from healthy cells and ectopic ESCs suggests such invasive behavior might be an inherent \nproperty of stromal endometrial menstrual condensates and could be critical not only for the pathophysiology \nof endometriosis but also for normal regeneration of endometrium.\nDirectional migration followed by matrix remodeling was not observed in the 12Z-spheroid or the 12Z: St-\nT1b co-culture groups, suggesting the stromal-epithelial interactions modulate stromal invasiveness. While we \ndid not investigate the MMP levels of the co-culture spheroids, previous research determined that the co-culture \nbetween endometrial Ishikawa epithelial and telomerase-immortalized stromal cells reduces the MMP2 levels in \nstromal cells both in the absence of hormonal stimulation and in the presence of 10 nM estradiol  concentration46.\nIn this paper, we further demonstrate that the endometrial spheroid-ECM platform can be used for drug \nscreening of small molecule drugs and micro-RNAs (Fig. 7). We show that the collagen I circular defect caused \nby stromal cells arises due to matrix degradation via MMPs rather than due to cellular contraction. Both eutopic \nand ectopic stromal cells had significantly upregulated MMP expression and the MMP inhibitor, NNGH, signifi-\ncantly reduced the size of in vitro stromal lesions on collagen I. These results are in good agreement with Nap and \ncolleagues that demonstrated that inhibiting MMP activity prevents the development of endometriotic lesions \nin a model combining chicken chorioallantoic membrane model and biopsies of menstrual stage endometrium \nobtained from healthy  donors15. Our results refine this model and show that while, in agreement with the previ-\nous  studies47, the MMP inhibitor significantly slows down the invasion of spreading of stromal cells on collagen it \nhas little effect on the collective migration of 12Z cells. Another signaling molecule we targeted is ROCK, which \nis a key regulator of the  cytoskeleton30,48. On Collagen I, ROCK inhibitor Y27632 treatment led to a rapid loss \nof the spheroid core structure compared to controls and Y27632 did not prevent Collagen I remodeling by ESCs \nsuggesting the matrix remodeling is not primarily driven by matrix contraction but rather by MMP proteolytic \naction. Y27632 further significantly increased the ‘fold change in area’ and cell numbers in vitro on Matrigel in \nall studied cell types. Similar increase in cellular spreading following the treatment with ROCK inhibitors have \nbeen described in microvascular endothelial  cells49, retinal pigment epithelial  cells50 and osteoblastic  cells51. It \nFigure 7.  The invasiveness of endometrial spheroids depends both on the cell type (stromal St-T1b and ESCs \nin brown, epithelial 12Z in blue) and ECM. Invasion and spreading is strongly enhanced by exposed collagen \nI. Migration on Matrigel is modulated by microRNAs and ROCK inhibitor increases invasion and migration \non this substrate. Invasion on collagen is MMP-dependent. Red marks signify experimental intervention. Blunt \narrow signifies that an inhibitor was added. Black arrows show the effect of the intervention. Dashed arrow \nsuggest weak effect.\n\n10\nVol:.(1234567890)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nneeds to be noted that Y27632 has a complex effect on  phenotype52,53. For example, prior studies demonstrated \nthat Y27632 reduces endometriosis associated fibrosis in vitro54.