{"paper_id":"e1ff1f9b-5415-42a0-bc68-438591736fee","body_text":"ARTICLE | Article in Press\nScientific Reports https://doi.org/10.1038/s41598-026-67394-0\nScientific Reports\nThis Article in Press is shared early to give you faster access to new research. It is citable and carries a permanent DOI. \nThe final edited version will replace it automatically.\nSalinomycin modulates viability, invasion, and \ninflammatory responses in primary endometriotic \nstromal cell cultures in association with Wnt/β-catenin \nsignaling\nSonia Sadeghpour · Morteza Ghasemnejad-Berenji · Farzad Maleki ·  \nMohammad Reza Pashaei · Hojat Ghasemnejad-Berenji\nReceived: 27 December 2025 / Accepted: 13 August 2026\n© The Author(s) 2026\nAbstract\nEndometriosis is a chronic inflammatory disorder characterized by aberrant stromal-cell behavior, including \nenhanced invasion, resistance to apoptosis, and sustained cytokine production. Dysregulation of Wnt/β-catenin \nsignaling has been linked to these pathological features, emphasizing its association with disease-related cellu -\nlar phenotypes. Identifying non-hormonal approaches that selectively target endometriotic stromal cells remains \nan important research focus. In this study, primary endometriotic stromal cell cultures derived from ectopic \nlesions of women with ovarian endometrioma were compared with primary control endometrial stromal cell \ncultures from women without endometriosis. Vimentin and CK18 were evaluated in separate flow-cytometry \ntubes; therefore, the designation of these cultures as stromal cultures is operational and does not imply com -\nplete lineage purity. Cells were treated with Salinomycin, and MTT-derived viable-cell/metabolic activity, \nMatrigel invasion, cytokine secretion, and selected molecular markers related to Wnt/β-catenin signaling, cell-\ncycle regulation, invasion, and apoptosis were evaluated using MTT assays, Transwell assays, ELISA, and \nquantitative real-time PCR. Salinomycin induced a dose- and time-dependent reduction in MTT signal, with \nprimary endometriotic cultures showing greater sensitivity than control cultures. At the selected experimental \nconcentration, salinomycin reduced the viable-cell/metabolic signal and invasion in primary endometriotic \nstromal cell cultures and decreased IL-6 and IL-8 secretion. These phenotypic changes were accompanied \nby increased β-catenin phosphorylation at Ser45, reduced AXIN2, Cyclin D1, and MMP-9 expression, and \nincreased Caspase-3 expression in ectopic cultures. Because MTT alone cannot distinguish reduced prolifera -\ntion from cytotoxicity or altered metabolism, the findings are interpreted as viability-associated and prolif -\neration-associated responses rather than direct proof of isolated growth arrest. These findings indicate that \nSalinomycin modulates disease-relevant features of primary endometriotic stromal cell cultures in association \nwith Wnt/β-catenin pathway-related changes and support further mechanistic investigation of this experimental \nnon-hormonal approach.\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nKeywords Endometriosis · Salinomycin · Wnt/β-catenin signaling  · Endometriotic stromal cells · \nInflammation · Cell invasion\nIntroduction\nEndometriosis is a chronic, estrogen-dependent inflammatory disorder in which endometrial-like tissue devel -\nops outside the uterine cavity 1,2. Although the disease is histologically benign, endometriotic lesions can show \nseveral tumor-like biological features, including adhesion, invasion, remodeling of surrounding tissue, and per -\nsistence at ectopic sites3. These biological properties are clinically important because endometriosis is frequently \nassociated with chronic pelvic pain, dysmenorrhea, dyspareunia, abnormal uterine bleeding, and infertility, all of \nwhich may substantially impair quality of life 4. The origin and progression of the disease are not explained by \na single mechanism. Retrograde menstruation remains a major theory, but lesion establishment also depends on \nimmune dysfunction, altered proliferation and apoptosis, aberrant endocrine responses, and genetic susceptibil -\nity2,5. In advanced disease, pelvic adhesions and fibrosis are common and may reflect repeated cycles of tissue \ninjury and repair within ectopic lesions6,7.\nAmong the signaling pathways implicated in endometriosis, Wnt/β-catenin signaling has received increas -\ning attention. Wnt signaling is a conserved network that regulates embryonic development, organogenesis, tis -\nsue homeostasis, stem-cell function, differentiation, migration, and tissue polarity8–11. In the canonical pathway, \nβ-catenin stabilization and nuclear translocation activate TCF/LEF-dependent transcriptional programs that influ-\nence cell fate and stromal-cell behavior. In endometriosis, dysregulated Wnt/β-catenin activity has been linked \nto fibrotic remodeling through the regulation of connective tissue growth factor, collagen I, α-smooth muscle \nactin, and fibronectin12. Other disease-related regulators, including FOXP1 and WEE1, have also been reported \nto promote fibrotic changes in endometriotic stromal cells through Wnt/β-catenin-associated mechanisms13,14.\nInflammation is another central feature of the endometriotic microenvironment. A broad range of inflammatory \nmediators, including IL-1β, IL-17 A, IL-6, IL-8, IL-10, TNF-α, and VEGF, has been implicated in endometrio -\nsis15. In particular, increased IL-6 and IL-8 concentrations have been reported in the peritoneal fluid of women \nwith active endometriosis16,17. IL-6 and IL-8 are of particular relevance because they are produced not only by \nimmune cells but also by endometrial and endometriotic cells 15,18. These mediators contribute to angiogenesis, \nimmune-cell recruitment, adhesion, proliferation-associated responses, and lesion progression 19–24. Therefore, \nstromal-cell behavior, inflammatory signaling, and tissue remodeling are closely connected processes in the biol-\nogy of endometriosis.\nCurrent treatment strategies include hormonal suppression and surgery. Although these approaches are useful \nfor many patients, hormonal therapy may not be suitable for women seeking fertility preservation, and recurrence \nor persistence of symptoms remains a major clinical problem25,26. These limitations have encouraged the evalu-\nation of non-hormonal experimental approaches that target disease-relevant cellular pathways. Because aberrant \nWnt/β-catenin signaling has been implicated in lesion growth, fibrosis, and invasive behavior, this pathway rep-\nresents a relevant target for investigating new pharmacological strategies in endometriosis research12,27,28.\nSalinomycin is a natural polyether carboxylic ionophore isolated from Streptomyces albus and has long been \nused in veterinary medicine 29. Beyond this use, it has been studied for its anti-tumor and pathway-modulat -\ning properties in several experimental models 29,30. Previous studies have shown that salinomycin can attenuate \nWnt/β-catenin signaling, including inhibition of Wnt-induced LRP6 phosphorylation and reduction of LRP6-\nassociated signaling31,32. It has also been associated with DNA-damage responses, reactive oxygen species gener-\nation, mitochondrial membrane depolarization, caspase-3 activation, PARP cleavage, oxidative stress, autophagy, \nmodulation of growth and migration, and p38 MAPK activation 33–39. These reported activities suggest that sali-\nnomycin may be useful for examining Wnt-related, inflammatory, and survival-associated responses in endome-\ntriotic stromal-cell models.