Figure 6. Functional analysis of inflammatory cue (IL1B) repsonses in co-cultures in synthetic hydrogel reveals the intitiation of the endometriotic phenotype mediated by cell-cell communication.

(A) 3D maximum intensity projections of PGR expresison in E2-only treated co-cultures in response to IL1B stimulation (B-C) IL1B stimulation (1 ng/mL) induces morphological changes in EEOs. Representative IF images (B) and quantification (C) of epithelial height (distance from base to apical edge of the epithelium, N=3). (D) Schematic of donor matched EEO monoculture and co-culture in IL1B treatment groups. (E) Represenative images of time-lapse co-cultures stimulated with IL1B reveals enlarged EEO diameter by day 7 of culture. Quantication of the mean fold-change of EEO diameter across 15-days of cultures from daily time-lapse images in co-cultures (black) and monocultures (red) across treatment conditions (E, PE, W) and IL1B-treated (E+, PE+) groups. Fold-change denotes the mean increase in diameter relative to day 1 of culture. Analysis compares the mean EEO growth rate between monoculture vs coculture (n=4; E + IL-1 p=0.0076; PE + IL1B p=0.0026). (G) Immunostaining analysis of DNA synthesis in co-cultures and EEO monocultures by EdU incorporation in response to hormones and IL1B treatment (N=3). (H-I) Quantification of proliferative profiles calculated as the number of EdU⁺ cells per organoid area (mm²) in (H) organoid monocultures and (I) co-cultures. (J) Working model of the cellular mechanisms driving epithelial and stromal communication in the endometrium under physiologic (hormones) and pathogenic (IL1B-treated) conditions. All images are of day-15 co-cultures in the synthetic ECM 3wt% 20kDa-PEG-MIX hydrogels. Significance is indicated as *p<0.05, **p<0.01, ***p<0.001.