\nAnother promising class of therapeutics targets microRNA  signaling35,55. Given that a typical micro-RNA \nhas tens of targets, sequencing studies need to be accompanied by reliable functional assays to be biologically \n meaningful56. In this study, we demonstrate that the spheroid assay can be used to reproducibly evaluate the \neffect of individual microRNAs on the complex, multicellular spreading of endometriosis-mimicking constructs \nover several days. We show that miR-200b treatment of 12Z cells resulted in a reduction of sprout formation, \nwhich may be indicative of a less invasive phenotype. Our previous 2D data suggest that miR-200b may have \nreverted the 12Z phenotype to an epithelial-state33, however, the paucity of RNA in the spheroids did not allow \nus to unequivocally confirm this hypothesis, as we saw only a non-significant increase in expression of the epi-\nthelial marker E-cadherin (Supplementary Figure S4). Our spheroid model further revealed that while miR-145 \nreduces the migrated area on Collagen I compared to controls (Supplementary Figure S3), results which are in \nagreement with previous in vitro 2D  assays34, the microRNA miR-145 up-regulated in ectopic lesions in vivo \nincreases 12Z sprouting on Matrigel in vitro. These findings were unexpected and investigating this into more \ndetail is beyond the main focus of this study. Nevertheless, there is an increasing appreciation that cells adopt \na host of invasive and migratory strategies that are highly context-dependent and enabled by distinct signal-\ning pathways. Liu and colleagues observed that miR-145 upregulation enhances angiogenesis, including the \nsprouting from aortic rings and linked this to the suppression of tropomodulin 3 (TMOD3)57 while we observed \nthat miR-145 inhibits proliferation and migration in breast cancer and endometriotic cells using the Transwell \nmigration and scratch  assays32,58. Therefore, miR-145 might influence cellular invasive behaviour not only in \ncell-type but also invasive/migratory-mode manner and ECM-substrate-dependent manner. While the major -\nity of oncological studies on miR-145 function suggest that it reduces invasive growth by targeting a variety of \nmRNAs, two studies in trophoblast cells have described invasion-promoting functions of miR-145, which were \nattributed to a targeting of mucin 1 (MUC1) and leukemia inhibitory factor receptor (LIFR),  respectively59,60. \nWe can only speculate that the 3D spheroid culture compared to 2D culture of 12Z cells may have altered the \nexpression patterns of miR-145 target mRNAs in a way that alters the response to this epigenetic regulator. For \nexample, miR-145 may target new mRNAs that are not expressed in the 2D setting (or vice versa), resulting in \na different net response. Overall, we demonstrate that the spheroid assay can be used as an additional assay to \nscreen for both small molecule and RNA-based therapeutics.\nA major limitation of our study is that it relies on cell lines that have been transformed and represent only \na limited subset of disease phenotypes and a more extensive primary cell pool will be required to confirm and \nfully elucidate the here reported findings. We also did not investigate the influence of decidualization. Nota -\nbly, our study did not incorporate primary endometrial epithelial cells with purely epithelial characteristics. \nAdditionally, the wider implementation of this assay for the study of endometriosis will rely on future advances \nin the molecular characterization of spheroids and high-throughput image analysis. Furthermore, automated \nimage analysis would significantly increase the throughput of this assay. In recent years, the quality of image \nprocessing algorithms has approached that of trained humans while significantly decreasing the time needed \nto evaluate individual  samples61. It needs to be noted that for such algorithms either large training datasets or \npre-defined criteria are needed. Given the wide array of spheroid phenotypical responses, we have only started \nto identify such criteria.