\n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nBased on this rationale, the present study investigated the effects of salinomycin on primary endometriotic \nstromal cell cultures derived from ectopic lesions and on primary control endometrial stromal cell cultures. \nWe evaluated MTT-derived viable-cell/metabolic activity, invasive behavior, IL-6 and IL-8 secretion, β-catenin \nphosphorylation at Ser45, and the expression of AXIN2, Cyclin D1, MMP-9, and Caspase-3. The aim was to \ncharacterize in vitro cellular responses associated with salinomycin exposure and Wnt/β-catenin signaling, while \navoiding overinterpretation of the findings as direct evidence of clinical therapeutic efficacy.\nMaterials and methods\nSample collection\nThis study was approved by the Research Ethics Committee of Urmia University of Medical Sciences (approval \nno. IR.UMSU.HIMAM.REC.1401.057). All experiments were conducted in accordance with relevant ethical \nguidelines and regulations, including the Declaration of Helsinki. Written informed consent was obtained from \nall participants before sample collection. Ectopic endometrial biopsies were collected from 10 women with ovar-\nian endometriosis (aged 29–43 years) who underwent laparoscopic surgery. Endometrial tissues were also col -\nlected from 10 women in the control group who had benign gynecological disorders, were confirmed to be free \nof endometriosis by laparoscopic evaluation, and underwent the same surgical procedure. Control biopsies were \nobtained from the functional layer at several sites in the fundal region of the uterine cavity using a biopsy catheter \n(Pipelle biopsy, Behrad Rouyesh Royan, Iran). All participants had regular menstrual cycles, had not used hor -\nmonal medications for at least 3 months before surgery, and had no history of malignant or autoimmune disease. \nAll biopsy samples were collected during the early secretory phase of the menstrual cycle (days 16–19), deter -\nmined from the date of the last menstrual period and confirmed by histopathological evaluation. All patients with \nendometriosis had stage III disease according to the American Society for Reproductive Medicine classification. \nSamples were immediately placed in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) \ncontaining 1% penicillin–streptomycin and transported to the laboratory on ice.\nCell isolation and culture\nPrimary endometriotic and control endometrial stromal cell cultures were established according to the protocol \ndescribed by Karamian et al.40 Briefly, ectopic endometriotic tissue samples from women with ovarian endome-\ntrioma and control endometrial biopsies from women without endometriosis were minced into small fragments \nand digested with 0.1% collagenase I (Gibco, New York, NY , USA) at 37 °C for 1 h. The digested suspension \nwas sequentially filtered through 70-µm and 40-µm cell strainers to reduce undigested tissue fragments and glan-\ndular epithelial components. The resulting single-cell suspension was seeded into T25 culture flasks containing \nDMEM/F12 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Gibco, Grand \nIsland, NY , USA) and incubated at 37 °C in a humidified atmosphere with 5% CO₂. After initial attachment, \nnon-adherent cells and debris were removed by medium replacement, and adherent fibroblast-like cells were \nexpanded for subsequent experiments. Primary cultures were established separately from individual participants \nand were not pooled. Although tissue samples were collected from 10 participants in each group, cultures from \nthree independent donors per group were used for the reported in vitro experiments. Each donor-derived culture \nconstituted one biological replicate ( n = 3 per group). Throughout the manuscript, the terms “primary endome -\ntriotic stromal cell cultures” and “primary control endometrial stromal cell cultures” are used as operational \ndesignations based on tissue source, the isolation procedure, adherent fibroblast-like morphology, and Vimentin \nexpression. These terms should not be interpreted as evidence of complete lineage purity. Vimentin and CK18 \nimmunophenotyping was used to characterize the cultures as described below.\n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nFlow cytometric analysis of vimentin and CK18 expression\nVimentin and CK18 expression was examined by flow cytometry to characterize the primary cell cultures. Vimen-\ntin was used as a stromal/mesenchymal-associated marker, whereas CK18 served as an epithelial-associated \nmarker. Cells were fixed with 4% paraformaldehyde and washed once with phosphate-buffered saline (PBS; \nSigma-Aldrich, P4417). To minimize nonspecific antibody binding, the cells were blocked with 10% (v/v) goat \nserum (Sigma-Aldrich, G9023) prepared in PBS for 30 min at room temperature. After removal of the blocking \nsolution, the cells were incubated with the corresponding primary antibodies, diluted 1:100 in PBS, for 4 h at \n37 °C. The cells were then washed once with PBS and incubated with the appropriate FITC-conjugated secondary \nantibodies at a dilution of 1:150 for 45 min at 37 °C in the dark. Following incubation, the samples were washed, \ncentrifuged, resuspended in PBS, and analyzed using a BD FACSCalibur flow cytometer. Vimentin and CK18 \nwere evaluated in separate tubes, and FITC-positive events were defined relative to the corresponding unstained \ncontrols. Because Vimentin and CK18 were analyzed in separate tubes, their co-expression within individual \ncells and the relative proportions of distinct cell populations could not be determined. Therefore, the Vimentin/\nCK18 profile was used only for descriptive characterization of the cultures and was not considered sufficient \nto establish complete lineage purity. The absence of additional stromal-cell markers, such as CD10, ER, and \nHOXA10, is acknowledged as a limitation of the study.\nMTT-based viable-cell/metabolic activity assessment\nThe effects of salinomycin on MTT-derived viable-cell/metabolic activity were evaluated using the MTT colo -\nrimetric assay. Primary endometriotic stromal cell cultures and primary control endometrial stromal cell cul -\ntures were seeded into 96-well plates at a density of 5 × 10³ cells per well in 100 µL of DMEM/F12 medium \nsupplemented with 10% fetal bovine serum and antibiotics. After overnight attachment, cells were treated with \nsalinomycin at 0.01, 0.1, 1, or 10 µM for 24, 48, or 72 h. Salinomycin was freshly prepared in DMSO, and the \nfinal DMSO concentration did not exceed 0.1%. Vehicle-treated cells received DMSO alone, and untreated cells \nserved as negative controls. At each time point, 10 µL of MTT solution (5 mg/mL in PBS) was added to each \nwell, followed by incubation for 3–4 h at 37 °C. The medium was then removed, and 100 µL of DMSO was added \nto dissolve the formazan crystals. Absorbance was measured at 570 nm with a reference wavelength of 630 nm \nusing a microplate reader. The MTT signal was expressed as a percentage of the vehicle-treated control group. \nExperiments were performed using primary cultures derived from three independent donors per group, with each \ndonor-derived culture representing one biological replicate ( n = 3 per group). Each