INHBA secreted by cancer-associated fibroblasts promotes cisplatin resistance and malignant progression of ovarian cancer via ENG-mediated suppression of apoptosis and induction of EMT

We integrated single-cell sequencing data using the Seurat package and assessed quality control (QC) parameters for all cells, including the number of expressed genes (nFeature_RNA), total mRNA molecules (nCount_RNA), mitochondrial gene ratio (percent_mito), hemoglobin gene ratio (percent_hb), and ribosomal gene ratio (percent_ribo). The results showed that most cells exhibited nFeature_RNA < 5000, nCount_RNA < 20,000, percent_mito < 20%, percent_hb < 1%, and percent_ribo < 40% (Fig. S1 A). Low-quality cells were then filtered according to the established QC criteria: percent_mito  3%, percent_hb < 1%, and nFeature_RNA < 6000. Sequencing depth correlation analysis revealed a correlation coefficient r  = 0.92 between nCount_RNA and nFeature_RNA (Fig. S1 B), confirming that the data quality met the requirements for downstream analyses. Highly variable genes were screened from the filtered cells, with the top 2000 genes selected for subsequent analyses (Fig. S1 C) and used for PCA dimensionality reduction. The Harmony algorithm was applied to correct batch effects caused by sample sources. Based on the variance explanation rate and cumulative contribution rate of principal components, the top 30 principal components were selected for cell clustering via the Louvain algorithm (resolution = 0.5). All cells were classified into 18 clusters by t-SNE and UMAP for two-dimensional visualization (Fig. S1 D, E). Combined with cell lineage-specific marker genes from published literature and the CellMarker database, the cells were annotated into six major types: ovarian epithelial cells, T cells, myeloid cells, CAFs, B cells, and endothelial cells (Fig.  1 A, B). The expression patterns of marker genes across all cell clusters were visualized using dot plots (Fig. S1 F). Student’s t-test was used to compare cell number differences between platinum-resistant and platinum-sensitive groups, which revealed a significant increase in the number of fibroblasts in the platinum-resistant group (Fig.  1 C). Fig. 1 Characterization of CAF subtypes, differential INHBA-ENG expression, and clinical sample validation in OC. ( A , B) Visualization of cell clustering for ovarian cancer single-cell transcriptome data: (A) t-SNE plot and (B) UMAP plot showing six major annotated cell types. (C) Proportional distribution of each cell type in platinum-sensitive and platinum-resistant samples. (D) UMAP clustering of CAFsshowing four distinct CAF subtypes. (E) Proportional distribution of different CAF subtypes in platinum-sensitive and platinum-resistant samples. (F) Expression distribution of INHBA in the single-cell UMAP landscape. (G) Average expression level of INHBA across each CAF subtype. (H) Violin plot showing INHBA expression distribution in each CAF subtype. (I) Expression localization of INHBA and ENG across different cell types. (J) Spearman’s correlation analysis of INHBA and ENG expression levels. (K-L) Representative IHC images and quantitative statistical analysis of (K) INHBA and (L) ENG protein expression in normal ovarian tissues, platinum-sensitive and platinum-resistant OC tissues ( n  = 3 per group; scale bar: 100 μm). (M) IF co-localization images of INHBA (red) and ENG (green) in normal ovarian and OC tissues (×40). Cell nuclei were counterstained with DAPI (blue). Normal ovarian tissues served as experimental blank controls. Exact P-values are provided in the figure; p  < 0.05 was considered statistically significant Characterization of CAF subtypes, differential INHBA-ENG expression, and clinical sample validation in OC. ( A , B) Visualization of cell clustering for ovarian cancer single-cell transcriptome data: (A) t-SNE plot and (B) UMAP plot showing six major annotated cell types. (C) Proportional distribution of each cell type in