\nOverall, our screening platform provides evidence that the physiological condensation of endometrial stromal \ncells into spheroids might play an important role in the development of a subset of endometriotic lesions. As \nsuch a directional invasive phenotype in vitro is unlikely to arise by chance, endometrial stromal condensation \nmight also have currently unknown but likely important biological role in the cyclical regeneration of normal \nendometrium. At the same time, our results show that the epithelial lesion-derived 12Z spheroids also rapidly \nmigrate on collagen I and stromal-epithelial interactions modulate the invasiveness of stromal cells. Previous \nstudies indeed revealed significant heterogeneity and variability among different endometriosis subtypes with \nseveral sub-types staining predominantly for stromal  markers62.\nIn conclusion, this study documents that endometrial stromal cell line St-T1b and primary endometriotic \nstromal cells engage in directional migration with significant collagen I remodeling when cultured in spheroid \nculture and that this behaviour is inhibited by the broad-spectrum MMP inhibitor NNGH. We anticipate that \nthis assay will be used to gain further insights into invasive processes involved in endometriosis and for the \nscreening of both small molecule and RNA-based drug candidates and their off-target effects.\nMethods\nCell culture. The 12Z ectopic epithelial cell  line17,29 was maintained in DMEM media (Sigma-Aldrich, cat. \nNo. D0819, Deisenhofen, Germany,) supplemented with 10% FBS (Biochrom GmbH, cat. no. S0615, Berlin, \nGermany) and 1% Pen/Strep (Sigma-Aldrich, cat. No. P4333). The St-T1b cell  line28 and primary ectopic lesion-\nderived stromal cells (ESCs) were maintained in 70% DMEM/18% MCDB 105 media (Sigma-Aldrich, cat. No. \n117-500) supplemented with 10% FBS, 1% Pen/Strep, 1% Glutamine and 5 µg/mL insulin (Sigma-Aldrich, cat. \nNo. 10516). Cells were routinely split twice a week. ESCs were prepared from ectopic lesions and characterized \nas previously  described63. Primary endometriotic stromal cells were prepared from a biopsy of a woman with \nendometriosis who underwent surgical treatment at the Department of Gynecology and Obstetrics of Münster \nUniversity Hospital in 2013, and stored as aliquoted stocks in liquid nitrogen, which were freshly thawed and \npassaged in routine culture two times prior to usage in the experiments described. The modified American Soci-\nety for Reproductive Medicine classification was used to assess  endometriosis64. For all ESC experiments, stroma \ncells derived from a lesion located at the pelvic wall (rASRM score II) of a 19-year-old patient were employed. \nThe study was carried out following the Declaration of Helsinki and approved by the local ethics commission \n\n11\nVol.:(0123456789)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\n(Ethikkommission der Ärztekammer Westfalen‐Lippe und der Medizinischen Fakultät der WWU; approval no. \n1 IX Greb 1 from 19 September 2001, updated 2012). The participant gave written informed consent.\nSpheroid formation. Spheroids were generated using the hanging drop  method65, where 20 µL drops each \ncontaining 20,000 cells were deposited on the top lid of a plastic Petri dish and the bottom chamber was filled \nwith sterile water or PBS (Sigma-Aldrich, cat. No. D1408). The spheroids were harvested after 4 days at 37 °C and \n7.5% or 5%  CO2. The co-culture spheroids were formulated at 1:1 12Z:St-T1b ratio.\nPreparation of collagen I and Matrigel. A 3 mg/mL collagen I hydrogel was formed by neutralizing and \ndiluting the stock solution of Collagen Type I Rat Tail matrix (Corning, Bedford, MA, USA, cat. No. 354236, \n4 mg/mL or 3.4. mg/mL batch) with 1 N NaOH (Applichem, cat. No. A1432, Darmstadt, Germany), 10 × PBS \n(Sigma-Aldrich, cat. No. D1408) and chilled deionized water. The amount of 1 N NaOH was calculated as 1 N \nNaOH volume = (volume of the stock collagen) × 0.023 mL. The amount of 10 × PBS was calculated as volume \n10 × PBS = (final volume)/10. Phenol red-free Basement Membrane Matrix Growth Factor Reduced Matrigel \n(Corning, cat. No. 356231) was thawed on ice prior to use. The gels were deposited into pre-chilled 96-wells at \n35–40 µL per well in 9.2–9.4 mg/mL Matrigel. Each 96-well plate was subsequently sealed with parafilm and \nthe gels were left to solidify for 30–60 min at 37 °C. For higher magnification confocal imaging, collagen and \nMatrigel were deposited on glass coverslips.