donor-derived culture was \nanalyzed in technical triplicate, and the technical replicate values were averaged to generate one value per donor \nbefore statistical analysis. IC₅₀ values were calculated by nonlinear regression analysis of dose–response curves \nusing GraphPad Prism version 6.0. For the endpoint MTT-based viable-cell activity assay, cells were seeded \nat 1 × 10⁴ cells per well and treated under the selected experimental condition. MTT staining and absorbance \nmeasurement were performed as described above, without repeating the full protocol. Because MTT reduction \nreflects cellular metabolic activity and is influenced by assay conditions in addition to cell number, the results \nwere interpreted as changes in viable-cell/metabolic signal rather than as direct evidence of proliferation or cyto-\ntoxicity41. The 24-, 48-, and 72-h measurements represent a time-course MTT response and not a direct prolifera-\ntion curve. Proliferation-associated effects were further evaluated by Cyclin D1 expression analysis. This assay \nwas performed based on the previously described protocol with minor modifications40.\nCell invasion assay\nThe invasive behavior of stromal cells was assessed using Matrigel-coated Transwell inserts with an 8-µm \npore membrane. Briefly, cells were collected by trypsinization, washed with phosphate-buffered saline (PBS), \ncentrifuged, and resuspended in a medium containing reduced serum. After determining cell numbers using a \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nhemocytometer, 1 × 10⁵ cells were placed into the upper compartment in DMEM/F12 supplemented with 2% fetal \nbovine serum. The lower compartment was filled with complete culture medium to create a chemotactic gradi -\nent. Cells were then incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. During the 24-h \ninvasion assay, cells were left untreated or exposed to vehicle or 1.1 µM salinomycin.\nAt the end of the incubation period, non-invasive cells remaining on the upper side of the membrane were care-\nfully removed using a sterile cotton swab. Cells that had migrated to the underside of the membrane were fixed \nand stained with 0.1% Giemsa. Invasive cells were quantified by counting five randomly selected microscopic \nfields per insert using a light microscope at 200× magnification. Data were expressed as the mean number of \ninvaded cells per field for each experimental group, as previously described40.\nELISA-based quantification of cytokines and phosphorylated β-catenin\nLevels of phosphorylated β-catenin at Ser45 were determined using the InstantOne™ ELISA kit (eBioscience, \nUSA). Cells were seeded into 96-well plates at a density of 3 × 10⁴ cells per well and allowed to adhere overnight. \nCells were subsequently lysed with 100 µL of 1× lysis buffer, and 50 µL of each cell lysate, together with the kit-\nprovided controls, was transferred into ELISA wells in triplicate. An equal volume (50 µL) of Antibody Cocktail \nwas then added, and plates were incubated for 1 h at room temperature with gentle agitation. After three washing \nsteps, 100 µL of detection reagent was applied for 20 min, followed by termination of the reaction with 100 µL \nof stop solution. Optical density was measured at 450 nm.\nConcentrations of secreted IL-6 and IL-8 in conditioned culture media were quantified using DuoSet® ELISA \nkits (R&D Systems, USA) in accordance with the manufacturer’s protocols. The assay sensitivities were 9.4 pg/\nmL for IL-6 and 31.2 pg/mL for IL-8. Total protein content in cell lysates was measured using the Bradford pro-\ntein assay (Bio-Rad) and used for data normalization. The cytokine-assay procedures were based on Karamian \net al. with minor modifications42. For each group, assays were performed using three independent donor-derived \ncultures (n = 3 biological replicates). Each donor-derived culture was analyzed in technical triplicate, and techni-\ncal replicate values were averaged before statistical analysis.\nRNA extraction and quantitative real-time PCR\nTotal RNA was isolated and purified from cultured cells using RiboEx reagent (GeneAll, Seoul, Korea). Comple-\nmentary DNA (cDNA) was synthesized from 500 ng of RNA using a cDNA synthesis kit (Qiagen, Hilden, Ger-\nmany). Quantitative real-time PCR (qPCR) was performed in technical triplicate for each donor-derived culture \nusing gene-specific primers and SYBR ® Green Master Mix (Ampliqon, Odense, Denmark) on a StepOnePlus \nReal-Time PCR System (Applied Biosystems, Foster City, CA, USA). Gene-expression levels were normal -\nized to GAPDH as the internal reference. Primer sequences are summarized in Table 1. The thermal cycling \nprotocol included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and \n60 °C for 40 s for combined annealing and extension. Relative expression levels of AXIN2, Caspase-3, Cyclin \nD1, and MMP-9 were calculated using the 2 − ΔΔCt method with REST-RG software (version 3). The resulting \nGene Sense primers Antisense primers\nCyclin D1 5 ´ -    A   T   T   A   G   G   T   T   C   C   A   T   C   C   T   T   T   A   C   G   T   G   - 3 ´5 ´ -    G   T   A   C   A   G   A   G   A   T   G   C   C   T   A   G   A   A   C   C   \nC   - 3 ´\nCaspase-3 111 5 ´ -    G   G   A   A   G   C   G   A   A   T   C   A   A   T   G   G   A   C   T   C   T   G   \nG − 3´\n5 ´ -    G   C   A   T   C   G   A   C   A   T   C   T   G   T   A   C   C   A   G   A   \nC   C − 3´\nMMP-9 5 ´ -    C   A   G   G   C   A   G   C   T   G   G   C   A   G   A   G   G   A   A   T   - 3 ´ 5 ´ -    T   T   C   G   A   C   T   C   T   C   C   A   C   G   C   A   T   C   T   C   - 3 ´\nAxin-2 5 ´ -       T   G   A   G   C   G   G   G   A   T   G   C   T   T   T   G   A   A   C      - 3 ´ 5 ´ -    A   T   C   C   T   G   T   C   T   C   T   G   T   G   C   A   T   T   G   C   T   \nG   - 3 ´\nGAPDH 5 ’ -    C   G   C   T   T   C   G   G   C   A   G   C   A   C   A   T   A   T   A   C   - 3 ’ 5 ’ -    A   A   A   T   A   T   G   G   A   A   C   G   C   T   T   C   A   C   G   A   - 3 ’\nTable 1 Sequences of the \nprimers used for mRNA \nquantitation by real-time \nRT-PCR.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\ndonor-level relative-expression values were used for the statistical analyses described below. A no-template con-\ntrol reaction without cDNA was included for each gene42.\nStatistical analyses\nData are presented as mean ± standard deviation (SD), as indicated in the corresponding figure legends. The indi-\nvidual donor-derived primary culture was considered the biological unit of analysis. For each assay, technical \nreplicate measurements were averaged to generate a single value for each donor and were not treated as inde -\npendent observations. Statistical analyses were therefore based on three independent donor-derived cultures per \ngroup (n = 3 biological replicates). For the dose- and time-response MTT experiment, the effects of salinomycin \nconcentration and exposure time were evaluated using two-way analysis of variance (ANOV A), followed by \nTukey’s multiple-comparisons test. For the endpoint MTT assay, cell-invasion assay, cytokine measurements, \nphospho-β-catenin ELISA, and gene-expression analyses, differences among experimental groups were evalu -\nated using one-way ANOV A followed by Tukey’s post hoc test. Statistical analyses were performed using SPSS \nsoftware, version 19.0 (IBM Corp., Armonk, NY , USA). A p value < 0.05 was considered statistically significant.