platinum-sensitive and platinum-resistant samples. (D) UMAP clustering of CAFsshowing four distinct CAF subtypes. (E) Proportional distribution of different CAF subtypes in platinum-sensitive and platinum-resistant samples. (F) Expression distribution of INHBA in the single-cell UMAP landscape. (G) Average expression level of INHBA across each CAF subtype. (H) Violin plot showing INHBA expression distribution in each CAF subtype. (I) Expression localization of INHBA and ENG across different cell types. (J) Spearman’s correlation analysis of INHBA and ENG expression levels. (K-L) Representative IHC images and quantitative statistical analysis of (K) INHBA and (L) ENG protein expression in normal ovarian tissues, platinum-sensitive and platinum-resistant OC tissues ( n  = 3 per group; scale bar: 100 μm). (M) IF co-localization images of INHBA (red) and ENG (green) in normal ovarian and OC tissues (×40). Cell nuclei were counterstained with DAPI (blue). Normal ovarian tissues served as experimental blank controls. Exact P-values are provided in the figure; p  < 0.05 was considered statistically significant Fibroblasts with confirmed annotations were further extracted for subpopulation analysis. The raw RNA expression matrix was retrieved using the DefaultAssay function, and the top 2000 highly variable genes were screened by variance stabilizing transformation (VST). After z-score normalization of gene expression values, PCA dimensionality reduction was performed. The elbow plot was used to evaluate the variance explanation rate, and the top 15 principal components were selected for clustering via the Louvain algorithm (resolution = 0.5). Fibroblast subpopulations were visualized by UMAP (Fig. S1 G). Marker genes for fibroblast subpopulations were screened with the criteria of a minimum expression proportion of 25% and a log fold change threshold of 0.25, and the 15 initial clusters were re-annotated into four CAF subtypes: inflammatory and growth factor-enriched CAFs (iCAFs) and myofibroblastic CAFs (myoCAF1, myoCAF2, myoCAF3) (Fig. S1 H). UMAP plots illustrated the spatial distribution characteristics of each CAF subtype (Fig.  1 D), with myoCAF1 identified as the predominant CAF subtype in the platinum-resistant group (Fig.  1 E). The expression distribution of the INHBA gene was visualized on UMAP plots using the FeatureDimPlot function, which revealed its predominant localization in fibroblasts (Fig.  1 F) and specific enrichment in the myoCAF1 subtype (Fig.  1 G). Further analysis via the FeatureStatPlot function combined with the Wilcoxon rank-sum test verified that INHBA had the highest expression proportion in myoCAF1 (Fig.  1 H). Sequencing data of epithelial cells and CAFs were merged to construct an analytical object, and cell-cell communication analysis was performed using NicheNet. The ligand-target matrix and human ligand-receptor network were loaded with only validated interaction pairs retained. Two niches (platinum-resistant and platinum-sensitive) were defined, and differentially expressed genes in sender/receiver cells were analyzed to complete ligand-receptor pair scoring, target gene analysis, and ligand activity analysis. The integrated data were weighted, ranked, and visualized. The results showed that compared with the sensitive group, INHBA was significantly highly expressed in myoCAF1 of the resistant group, and its corresponding ligand gene in epithelial cells was ENG (Fig. S1 I). Expression values of INHBA and ENG were extracted from the RNA detection matrix of the Seurat object; localization analysis demonstrated that INHBA was mainly expressed in fibroblasts (red), while ENG was enriched in endothelial cells and epithelial cells (green) (Fig.  1 I). Spearman’s rank correlation coefficient analysis indicated a significant positive correlation between the expression of INHBA and ENG (Fig.  1 J). IHC (Fig.  1 K, L) and IF co-localization (Fig.  1 M) assays for INHBA and ENG were performed on 50 