\nSpheroid response to collagen I/Matrigel. Following gel formation, the wells in a 96-well plate were \nfilled with 50 µL of phenol-red free DMEM (Gibco, cat. No. 21063-029, Darmstadt, Germany) supplemented \nwith 5% charcoal-treated FBS (Biochrom GmbH, cat. no. S0615) and 5 µg/mL insulin solution (Sigma-Aldrich, \ncat. No. 10516). Subsequently, one to three spheroids per well were manually added to individual wells. The \nmedia were changed every 3–5 days and the samples were kept in an incubator at 37 °C and 7.5%  CO2. The \nspheroids were imaged on day 1, 3, 5 and 7.\nMetabolic activity measurement. Viability was assessed using the CellTiter-Glo 3D Viability assay (Pro-\nmega, cat. No. G9681, Walldorf, Germany). Spheroids and surrounding medium were collected after four days \nand transferred to an opaque-walled 96-well plate. A volume of CellTiter-Glo Reagent equal to the volume of cell \nculture medium was added. The mix was incubated according to manufacturer instructions and luminescence \nin the form of relative light units (RLUs) was recorded using a CLARIOstar Plus (BMG Labtech, Ortenberg, \nGermany).\nInhibitors. The effects of three inhibitors on spheroid spreading were evaluated. The MMP inhibitor NNGH \n(Merck, cat. No. SML0584, Darmstadt, Germany) was stored at 15 mM in DMSO and dissolved to the final con-\ncentration of 15 µM in media and the ROCK inhibitor Y27632 (Sigma-Aldrich, cat. No. Y0503, 10 mM stock) \nat 10 µM. In all experiments, spheroids were added directly to inhibitor-containing media. Inhibitor-containing \n5% charcoal-treated FBS/insulin media were exchanged every 3 days.\nmicroRNA transfection. The transfection with negative control microRNA (Scr. miR), miR-200b and \nmiR-145 (Table 1) was performed in a 6-well plate on 60–70% confluent cells. Before transfection, the growth \nmedia were exchanged for Opti-MEM I Reduced Serum Media (Gibco, cat. no. 31985-070, Thermo-Scientific, \nGermany). The transfection with 20 nM microRNA of interest (Table  1) was conducted using the Dharmafect \nreagent (Dharmacon, cat. no. T-2001-03, Lafayette, CO, USA). The cells were incubated with the transfection \nmixture for 24 h when the media were exchanged for full growth media. MiR spheroids were fabricated 48 h after \nthe addition of transfection media.\nLive cell staining and immunostaining. The F-actin cytoskeleton was visualized using Phalloidin Cru-\nzFluor 594 Conjugate (Santa Cruz Biotechnology, cat. No. sc-363795, Santa Cruz, CA, USA) at 1:1000 dilution. \nThe nuclei were visualized using DAPI (Sigma-Aldrich, cat. No. D9564) diluted at 1:50,000. The cells were fixed \nusing 3.7% formaldehyde (Merck, cat. No. 1.04003.1000, Darmstadt, Germany) at 37 °C for 15 min. Following \nwashing with PBS (Sigma-Aldrich, cat. No. D1408), the cells were permeabilized with 0.1% Triton-X (Riedel-de-\nHaen, cat. No. AG 56029, Seelze, Germany) for 5 min. Hydrogels in a 96-well plate were stained by adding 25 µL \nof the 1:1000 phalloidin dye and incubated for 1 h at 37 °C. Live cells were stained either with CellTracker Green \nCMFDA (Thermo Fischer, cat. No. C2925) or CellTracker Red CMTPX (Thermo Fischer, cat. No. C34552) at \na concentration of 5 µM according to manufacturer’s instructions prior to mixing two cell types to form a co-\nculture.\nTable 1.  MicroRNAs used in the study.\nMiR Specifications Cat. number Manufacturer\nScr. miR Pre-miR Negative Control 2 AM17111 Ambion, Darmstadt, Germany\nmiR-200b hsa-miR-200b-3p: MC 10492, mirVana, miRNA mimic 4464066 Ambion\nmiR-145 hsa-miR-145, Pre-miR miRNA Precursor AM17100 Ambion\n\n12\nVol:.