\nResults\nCharacterization of isolated primary cell cultures\nFlow cytometry was used to assess Vimentin and CK18 expression in the isolated primary cell cultures. Vimentin \nwas examined as a stromal/mesenchymal-associated marker, whereas CK18 was examined as an epithelial-asso-\nciated marker. Vimentin staining showed 85.0% FITC-positive events compared with 3.85% in the corresponding \ncontrol (Fig. 1a–c). CK18 staining showed 79.6% FITC-positive events compared with 5.61% in the correspond-\ning control (Fig. 1d–f). Because Vimentin and CK18 were assessed in separate tubes, these findings do not distin-\nguish marker co-expression within the same cells from the presence of distinct cell populations. Thus, the marker \nprofile supported the operational designation of the cultures as primary endometriotic and control endometrial \nstromal cell cultures but did not establish complete lineage purity.\nSalinomycin reduced MTT-derived viable-cell/metabolic activity\nA time-course analysis of salinomycin effects on MTT-derived viable-cell/metabolic activity was performed in \nprimary endometriotic stromal cell cultures using concentrations of 0.01, 0.1, 1, and 10 µM over 24, 48, and 72 h. \nSalinomycin produced a progressive reduction in the MTT signal with increasing concentration and exposure \ntime, with the strongest reduction observed after 72 h (Fig. 2). Based on the dose- and time-dependent response \nanalysis, 1.1 µM salinomycin was selected for subsequent endpoint experiments. These measurements were not \ninterpreted as a direct proliferation curve.\nThe endpoint MTT assay was then used to compare primary control endometrial and endometriotic stromal \ncell cultures under baseline and treatment conditions. Baseline MTT absorbance was lower in endometriotic \nstromal cell cultures than in control endometrial stromal cell cultures (Fig. 3A). Because the MTT assay mainly \nreflects viable-cell metabolic activity, this difference was interpreted as a change in MTT-derived signal rather \nthan as direct evidence of altered proliferation. In primary endometriotic stromal cell cultures, treatment with \n1.1 µM salinomycin for 72 h markedly reduced the MTT-derived signal compared with vehicle-treated cells \n(Fig. 3B). A smaller but statistically significant reduction was also observed in primary control endometrial stro-\nmal cell cultures (Fig. 3C). Overall, these findings show that salinomycin reduced viable-cell/metabolic activity, \nwith a stronger effect in endometriotic stromal cell cultures.\n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nSalinomycin reduced invasive behavior in primary stromal cell cultures\nThe invasive behavior of primary control endometrial and endometriotic stromal cell cultures was evaluated \nusing Matrigel-coated Transwell inserts. Representative images showed invaded cells under control, vehicle-\ntreated, and salinomycin-treated conditions (Fig. 4). Quantification was performed after the 24-h Transwell inva-\nsion assay by counting invaded cells in randomly selected microscopic fields. At baseline, endometriotic stromal \ncell cultures showed a higher number of invaded cells per field than control endometrial stromal cell cultures \n(Fig. 5A). Exposure to 1.1 µM salinomycin reduced invasion in primary control endometrial stromal cell cultures \ncompared with vehicle-treated cells (Fig. 5B). The reduction was more pronounced in primary endometriotic \nstromal cell cultures (Fig. 5C). These results indicate that salinomycin attenuated invasive behavior under the \nexperimental conditions, particularly in endometriotic stromal cell cultures.\nSalinomycin increased phospho-β-catenin Ser45 signal in endometriotic stromal cell cultures\nPhospho-β-catenin at Ser45 was measured by ELISA and reported as optical density values. At baseline, primary \nendometriotic stromal cell cultures showed a lower phospho-β-catenin Ser45 signal than primary control endo -\nmetrial stromal cell cultures (Fig. 6A). Treatment with 1.1 µM salinomycin for 72 h increased the phospho-β-\ncatenin Ser45 signal in primary endometriotic stromal cell cultures compared with vehicle-treated cells (Fig. 6B). \nIn contrast, salinomycin did not produce a significant change in primary control endometrial stromal cell cultures \n(Fig. 6C). These data suggest that salinomycin was associated with altered β-catenin regulation, mainly in endo-\nmetriotic stromal cell cultures.\nFig. 1 Flow-cytometric characterization of isolated primary cell cultures. Vimentin (a–c) and CK18 (d–f) were evaluated as \nstromal/mesenchymal-associated and epithelial-associated markers, respectively. Vimentin staining yielded 85.0% FITC-\npositive events versus 3.85% in the corresponding control, whereas CK18 staining yielded 79.6% versus 5.61%. Because \nVimentin and CK18 were assessed in separate tubes, these data do not establish single-cell co-expression or complete lineage \npurity.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nSalinomycin reduced IL-6 and IL-8 secretion\nSecreted IL-6 and IL-8 levels were measured in conditioned culture media by ELISA and normalized to total \nprotein content. Baseline IL-6 secretion was comparable between untreated primary control endometrial stromal \ncell cultures and primary endometriotic stromal cell cultures from the same experimental set (Fig. 7A). After \ntreatment with 1.1 µM salinomycin for 72 h, IL-6 secretion was reduced in primary control endometrial stromal \ncell cultures compared with vehicle-treated cells (Fig. 7B). A stronger reduction was observed in primary endo-\nmetriotic stromal cell cultures (Fig. 7C). For IL-8, the initial baseline comparison showed higher secretion in \nprimary endometriotic stromal cell cultures than in primary control endometrial stromal cell cultures (Fig. 8A). \nUnder endpoint treatment conditions, salinomycin produced a modest reduction in IL-8 secretion in primary \ncontrol endometrial stromal cell cultures (Fig. 8B). In primary endometriotic stromal cell cultures, salinomycin \nmarkedly reduced IL-8 secretion compared with vehicle-treated cells (Fig. 8C). Together, these findings indicate \nthat salinomycin reduced pro-inflammatory cytokine secretion, with a more evident effect in endometriotic stro-\nmal cell cultures.\nSalinomycin altered expression of proliferation-, invasion-, and apoptosis-associated markers\nCyclin D1 mRNA expression was evaluated as a proliferation-associated marker. Baseline Cyclin D1 expression \ndid not differ significantly between primary control endometrial stromal cell cultures and primary endometriotic \nFig. 2 Dose- and time-dependent effects of salinomycin on MTT-derived viable-cell/metabolic activity in primary endo -\nmetriotic stromal cell cultures. Cells were exposed to 0.01, 0.1, 1, or 10 µM salinomycin for 24, 48, or 72 h. MTT signal \nis expressed as a percentage of the vehicle-treated control and represents viable-cell metabolic activity rather than a direct \nmeasure of proliferation. Data are mean ± SD from three independent donor-derived cultures ( n = 3 biological replicates). \nEach culture was measured in technical triplicate, and replicate wells were averaged before analysis.