epithelial ovarian cancer samples (34 platinum-sensitive, 16 platinum-resistant) and 20 normal ovarian samples. The results confirmed that INHBA and ENG were weakly expressed in normal ovarian tissues, while their protein expression levels in epithelial ovarian cancer tissues were significantly higher than those in normal ovarian tissues ( p  < 0.001); additionally, their protein expression levels in platinum-resistant samples were markedly higher than those in platinum-sensitive samples ( p <0.001). TSA-IF co-localization assays further validated the above findings (Fig.  1 M). Primary CAFs and NFs were isolated from ovarian tumor tissues and adjacent normal tissues, respectively. Morphological assessment revealed an elongated, spindle‑shaped appearance in both cell types (Fig. 2 A). Expression of the mesenchymal marker Vimentin was detected in all isolates by immunocytochemistry (ICC, Fig. 2 B), immunofluorescence (IF, Fig. 2 C), and Western blotting (Fig. 2 D), with no significant intergroup difference ( p = 0.56). In contrast, consistent with the activated phenotype of CAFs, FAP and α‑SMA were robustly expressed in CAFs but absent in NFs, yielding a highly significant between-group difference [ 33 ]. These findings verify the successful isolation of activated CAFs and quiescent NFs. Fig. 2 Isolation and phenotypic validation of primary CAFs and NFs. (A) Light microscopic observation of the in vitro morphology of CAFs (derived from ovarian tumor tissues) and NFs (derived from normal ovarian tissues) (scale bar = 100 μm). (B) ICC was performed to detect the expression of Vimentin, FAP, and α-SMA in cells (scale bar = 100 μm). (C) IF assay was used to examine the expression of Vimentin, FAP, and α-SMA (cell nuclei were stained with DAPI; scale bar = 100 μm). (D) Western blot analysis was conducted to determine the protein expression levels of Vimentin, FAP, and α-SMA, with β-actin serving as the internal loading control.Data are presented as mean ± SD from 3 independent experiments ( n  = 3 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant.NFs cells serve as the control group Isolation and phenotypic validation of primary CAFs and NFs. (A) Light microscopic observation of the in vitro morphology of CAFs (derived from ovarian tumor tissues) and NFs (derived from normal ovarian tissues) (scale bar = 100 μm). (B) ICC was performed to detect the expression of Vimentin, FAP, and α-SMA in cells (scale bar = 100 μm). (C) IF assay was used to examine the expression of Vimentin, FAP, and α-SMA (cell nuclei were stained with DAPI; scale bar = 100 μm). (D) Western blot analysis was conducted to determine the protein expression levels of Vimentin, FAP, and α-SMA, with β-actin serving as the internal loading control.Data are presented as mean ± SD from 3 independent experiments ( n  = 3 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant.NFs cells serve as the control group To assess INHBA expression in NFs and CAFs, ICC and IF analyses demonstrated markedly higher levels in CAFs (Fig. 3 A, B). RT-qPCR and WB confirmed this elevation at both mRNA and protein levels (Fig. 3 C, D). Given its secretory nature [ 34 ], we measured INHBA concentrations in conditioned media and found values of approximately 1000 ng/L in CAF supernatants compared with about 500 ng/L in NF supernatants(Fig. 3 E). Fig. 3 INHBA is upregulated in CAFs compared with NFs. (A) ICC and (B) IF: Representative images of INHBA in NFs and CAFs. (C) RT-qPCR analysis of INHBA mRNA levels. (D) WB and quantitative analysis of INHBA protein. (E) ELISA detection of INHBA in cell supernatants.Data are presented as mean ± SD from 3 independent experiments ( n  = 3 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant. NFs cells serve as the control group INHBA is upregulated in CAFs compared with NFs. (A) ICC and (B) IF: Representative images of INHBA in NFs and CAFs. (C) RT-qPCR analysis of INHBA mRNA levels. (D) WB and quantitative analysis of INHBA