(1234567890)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nImaging. Cells were analysed for morphological and cytoskeletal markers. The bright-field images were \nobtained using either an Axiovert100 (Carl Zeiss, Jena, Germany) or an inverted microscope (Leica, Wetzlar, \nGermany) using 5 ×, 10 × and 20 × objectives. Confocal imaging was performed on fixed stained samples in a \n96 well plate. Samples were imaged with the Zeiss LSM 880 inverted confocal microscope (10 ×, 0.45 NA) (Carl \nZeiss, Jena, Germany) equipped with ZEN 2 software and using 11.04 µm z-stack intervals and sequential scan-\nning (514 nm argon laser, 405 nm diode laser, Bright field). The number of sections was adjusted based on the \nsample thickness.\nImage analysis. All images were analysed in  FIJI66. Confocal images are depicted as maximal intensity \nprojections. The spheroid area was measured manually by tracing the spheroids using the freehand tool and \nmeasure function on Bright-field images of spheroids on Petri Dishes, glass slides or in a 96-well plate. Fold \nincrease in area was calculated as the spheroid area on a given day divided by spheroid size on day 0 or day 1. If \non day 1 any protrusions were present and the spheroid was used as a reference size for the given experiment, \nthe protrusions on day 1 were excluded from the analysis to better reflect the size of the original spheroid core. \nThe parameter directionality was calculated as the ratio between the distance in pixels between the centre of \nthe overall migrated area and the centre of the spheroid, divided by the semi-major axis of the overall migrated \narea of the spheroid (Fig.  4E). The number of sprouts per image was counted manually and the sprouting area \nwas calculated as the total area occupied by an expanding spheroid with sprouts minus the area occupied by the \nspheroid without any protrusions (Fig. 6F).\nRNA extraction and cDNA synthesis. mRNA isolation was performed with InnuPREP RNA mini kit \n(Analytikjena, cat. no. 845-KS-2040250, Jena, Germany) according to the supplier’s protocols. The quantity of \nRNA was measured on an Eppendorf BioPhotometer (Eppendorf, Hamburg, Germany) and considered pure if \nthe absorbance at 260 nm/280 nm was more than 1.8. The concentration of 0.4 µg RNA/10 µL of  dH2O was used. \ncDNA synthesis was performed using High Capacity kit (Applied Biosystems, cat. No. 4368814, Foster City, \nCA, USA) according to the manufacturer’s instructions on a TGradient thermocycler (Biometra, Göttingen, \nGermany).\nPCR. Quantitative RT-PCR analysis was performed using 20 ng cDNA per reaction using the Taqman Uni-\nversal PCR Master Mix (Thermo Fisher, cat. No. 4304437) and SYBR Green PCR Master Mix (Thermo Fisher, \ncat. No. 4344463). Gene expression values were calculated using the mean  Ct values of the samples. The expres-\nsion of target genes was normalized to the housekeeping gene ACT, and then to St-T1b cells line  (2−ΔΔCt ). The \nprimers were synthesized by Biolegio (Nijmegen, The Netherlands) and are listed in Tables 2 and 3.\nStatistical analysis. Data were analysed using GraphPad Prism8 (GraphPad Software, San Diego, USA). \nNormal distribution was tested using the Shapiro–Wilk test. A two-tailed unpaired Student’s t tests were used \nto analyse statistical significance between two conditions in an experiment. For experiments with three or more \ncomparisons, an ordinary one-way ANOV A with a Tukey’s multiple comparisons test was used. For data that \nwere not normally distributed, the Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used. A \ntwo-way repeated-measures (RM) ANOV A with Šidák’s multiple comparisons test was used to evaluate the effect \nof Matrigel and collagen I on spheroid size over time. Significance values were chosen as *p < 0.05; **p < 0.01; \n***p < 0.001, ****p < 0.0001. Error bars represent the mean ± s.d or mean + s.d. All figure panels were assembled \nin Inkscape 0.92.