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nstromal cell cultures (Fig. 9A). Salinomycin treatment did not significantly alter Cyclin D1 expression in primary \ncontrol endometrial stromal cell cultures (Fig. 9B). In contrast, treatment with 1.1 µM salinomycin for 72 h sig-\nnificantly reduced Cyclin D1 mRNA expression in primary endometriotic stromal cell cultures compared with \nvehicle-treated cells (Fig. 9C). These results provide marker-based support for reduced proliferation-associated \nsignaling in endometriotic stromal cell cultures. MMP-9 mRNA expression was assessed as an invasion-associ -\nated marker. At baseline, MMP-9 expression did not show a statistically significant difference between primary \ncontrol endometrial and endometriotic stromal cell cultures (Fig. 10A). Salinomycin did not significantly change \nMMP-9 expression in primary control endometrial stromal cell cultures (Fig. 10B). However, in primary endo-\nmetriotic stromal cell cultures, salinomycin significantly reduced MMP-9 mRNA expression compared with \nvehicle-treated cells (Fig. 10C). This decrease in MMP-9 expression was consistent with the reduced invasive \nbehavior observed in the Transwell assay. Caspase-3 mRNA expression was measured as an apoptosis-associated \nmarker. Baseline Caspase-3 expression was significantly lower in primary endometriotic stromal cell cultures \nthan in primary control endometrial stromal cell cultures (Fig. 11A). Salinomycin did not significantly alter \nFig. 3 Effects of salinomycin on MTT-derived viable-cell/metabolic activity in primary control endometrial and endome -\ntriotic stromal cell cultures. (A) Baseline MTT absorbance in control endometrial and endometriotic cultures. ( B, C) MTT \nabsorbance after treatment with vehicle or 1.1 µM salinomycin for 72 h in endometriotic and control endometrial cultures, \nrespectively. Absorbance was measured at 570 nm. MTT is a viable-cell metabolic readout and does not directly distinguish \nproliferation inhibition from cytotoxicity. Data are mean ± SD from three independent donor-derived cultures per group \n(n = 3 biological replicates). Each culture was measured in technical triplicate, and replicate wells were averaged before \nanalysis. *p < 0.05; ***p < 0.001. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic \nstromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell \nculture; SAL, salinomycin.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nCaspase-3 expression in primary control endometrial stromal cell cultures (Fig. 11B). In primary endometriotic \nstromal cell cultures, treatment with 1.1 µM salinomycin for 72 h significantly increased Caspase-3 mRNA \nexpression compared with control and vehicle-treated cells (Fig. 11C). These findings suggest that the reduction \nin MTT-derived viable-cell/metabolic signal may partly reflect apoptosis-associated transcriptional responses in \nendometriotic stromal cell cultures.\nSalinomycin reduced AXIN2 expression\nAXIN2 mRNA expression was assessed as a Wnt/β-catenin pathway-associated transcriptional marker. At base-\nline, AXIN2 expression was higher in primary endometriotic stromal cell cultures than in primary control endo-\nmetrial stromal cell cultures (Fig. 12A). Treatment with 1.1 µM salinomycin for 72 h significantly reduced \nAXIN2 mRNA expression in primary control endometrial stromal cell cultures (Fig. 12B). A more pronounced \nreduction was observed in primary endometriotic stromal cell cultures (Fig. 12C). Together with the increase \nin phospho-β-catenin Ser45 signal, the reduction in AXIN2 expression is consistent with attenuation of Wnt/β-\ncatenin pathway-associated transcriptional activity following salinomycin exposure, particularly in primary \nendometriotic stromal cell cultures.\nFig. 4 Representative images of Matrigel Transwell invasion assays in primary control endometrial and endometriotic stro-\nmal cell cultures. CESCs (top row) and EESCs (bottom row) are shown under control, vehicle-treated, and 1.1 µM salinomy-\ncin-treated conditions during the 24-h assay. Invaded cells on the lower membrane surface were fixed, stained with Giemsa, \nand imaged at 200× magnification. Quantification is shown in Fig. 5. Scale bar, 100 μm. CESC, primary control endometrial \nstromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nDiscussion\nEndometriosis is increasingly viewed as a chronic inflammatory disorder in which ectopic endometrial-like \nlesions acquire abnormal stromal-cell behaviors, including enhanced survival, invasive capacity, tissue remodel-\ning, and sustained inflammatory activity 43–45. Although the disease is benign, several of these cellular features \noverlap with tumor-like biological processes, particularly adhesion, invasion, and resistance to apoptosis 3,44,45. \nDevelopmental signaling pathways, including Wnt/β-catenin signaling, have been implicated in these disease-\nassociated phenotypes and may contribute to lesion persistence, fibrotic remodeling, and altered stromal-cell \nfunction28,45. Because current hormone-based treatments may not be suitable for all patients, particularly those \nseeking fertility preservation, there remains a need to investigate non-hormonal experimental approaches that \ntarget disease-relevant cellular pathways46.\nIn the present study, we examined the effects of salinomycin in primary control endometrial stromal cell cul -\ntures and primary endometriotic stromal cell cultures. These terms are retained as operational designations based \non tissue source, isolation procedure, adherent fibroblast-like morphology, and Vimentin expression. However, \nthe substantial CK18 signal and separate-tube analysis preclude claims of complete lineage purity or single-cell \nVimentin/CK18 co-expression. Within this experimental framework, salinomycin reduced MTT-derived viable-\ncell/metabolic activity, invasive behavior, inflammatory cytokine secretion, and selected molecular markers \nFig. 5 Effects of salinomycin on invasion by primary control endometrial and endometriotic stromal cell cultures. (A) Base-\nline invasion in control endometrial and endometriotic cultures. (B, C) Invasion after exposure to vehicle or 1.1 µM salino-\nmycin in control endometrial and endometriotic cultures, respectively, during the 24-h assay. Invaded cells were counted in \nfive randomly selected fields per insert. Data are mean ± SD from three independent donor-derived cultures per group (n = 3  \nbiological replicates). Each culture was analyzed in technical triplicate, and replicate measurements were averaged before \nanalysis. *p < 0.05; **p < 0.01. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic \nstromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell \nculture; SAL, salinomycin.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nassociated with proliferation, invasion, apoptosis, and Wnt/β-catenin-related signaling. These effects were gener-\nally more pronounced in primary endometriotic cultures than in control endometrial cultures.