protein. (E) ELISA detection of INHBA in cell supernatants.Data are presented as mean ± SD from 3 independent experiments ( n  = 3 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant. NFs cells serve as the control group To establish the optimal cisplatin concentration for subsequent functional assays, SKOV3 and A2780 cells (cisplatin‑sensitive) alongside their resistant derivatives (SKOV3‑DDP and A2780‑DDP) were treated with 0–10 µg/mL cisplatin for 48 h. CCK‑8 assays revealed that sensitive cells exhibited significantly lower viability and IC₅₀ values (SKOV3, 3.58 µg/mL; A2780, 2.67 µg/mL) compared with resistant cells (SKOV3‑DDP, 14.26 µg/mL; A2780‑DDP, 13.07 µg/mL) (Fig.  4 A), confirming phenotypic stability. Considering the pronounced cytotoxicity in sensitive cells and the distinct responsiveness of resistant cells, 3 µg/mL cisplatin was selected as the working concentration to ensure clear discrimination between experimental groups. To investigate whether CAFs modulate cisplatin sensitivity via paracrine INHBA, INHBA‑knockout (KO) CAFs were generated, and knockout efficiency was validated by RT‑qPCR and Western blotting, which demonstrated a significant reduction in INHBA expression in the KO1 clone compared with non‑targeting control CAFs (NC‑CAFs) (Fig.  4 B, C). This clone was subsequently employed for all functional assays. Conditioned medium (CM) from NFs, NC‑CAFs, and INHBA‑KO CAFs was applied to SKOV3 and A2780 cells treated with 3 µg/mL cisplatin. Medium from NFs did not significantly alter cellular viability or behavior relative to the blank control ( p  > 0.05). In contrast, NC‑CAFs‑CM markedly diminished cisplatin sensitivity, as evidenced by attenuated cytotoxicity (Fig.  4 D), enhanced proliferation (Fig.  4 E), suppressed apoptosis (Fig.  4 F), and increased migratory and invasive capacities (Fig.  4 G, H). INHBA‑KO CAFs‑CM produced comparable but significantly attenuated effects across all assays (all p  < 0.05 vs. NC‑CAFs, one‑way ANOVA with post hoc Tukey’s test). Western blot analysis of apoptosis‑ and EMT‑related proteins corroborated these findings. Relative to controls, NC‑CAFs‑CM significantly upregulated BCL‑2, downregulated BAX and cleaved caspase‑3, increased N‑cadherin and Vimentin levels, and reduced E‑cadherin expression at the protein level (Fig.  4 I). NFs‑CM induced negligible changes, whereas INHBA‑KO CAFs‑CM caused only minor alterations that were significantly less pronounced than those in the NC‑CAFs group. Fig. 4 CAF-derived INHBA Regulates Cisplatin Sensitivity of OC Cells. (A) CCK-8 assays were used to measure cell viability and calculate the IC 50 values of cisplatin-sensitive (SKOV3, A2780) and cisplatin-resistant (SKOV3-DDP, A2780-DDP) OC cell lines after 48 h exposure to 0–10 µg/mL cisplatin. (B , C) I NHBA knock-out efficiency in CAFs was verified by (B) RT-qPCR and (C) Western blot. (D-H) In the presence of 3.0 µg/mL cisplatin, the effects of conditioned medium from blank control, NFs, NC-CAFs, and INHBA-KO-CAFs on OC cells were assessed as follows: (D) cytotoxicity by CCK-8, (E) proliferation by Colony formation assay, (F) apoptosis by Annexin V/PI flow cytometry, (G) invasion and (H) migration by transwell assays. (I) In the presence of 3.0 µg/mL cisplatin, WB was used to evaluate the effects of conditioned medium from blank control, NFs, NC-CAFs, and INHBA-KO-CAFs on the expression of apoptosis- and EMT-related markers in OC cells. Data are presented as mean ± SD from 3 independent experiments ( n  = 3 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant. Corresponding normal/negative/blank controls were set for all experimental groups CAF-derived INHBA Regulates Cisplatin Sensitivity of OC Cells. (A) CCK-8 assays were used to measure cell viability and calculate the IC 50 values of cisplatin-sensitive (SKOV3, A2780) and cisplatin-resistant (SKOV3-DDP, A2780-DDP) OC cell lines after 48 h exposure to 0–10 µg/mL cisplatin. (B , C) I NHBA knock-out efficiency in