Fig 2: Effects of matrix stiffness, with or without TGF-ß1 treatment, on the PGE2 signaling pathway in endometrial stromal cells. (A) Relative fold change in gene expression of COX-2, EP2, and EP4 in endometrial stromal cells grown on soft substrates (5-kPa PGS), rigid substrates (30-kPa PGS), and plastic for three days, with or without TGF-ß1 treatment (n = 7). Values are normalized to the GAPDH expression. All data were expressed as fold change relative to the cells grown at 5-kPa PGS without TGF-ß1 treatment. (B) Left panel: Detection of protein levels of COX-2, EP2, and EP4 by immunoblotting of lysates of endometrial stromal cells treated under the indicated conditions. Right panel: Relative fold change in the protein levels of COX-2, EP2, and EP4 in endometrial stromal cells grown on soft (5-kPa PGS) and rigid substrates (30-kPa PGS) for three days, with or without TGF-ß1 treatment (n = 4–5). The GAPDH expression levels were served as a loading control. All data were expressed as fold change in protein expression relative to cells grown at 5-kPa PGS without TGF-ß1 treatment. Symbols of statistical significance levels: *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not statistically significant (ie, p > 0.05). Data are represented in means ± SDs

Fig 3: (A) Representative photomicrographs of histochemistry (Masson trichrome) and immunohistochemistry analysis of COX-2, EP2, and EP4 in ovarian endometrioma tissue samples from adolescent patients (left panel) and adult patients (middle panel), along with data summary (right panel). For both the left and middle panels, different rows show different markers as indicated. Magnification: ×400. Scale bar = 50 µm. On the right panel, the results are summarized by the boxplot. The results for COX-2, EP2, and EP4 are for the stromal and the epithelial components. COX-2, cyclooxygenase-2; EP2 and EP4, E-series of prostaglandin receptors type 2 and type 4, respectively. (B) Scatter plots showing the correlations between the extent of lesional fibrosis and the staining levels of COX-2, EP2, and EP4 in the stromal (upper panel) and the epithelial components (lower panel). In each plot, each dot represents one data point from one patient, and the dashed line represents the regression line. Pearson's correlation coefficient, along with its statistical significance level, also is shown. Symbols for statistical significance levels: NS: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001

Fig 4: Summary result of immunohistochemistry and Masson trichrome staining in human samples showing the dynamic changes as endometriotic lesions progress over time. Dynamic changes in the extent of fibrosis in tissues/lesions in different groups of mice (A). Dynamic changes in the immunostaining of COX-2 the stromal (B) and epithelial (C) components in tissues/lesions in different groups of mice. Dynamic changes in the immunostaining of EP2 the stromal (D) and epithelial (E) components in tissues/lesions in different groups of mice. Dynamic changes in the immunostaining of EP4 the stromal (F) and epithelial (G) components in tissues/lesions in different groups of mice. In all panels, the data are represented by the means ± SDs, and Kruskal's rank test was used. Symbols of statistical significance levels: *P < 0.05; **P < 0.01; ***P < 0.001; NS: not statistically significant (P > 0.05)

Fig 5: Roles of TAMMs in supporting organoid culture in vitro and tumor growth in vivo(A and B) Images of growth changes in the co-culture of adenoma-derived organoids with or without M-MDCs and with or without adding EP4 and COX-2 inhibitors (A), and the quantification of organoid mass (= No. × area) under different conditions. This quantification was based on two independent experiments each with multiple replicates. Each experiment was normalized to the mean of the crypt-only group, and then two experiments were subject to statistical analysis. Two-way ANOVA with post hoc test was used to compare group means. (B). For normalized mass data: we fit a two-way ANOVA model with all data from two experiments. Then, post-hoc t-tests were performed to test specific comparisons of interest. P-values were adjusted using the sidak method. The same experiment was repeated two times at different times and data are Mean+/- SEM.(C and D) IF assay of crypt budding and p-ßcatS552 detection (C), and western blot analyses of the p-ßcatS552 protein level in organoid culture with or without adding inhibitors (D).(E and F) Flow cytometry analysis of RMs (E) and TAMs (F) and inhibition of Cox-2 reduced both RMs and TAMs. T-tests with Means +/- SD.(G) Dot plot showing gene expression associated with RM and TAMs.(H) Adenoma growth was substantially reduced by Clodrosome compared with Encapsome.(I–K) Clodrosome caused significant depletion of CD11bintCX3CR1+ cells, which was shown to be Ly6c-MHCII+, thus fitting the definition of TAMs (B). H-K, T-tests with Welch’s correction Means +/-SD.