\nTable 2.  Sybr Green PCR primers.\nForward Reverse\nACTB TCA AGA TCA TTG CTC CTC CTGAG ACA TCT GCT GGA AGG TGG ACA \nRAC1 CGC CTC CTG TAG TCG CTT TG CAC GCT GTA TTC TCG CCA GTG \nMMP14 CCA TTG GGC ATC CAG AAG AGAGC GGA TAC CCA ATG CCC ATT GGCCA \nMMP2 GCC GTG TTT GCC ATC TGT TT CTG CAG GGA GCA GAG ATT CG\nVIM TCA GCA TCA CGA TGA CCT TGAA CTG CAG AAA GGC ACT TGA AAGC \nCDH2 TTC TGA CAA CAG CTT TGC CTCTG TTT ATT CAG AAC GCT GGG GTCA \nCDH1 CAA AGC CCA GAA TCC CCA AG CAC ACC TGG AAT TGG GCA AA\nTable 3.  PCR primers Taqman.\nActin hs99999903 m1\nROCK2 hs00153074 m1\n\n13\nVol.:(0123456789)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nReceived: 18 October 2019; Accepted: 29 January 2021\nReferences\n 1. Zondervan, K. T. et al. Endometriosis. Nat. Rev. Dis. Prim. 4, 9 (2018).\n 2. Y oung, V . J., Brown, J. K., Saunders, P . T. K. & Horne, A. W . The role of the peritoneum in the pathogenesis of endometriosis. Hum. \nReprod. Update 19, 558–569 (2013).\n 3. Boretto, M. et al. Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug \nscreening. Nat. Cell Biol. 21, 1041–1051 (2019).\n 4. Abu-Asab, M., Zhang, M., Amini, D., Abu-Asab, N. & Amri, H. Endometriosis gene expression heterogeneity and biosignature: \nA phylogenetic analysis. Obstet. Gynecol. Int. 2011, 1–12 (2011).\n 5. Sampson, J. A. 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Methods 9, 676–682 (2012).\nAcknowledgements\nWe would like to acknowledge Anna Starzinski-Powitz for the generous gift of 12Z cells, and Birgit Gellersen \nfor the generous gift of the St-T1b cell line, Birgit Pers and Dorothea Godulla for expert technical assistance, \nNiki Loges for help with confocal microscopy, Timo Strünker for providing access to equipment, and Peter \nFriedl for helpful discussions. This research was supported by a WiRe—Women in Research Fellowship and a \nWWU Fellowship of the University of Münster (to AS) and European Commission (REA) EU H2020 ‐MSCA-\nRISE‐2015 Grant 691058 MOMENDO (to MG). We acknowledge funding by the Open Access Publishing Fund \nof Münster University.\nAuthor contributions\nA.S. and V .F . performed the majority of the experiments and analysed the data. A.S. drafted the figures and wrote \nthe manuscript draft. M.N., Y .S. and M.-K.W . helped to establish the 3D system by assisting with experiments, \nand by providing unpublished data and expertise in 3D culture. K.B. performed confocal immunofluorescence \nmicroscopy. S.D.S. and L.K. provided patient tissues and documented clinical data. B.G. provided resources, \nadvice and expertise in 3D and primary cell culture and confocal immunofluorescence microscopy. L.K. provided \nresources and general support. M.G. oversaw and coordinated the study and wrote the manuscript M.G. and \nA.S. conceived the study. All authors reviewed the manuscript.\nFunding\nOpen Access funding enabled and organized by Projekt DEAL.\nCompeting interests \nThe authors declare no competing interests.\n\n15\nVol.:(0123456789)Scientific Reports |         (2021) 11:4115  | https://doi.org/10.1038/s41598-021-83645-8\nwww.nature.com/scientificreports/\nAdditional information\nSupplementary Information The online version contains supplementary material available at https:// doi. org/ \n10. 1038/ s41598- 021- 83645-8.\nCorrespondence and requests for materials should be addressed to A.S. or M.G.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access  This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the \nCreative Commons licence, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from \nthe copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.\n© The Author(s) 2021","source_license":"CC0","license_restricted":false}