CAFs was verified by (B) RT-qPCR and (C) Western blot. (D-H) In the presence of 3.0 µg/mL cisplatin, the effects of conditioned medium from blank control, NFs, NC-CAFs, and INHBA-KO-CAFs on OC cells were assessed as follows: (D) cytotoxicity by CCK-8, (E) proliferation by Colony formation assay, (F) apoptosis by Annexin V/PI flow cytometry, (G) invasion and (H) migration by transwell assays. (I) In the presence of 3.0 µg/mL cisplatin, WB was used to evaluate the effects of conditioned medium from blank control, NFs, NC-CAFs, and INHBA-KO-CAFs on the expression of apoptosis- and EMT-related markers in OC cells. Data are presented as mean ± SD from 3 independent experiments ( n  = 3 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant. Corresponding normal/negative/blank controls were set for all experimental groups To identify the downstream effector of INHBA in OC progression and chemoresistance, we examined ENG expression in SKOV3 and A2780 cells exposed to CM from different sources. WB (Fig. 5 A) showed that, relative to the blank control, NC-CAF-CM significantly up-regulated ENG, whereas NFs-CM had no effect, and INHBA KO-CAF-CM induced only a modest increase that was markedly lower than that of NC-CAF-CM, indicating that INHBA is the principal CAF-derived regulator of ENG expression. Molecular docking predicted INHBA–ENG interaction(Fig. 5 B). The detailed experimental results showed that the binding energy of the INHBA-ENG complex was − 12.3 kcal/mol, which is far lower than the strong binding threshold of -7.0 kcal/mol [ 35 ].Furthermore, the complex appears to be stabilized by hydrogen bonds (yellow dashed lines) and electrostatic interactions (purple dashed lines), suggesting a high-affinity interaction. To validate this predicted interaction, CoIP assays were performed. The results confirmed that INHBA physically interacts with ENG (Fig. 5 C), providing direct evidence for their binding. GEPIA analysis of TCGA and GTEx data [ 36 ] further revealed a positive correlation between INHBA and ENG expression in OC tissues (Pearson r = 0.33, p < 0.001,Spearman r = 0.33, P < 0.001,Fig. 5 D). Gene set variation analysis of the TCGA cohort showed that ENG-related pathways, particularly EMT, were significantly enriched (Fig. 5 E), providing clinical support for the functional link between INHBA and ENG. To corroborate in vitro findings, a subcutaneous xenograft tumor model was established. On day 6, tumors co-inoculated with SKOV3 cells and NC-CAFs were significantly larger than those in the SKOV3 alone or SKOV3 + INHBA-KO-CAFs groups (Fig. 5 F–H). Upon intraperitoneal cisplatin (PT) administration, tumors in the SKOV3 and SKOV3 + INHBA-KO-CAFs groups regressed—significant shrinkage was observed after 6 and 18 days of PT, respectively—whereas tumors in the SKOV3 + NC-CAFs group continued to enlarge despite reduced growth at day 18. Tumor weights on day 28 mirrored these trends. Immunohistochemical analysis (Fig. 5 I) demonstrated high expression levels of INHBA, ENG and Ki67 in the SKOV3 + NC-CAFs group. INHBA was predominantly localized to the extracellular matrix, ENG to the stromal compartment, and Ki67 to the cell nucleus, which was consistent with our in vitro findings. Fig. 5 INHBA Regulates OC Growth and Cisplatin Resistance via ENG In Vitro & In Vivo Validation. (A) WB analysis of ENG expression in SKOV3 and A2780 cells after treatment with conditioned medium from blank control, NFs, NC-CAFs, or INHBA KO-CAFs. (B) Molecular docking predicted the interaction between INHBA and ENG. (C) Co-immunoprecipitation validation of the physical interaction between INHBA and ENG. (D) GEPIA database analysis of the expression correlation between INHBA and ENG in ovarian cancer tissues. (E) Correlations of ENG with GOBP (bottom left) and KEGG (top right) pathway enrichment scores. (F-H) Subcutaneous xenografts were established in three groups: SKOV3, SKOV3 + NC-CAFs, and SKOV3 + INHBA-KO-CAFs. (F) Representative tumors at