\nThe reduction in MTT-derived signal should be interpreted carefully. The MTT assay primarily reflects cel -\nlular metabolic activity and does not, by itself, distinguish reduced proliferation from reduced metabolic activity, \nreduced cell number, or cell death41. In the present study, baseline MTT absorbance was lower in endometriotic \nstromal cell cultures than in control cultures, and salinomycin produced a stronger reduction in MTT-derived \nFig. 7 Effects of salinomycin on IL-6 secretion by primary control endometrial and endometriotic stromal cell cultures. IL-6 \nin conditioned media was quantified by ELISA and normalized to total protein in the corresponding cell lysates. ( A) Base-\nline IL-6 secretion in untreated control endometrial and endometriotic cultures. ( B, C) IL-6 secretion after treatment with \nvehicle or 1.1 µM salinomycin for 72 h in control endometrial and endometriotic cultures, respectively. Data are mean ± SD \nfrom three independent donor-derived cultures per group ( n = 3 biological replicates). Each culture was analyzed in techni -\ncal triplicate, and replicate measurements were averaged before analysis. **p < 0.01; ***p < 0.001. Normal, primary control \nendometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial \nstromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin.\n \nFig. 6 Effects of salinomycin on phospho-β-catenin (Ser45) in primary control endometrial and endometriotic stromal cell \ncultures. Phospho-β-catenin (Ser45) was measured by ELISA and reported as optical-density values. (A) Baseline phospho-\nβ-catenin (Ser45) signal in control endometrial and endometriotic cultures. (B, C) Signal after treatment with vehicle or 1.1 \nµM salinomycin for 72 h in endometriotic and control endometrial cultures, respectively. Data are mean ± SD from three \nindependent donor-derived cultures per group ( n = 3 biological replicates). Each culture was analyzed in technical tripli -\ncate, and replicate measurements were averaged before analysis. *p < 0.05; **p < 0.01. Normal, primary control endometrial \nstromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell \nculture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nsignal in endometriotic stromal cell cultures. This finding suggests differential responsiveness to salinomycin \nunder the present in vitro conditions, but it should not be interpreted as direct evidence of reduced proliferation \nalone. The observed decrease in Cyclin D1 mRNA expression in salinomycin-treated endometriotic stromal cell \ncultures provides marker-based support for a reduction in proliferation-associated signaling. Reduced prolif -\neration-, migration-, and invasion-related responses have been reported after pharmacological disruption of the \nTCF/β-catenin complex in endometrial and endometriotic cell models, supporting the relevance of this pathway \nFig. 9 Effects of salinomycin on Cyclin D1 mRNA expression in primary control endometrial and endometriotic stromal \ncell cultures. Relative Cyclin D1 mRNA expression was quantified by real-time PCR and normalized to GAPDH; Cyclin \nD1 was evaluated as a proliferation-associated marker. ( A) Baseline expression in control endometrial and endometriotic \ncultures. (B, C) Expression after treatment with vehicle or 1.1 µM salinomycin for 72 h in control endometrial and endome-\ntriotic cultures, respectively. Data are mean ± SD from three independent donor-derived cultures per group (n = 3 biological \nreplicates). Each culture was analyzed in technical triplicate, and replicate measurements were averaged before analysis. \n**p < 0.01 versus vehicle. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal \ncell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; \nSAL, salinomycin.\n \nFig. 8 Effects of salinomycin on IL-8 secretion by primary control endometrial and endometriotic stromal cell cultures. IL-8 \nin conditioned media was quantified by ELISA and normalized to total protein in the corresponding cell lysates. ( A) Base-\nline IL-8 secretion in untreated control endometrial and endometriotic cultures. ( B, C) IL-8 secretion after treatment with \nvehicle or 1.1 µM salinomycin for 72 h in control endometrial and endometriotic cultures, respectively. Data are mean ± SD \nfrom three independent donor-derived cultures per group ( n = 3 biological replicates). Each culture was analyzed in techni -\ncal triplicate, and replicate measurements were averaged before analysis. * p < 0.05; ***p < 0.001. Normal, primary control \nendometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial \nstromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nin regulating disease-associated cellular behavior27. Direct cell counting, Ki67 or PCNA analysis, and BrdU/EdU \nincorporation would provide more direct evidence of proliferation inhibition and should be considered in future \nstudies.\nInvasion is a key feature of lesion establishment and tissue remodeling in endometriosis. In the present study, \nprimary endometriotic stromal cell cultures showed higher basal invasive behavior than control endometrial \nstromal cell cultures in the Transwell assay. Salinomycin reduced invasion in both cell types, with a stronger \nFig. 11 Effects of salinomycin on Caspase-3 mRNA expression in primary control endometrial and endometriotic stromal \ncell cultures. Relative Caspase-3 mRNA expression was quantified by real-time PCR and normalized to GAPDH; Caspase-3 \nmRNA was evaluated as an apoptosis-associated transcriptional marker. (A) Baseline expression in control endometrial and \nendometriotic cultures. (B, C) Expression after treatment with vehicle or 1.1 µM salinomycin for 72 h in control endometrial \nand endometriotic cultures, respectively. Data are mean ± SD from three independent donor-derived cultures per group (n = 3  \nbiological replicates). Each culture was analyzed in technical triplicate, and replicate measurements were averaged before \nanalysis. * p < 0.05; ** p < 0.01; *** p < 0.001, as indicated by comparison brackets. Normal, primary control endometrial \nstromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell \nculture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin.\n \nFig. 10 Effects of salinomycin on MMP-9 mRNA expression in primary control endometrial and endometriotic stromal cell \ncultures. Relative MMP-9 mRNA expression was quantified by real-time PCR and normalized to GAPDH. ( A) Baseline \nexpression in control endometrial and endometriotic cultures. ( B, C) Expression after treatment with vehicle or 1.1 µM \nsalinomycin for 72 h in control endometrial and endometriotic cultures, respectively. Data are mean ± SD from three inde-\npendent donor-derived cultures per group ( n = 3 biological replicates). Each culture was analyzed in technical triplicate, \nand replicate measurements were averaged before analysis. * p < 0.05 versus vehicle. Normal, primary control endometrial \nstromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell \nculture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nreduction in endometriotic stromal cell cultures. This functional change was accompanied by reduced MMP-9 \nmRNA expression in salinomycin-treated endometriotic stromal cell cultures. Because MMP-9 contributes to \nextracellular matrix degradation and stromal invasiveness in endometriosis, its downregulation is consistent with \nthe observed reduction in invasion47. However, baseline MMP-9 expression may vary according to tissue origin, \ndisease context, and menstrual-cycle phase 47,48. Therefore, the present findings are best interpreted as show -\ning that salinomycin exposure was associated with reduced invasive behavior and decreased expression of an \ninvasion-associated marker, rather than establishing MMP-9 as the sole mediator of the anti-invasive response.