day 28. (G) Tumor volumes recorded every 3 days from day 0 to day 28 under continued PT. (H) Tumor weights measured at day 28. (I) IHC of xenograft sections for INHBA, ENG, and Ki67 expression and localization(Scale bar: 100 μm, 50 μm).Data are presented as mean ± SD from 3 independent experiments ( n  = 3–5 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant INHBA Regulates OC Growth and Cisplatin Resistance via ENG In Vitro & In Vivo Validation. (A) WB analysis of ENG expression in SKOV3 and A2780 cells after treatment with conditioned medium from blank control, NFs, NC-CAFs, or INHBA KO-CAFs. (B) Molecular docking predicted the interaction between INHBA and ENG. (C) Co-immunoprecipitation validation of the physical interaction between INHBA and ENG. (D) GEPIA database analysis of the expression correlation between INHBA and ENG in ovarian cancer tissues. (E) Correlations of ENG with GOBP (bottom left) and KEGG (top right) pathway enrichment scores. (F-H) Subcutaneous xenografts were established in three groups: SKOV3, SKOV3 + NC-CAFs, and SKOV3 + INHBA-KO-CAFs. (F) Representative tumors at day 28. (G) Tumor volumes recorded every 3 days from day 0 to day 28 under continued PT. (H) Tumor weights measured at day 28. (I) IHC of xenograft sections for INHBA, ENG, and Ki67 expression and localization(Scale bar: 100 μm, 50 μm).Data are presented as mean ± SD from 3 independent experiments ( n  = 3–5 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant To validate the INHBA–ENG axis in cisplatin-resistant OC cells, ENG expression levels were first quantified by RT-qPCR and Western blotting. Significantly higher ENG expression was detected in cisplatin-resistant cell lines (SKOV3/DDP, A2780/DDP) compared with their parental sensitive counterparts (SKOV3, A2780) (Fig.  6 A, B). Based on these results, lentiviral short hairpin RNA (shRNA) was used to knock down ENG expression in SKOV3/DDP and A2780/DDP cells. RT-qPCR and Western blotting confirmed efficient silencing at both the mRNA and protein levels (Fig.  6 C, D), and the stable shENG#1 clone was selected for subsequent experiments.To identify the optimal concentration of recombinant INHBA (rINHBA), shENG-expressing SKOV3 and A2780 cells were treated with 0–10 µg/mL rINHBA and assessed for proliferation at 0, 24, and 48 h using CCK-8. rINHBA stimulated cell growth in a dose-dependent manner, peaking at 8 µg/mL, which was therefore chosen for subsequent assays (Fig.  6 E).Under 3 µg/mL cisplatin, functional assays were performed to evaluate the effects of ENG knockdown and rINHBA intervention. CCK-8 and colony-formation assays showed that cisplatin-resistant cells with ENG knockdown exhibited significantly higher sensitivity to cisplatin cytotoxicity than the sh-NC group (Fig.  6 F, G). Apoptosis assays revealed that apoptosis levels in the ENG-knockdown group were markedly elevated (Fig.  6 H). Invasion and Migration assays confirmed that the migratory and invasive capacities of cells in the ENG-knockdown group were significantly weaker than those in the sh-NC group (Fig.  6 I, J). Furthermore, addition of 8 µg/mL rINHBA to ENG-knockdown resistant cells markedly reversed the above phenomena of enhanced sensitivity, increased apoptosis, and reduced invasion and migration. WB analysis of apoptosis- and EMT-related proteins further validated the mechanism. Under cisplatin treatment, compared with the sh-NC group, ENG-knockdown resistant cells exhibited significant down-regulation of the anti-apoptotic protein BCL-2 and the mesenchymal markers Vimentin and N-cadherin, along with significant up-regulation of the pro-apoptotic protein BAX, the apoptosis-execution molecule Cleaved caspase-3, and the epithelial marker E-cadherin, indicating that ENG knockdown effectively reverses cisplatin resistance in OC cells. Meanwhile, addition of rINHBA to shENG cells not only restored ENG expression levels but also reversed the above trends in apoptosis- and EMT-related