\nThe Wnt/β-catenin pathway has been implicated in endometriosis pathogenesis and in the regulation of stro -\nmal-cell survival, invasion, and fibrosis-related responses 28. In this study, primary endometriotic stromal cell \ncultures showed lower basal phospho-β-catenin Ser45 signal and higher AXIN2 mRNA expression than con -\ntrol cultures. Phosphorylation of β-catenin at Ser45 creates a priming site for subsequent GSK-3β-dependent \nphosphorylation and degradation, whereas AXIN2 is a Wnt-responsive transcriptional target that functions as \na negative-feedback regulator49,50. Salinomycin increased phospho-β-catenin Ser45 signal and reduced AXIN2 \nexpression, particularly in endometriotic stromal cell cultures. Together, these changes support an association \nbetween salinomycin exposure and attenuation of Wnt/β-catenin pathway-associated transcriptional activity. \nNevertheless, because the study did not include Wnt reporter assays, pathway rescue experiments, or targeted \ngain/loss-of-function approaches, these data should be interpreted as pathway-associated evidence rather than \ndefinitive proof of causal Wnt inhibition. AXIN2 can also have context-dependent functions; in colorectal cancer \nmodels, elevated AXIN2 promoted a Snail1-dependent epithelial-mesenchymal transition program and invasive/\nmetastatic behavior51. Therefore, the reduction in AXIN2 after salinomycin exposure provides molecular support \nfor altered Wnt-associated signaling but does not establish a cell-specific causal mechanism in endometriosis.\nInflammation is central to the endometriotic microenvironment. IL-6 and IL-8 contribute to immune-cell \nrecruitment, angiogenesis, adhesion, stromal-cell activation, and lesion progression 15,18–24. In the present data -\nset, baseline IL-6 secretion was comparable between control and endometriotic stromal cell cultures, whereas \nbaseline IL-8 secretion was higher in endometriotic stromal cell cultures. Salinomycin reduced both IL-6 and \nIL-8 secretion, with a more pronounced reduction in endometriotic stromal cell cultures. These findings sug -\ngest that salinomycin may modulate inflammatory output in primary stromal-cell cultures. This observation \nFig. 12 Effects of salinomycin on AXIN2 mRNA expression in primary control endometrial and endometriotic stromal cell \ncultures. Relative AXIN2 mRNA expression was quantified by real-time PCR and normalized to GAPDH. ( A) Baseline \nexpression in control endometrial and endometriotic cultures. ( B, C) Expression after treatment with vehicle or 1.1 µM \nsalinomycin for 72 h in control endometrial and endometriotic cultures, respectively. Data are mean ± SD from three inde-\npendent donor-derived cultures per group (n = 3 biological replicates). Each culture was analyzed in technical triplicate, and \nreplicate measurements were averaged before analysis. * p < 0.05, as indicated by comparison brackets. Normal, primary \ncontrol endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endo -\nmetrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nis compatible with reported crosstalk between Wnt/β-catenin signaling and inflammatory pathways, including \nNF-κB-dependent responses, although the present study did not directly test this interaction52.\nResistance to apoptosis is another important feature of ectopic endometrial stromal cells 28,45,53. Consistent \nwith this concept, basal Caspase-3 mRNA expression was lower in primary endometriotic stromal cell cultures \nthan in primary control endometrial stromal cell cultures. Salinomycin increased Caspase-3 mRNA expression in \nendometriotic stromal cell cultures, whereas no significant change was observed in control cultures. This finding \nsuggests activation of apoptosis-associated transcriptional responses after salinomycin exposure. Importantly, \nbecause apoptosis was evaluated only at the mRNA level, these data cannot be taken as definitive evidence of \nfunctional apoptosis. Confirmation by protein-level or functional assays, such as cleaved caspase-3 detection, \ncaspase activity assays, Annexin V/PI staining, or TUNEL analysis, would be required in future studies. Pro-\napoptotic effects of salinomycin have been demonstrated in cells with dysregulated Wnt signaling 31, supporting \na possible association between Wnt-pathway status and salinomycin responsiveness.\nWhen compared with previous studies targeting Wnt/β-catenin signaling in endometriosis, our findings are \ndirectionally consistent but mechanistically less definitive. Matsuzaki and Darcha showed that pharmacological \ndisruption of the Tcf/β-catenin complex using PKF 115–584 reduced proliferation-, migration-, and invasion-\nrelated responses in endometrial and endometriotic epithelial and stromal cell models, with additional effects \non MMP-2/MMP-9 activity 27. In a related study, the same group reported that targeting Wnt/β-catenin signal -\ning reduced fibrosis-associated markers and collagen gel contraction in endometrial and endometriotic stromal \ncells, and also affected fibrosis progression in a xenograft model of endometriosis12. Our data extend this general \nconcept by showing that salinomycin exposure was accompanied by reduced invasive behavior, lower MMP-9 \nexpression, increased β-catenin Ser45 phosphorylation, and decreased AXIN2 expression in primary endometri-\notic stromal cell cultures. However, unlike studies using pathway-specific antagonists, β-catenin knockdown, or \nin vivo models, the present study relied on pathway-associated markers rather than direct functional interrogation \nof Wnt activity. Therefore, the observed molecular changes should be interpreted as being consistent with attenu-\nation of Wnt/β-catenin-associated transcriptional activity, rather than as conclusive evidence of causal pathway \ninhibition.\nThe salinomycin-related findings should also be considered in light of studies performed in malignant gyne -\ncological and non-gynecological models. Salinomycin has been reported to inhibit Wnt signaling by blocking \nWnt-induced LRP6 phosphorylation and promoting LRP6 downregulation, and additional studies have shown \nsuppression of LRP6-associated Wnt/β-catenin signaling in breast and prostate cancer cells31,32. In gynecological \nmalignancy models, salinomycin reduced proliferation, migration, and invasion in human endometrial cancer \nstem-like cells and repressed epithelial-mesenchymal transition-associated responses in epithelial ovarian cancer \ncells through Wnt/β-catenin-related mechanisms54,55. These reports are broadly compatible with our observations \nin primary endometriotic stromal cell cultures, particularly the reduction in MTT-derived viable-cell/metabolic \nactivity, invasion, Cyclin D1, MMP-9, and AXIN2, together with increased Caspase-3 mRNA expression. Nev-\nertheless, direct extrapolation from malignant models to endometriosis should be avoided. Endometriosis is a \nbenign, hormone-responsive inflammatory disorder, and stromal-cell responses in primary cultures may differ \nsubstantially from malignant or cancer stem-like cell systems. Thus, the present findings should be viewed as \npreclinical, pathway-associated observations that require validation using expanded stromal-cell markers, direct \nWnt reporter or rescue experiments, functional apoptosis assays, and in vivo endometriosis models.\nSeveral limitations should be considered when interpreting these findings. First, cell characterization was \nbased on the isolation strategy, adherent fibroblast-like morphology, and Vimentin/CK18 profiling. Vimentin \nand CK18 were detected in separate tubes (85.0% and 79.6% FITC-positive events, respectively), and their co-\nexpression at the single-cell level was not assessed. The substantial CK18 signal may reflect epithelial-cell con-\ntamination, dual-marker expression, or distinct cell populations; the present data cannot distinguish among these \npossibilities. Additional markers such as CD10, ER, PR, HOXA10, or IFITM1 were not evaluated. Therefore, the \nterms “primary endometriotic stromal cell cultures” and “primary control endometrial stromal cell cultures” are \noperational designations and should not be interpreted as proof of a completely lineage-pure stromal population \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nor as identification of multipotent mesenchymal stromal cells. Second, MTT-based assays were used as viable-\ncell/metabolic readouts and do not directly measure proliferation or distinguish reduced proliferation from altered \nmetabolism or loss of viable cells 41. Direct cell-counting curves, Ki67/PCNA analysis, and BrdU/EdU incor -\nporation were not performed. Third, Caspase-3 was assessed at the mRNA level only, and functional apoptosis \nwas not directly confirmed. Fourth, the mechanistic interpretation of Wnt/β-catenin involvement was based on \nphospho-β-catenin Ser45 and AXIN2 expression; direct pathway activity was not tested using reporter or rescue \nexperiments. Finally, the study was conducted in vitro and did not address systemic toxicity, pharmacokinetics, \ntissue selectivity, or in vivo efficacy.\nTaken together, the present findings indicate that salinomycin modulates several disease-relevant cellular \nresponses in primary endometriotic stromal cell cultures, including MTT-derived viable-cell/metabolic activity, \ninvasive behavior, inflammatory cytokine secretion, apoptosis-associated gene expression, and Wnt/β-catenin \npathway-associated markers. The stronger responses observed in endometriotic stromal cell cultures suggest dif-\nferential sensitivity under the present in vitro conditions. These results support further investigation of Salinomy-\ncin and Wnt-directed pathway modulation as experimental, non-hormonal research approaches in endometriosis \nbiology.\nConclusion\nIn this study, Salinomycin modulated several disease-relevant cellular responses in primary endometriotic stro -\nmal cell cultures under in vitro conditions. Salinomycin reduced MTT-derived viable-cell/metabolic activity, \ninvasive behavior, IL-6 and IL-8 secretion, and the expression of the proliferation- and invasion-associated mark-\ners Cyclin D1 and MMP-9. These effects were generally more pronounced in primary endometriotic cultures than \nin primary control endometrial cultures. At the molecular level, Salinomycin increased Phospho-beta-Catenin \n(Ser45) signal and reduced AXIN2 expression, findings consistent with modulation of Wnt/β-catenin pathway-\nassociated transcriptional activity. Salinomycin also increased Caspase-3 mRNA expression in endometriotic \ncultures, suggesting an apoptosis-associated transcriptional response. However, because the study was limited \nto an in vitro model and did not include expanded cell-lineage validation, direct proliferation assays, functional \napoptosis assays, Wnt reporter assays, or pathway-specific rescue experiments, the findings should be interpreted \nas pathway-associated experimental evidence rather than proof of therapeutic efficacy. Further studies using \nexpanded phenotypic validation, functional mechanistic assays, and in vivo endometriosis models are required \nbefore cell-specific mechanisms or therapeutic relevance can be established.\nAcknowledgements The authors gratefully acknowledge the technical and administrative support provided by the Repro -\nductive Health Research Center and the Clinical Research Institute of Urmia University of Medical Sciences. We also thank \nall participants who provided tissue samples for this study. During the preparation of this manuscript, the authors used Chat-\nGPT to improve language clarity and readability. After using this tool, all authors carefully reviewed, revised, and approved \nthe final content and take full responsibility for the accuracy, integrity, and scientific content of the publication.\nAuthor contributions HGB conceptualized and designed the study, performed data curation, and wrote the original draft. \nFM analyzed the data and reviewed and edited the manuscript. MGB, SS contributed to the investigation and manuscript \nediting. MP was involved in methodology development, validation, and manuscript editing. All authors have read and ap -\nproved the final version of the manuscript.\nFunding No funding was received for conducting this study.\nData availability The data used and/or analyzed during thecurrent study are available from the corresponding author on \nreasonable request.\n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-67394-0\nDeclarations\nCompeting interests The authors declare no competing interests.\nDeclaration of generative AI and AI-assisted technologies in the writing process  During the preparation of this work, the \nauthors used ChatGPT to improve the language of the manuscript. After using this tool, the authors reviewed and edited the \ncontent as needed and take full responsibility for the content of the publication.\nOpen Access  This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 Interna-\ntional License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as \nlong as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, \nand indicate if you modified the licensed material. You do not have permission under this licence to share adapted material \nderived from this article or parts of it. The images or other third party material in this article are included in the article’s Cre-\native Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s \nCreative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you \nwill need to obtain permission directly from the copyright holder. To view a copy of this licence, visit  h t t p : / / c r e a t i v e c o m m o \nn s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 /     .  \nReferences\n 1. As-Sanie, S. et al. Endometriosis: a review. Jama 334 (1), 64–78 (2025).\n 2. Pašalić, E., Tambuwala, M. M. & Hromić-Jahjefendić, A. Endometriosis: Classification, pathophysiology, and treatment \noptions. Pathology-Research Pract. 251, 154847 (2023).\n 3. Wilbur, M. A., Shih, I. M., Segars, J. H. & Fader, A. N. Cancer implications for patients with endometriosis. In Seminars \nin reproductive medicine V ol. 35, No. 01, 110–116 (Thieme Medical, 2017).\n 4. Smolarz, B., Szyłło, K. & Romanowicz, H. 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OncoTargets Therapy 28, 1317–1325 (2017).\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional \naffiliations.\nAuthors and Affiliations\nSonia Sadeghpour1 · Morteza Ghasemnejad-Berenji1 · Farzad Maleki2 ·  \nMohammad Reza Pashaei2 · Hojat Ghasemnejad-Berenji1,3\n Hojat Ghasemnejad-Berenji\nh_ghasem_nejad@yahoo.com\n1 Reproductive Health Research Center, Clinical Research Institute, Urmia University of Medical Sciences, Urmia, \nIran\n2 Patient Safety Research Center, Clinical Research Institute, Urmia University of Medical Sciences, Urmia, Iran\n3 Reproductive Biology, Reproductive Health Research Center, Clinical Research Institute, Urmia University of \nMedical Sciences, Urmia, Iran","source_license":"CC0","license_restricted":false}