proteins, consistent with the previous experimental results (Fig.  6 K). Fig. 6 INHBA modulates apoptosis and EMT in cisplatin-resistant OC cells via ENG. (A , B) ENG expression was quantified by (A) RT-qPCR and (B) WB in cisplatin-resistant (SKOV3/DDP, A2780/DDP) and parental (SKOV3, A2780) cells. (C , D) Lentiviral shRNA was used to knock down ENG in SKOV3/DDP and A2780/DDP cells, and silencing efficiency was confirmed by (C) RT-qPCR and (D) WB. (E) CCK-8 assays were used to measure proliferation of shENG-stable SKOV3-DDP and A2780-DPP cells treated with 0–10 µg/mL rINHBA for 48 h. (F–K) Under 3 µg/mL cisplatin, three groups of resistant cells (ENG-knockdown, sh-NC, ENG-knockdown + 8 µg/mL rINHBA) were simultaneously analysed: (F) CCK-8 and (G) colony formation assays evaluated cisplatin cytotoxicity, (H) apoptosis was assessed by Annexin V-PE/7-AAD flow cytometry, (I) invasion and (J) migration assays were measured in transwell assays, (K) WB assessed apoptosis- and EMT-related proteins under the same conditions.Data are presented as mean ± SD from 3 independent experiments ( n  = 3 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant. Corresponding normal/negative/blank controls were set for all experimental groups INHBA modulates apoptosis and EMT in cisplatin-resistant OC cells via ENG. (A , B) ENG expression was quantified by (A) RT-qPCR and (B) WB in cisplatin-resistant (SKOV3/DDP, A2780/DDP) and parental (SKOV3, A2780) cells. (C , D) Lentiviral shRNA was used to knock down ENG in SKOV3/DDP and A2780/DDP cells, and silencing efficiency was confirmed by (C) RT-qPCR and (D) WB. (E) CCK-8 assays were used to measure proliferation of shENG-stable SKOV3-DDP and A2780-DPP cells treated with 0–10 µg/mL rINHBA for 48 h. (F–K) Under 3 µg/mL cisplatin, three groups of resistant cells (ENG-knockdown, sh-NC, ENG-knockdown + 8 µg/mL rINHBA) were simultaneously analysed: (F) CCK-8 and (G) colony formation assays evaluated cisplatin cytotoxicity, (H) apoptosis was assessed by Annexin V-PE/7-AAD flow cytometry, (I) invasion and (J) migration assays were measured in transwell assays, (K) WB assessed apoptosis- and EMT-related proteins under the same conditions.Data are presented as mean ± SD from 3 independent experiments ( n  = 3 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant. Corresponding normal/negative/blank controls were set for all experimental groups A subcutaneous xenograft tumor model was established using shENG SKOV3/DDP cells to validate these in vitro findings in vivo. During cisplatin therapy, ENG knockdown suppressed tumor growth and reduced tumor weight, whereas 8 µg/ml rINHBA restored growth and reversed cisplatin-induced tumor shrinkage (Fig.  7 A–C).These in vivo data demonstrate that ENG knockdown sensitizes ovarian cancer cells to cisplatin, whereas rINHBA abrogates this sensitizing effect. WB of tumor lysates (Fig.  7 D) and IHC for ENG and Ki67 (Fig.  7 E) further confirmed that INHBA promotes EMT and inhibits apoptosis in an ENG-dependent manner, thereby enhancing cisplatin resistance in vivo. Fig. 7 INHBA reduces cisplatin sensitivity of OC via activating ENG in vivo. (A-C) An subcutaneous xenograft model was established using shENG SKOV3/DDP cells and divided into three groups: ENG-knockdown, ENG-knockdown + 8 µg/mL rINHBA, and control. During cisplatin treatment, (A) tumor growth, (B) volume, and (C) weight were monitored. (D) Tumor lysates from each group were subjected to WB to quantify apoptosis- and EMT-related proteins. (E) IHC was performed on tumor sections to assess ENG and Ki67 expression and localization(Scale bar: 100 μm, 50 μm).Data are presented as mean ± SD from 3 independent experiments ( n  = 3–5 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant. Corresponding normal/negative/blank controls were set for all experimental groups INHBA reduces cisplatin sensitivity of OC via activating ENG in vivo. (A-C) An subcutaneous xenograft model was established using shENG SKOV3/DDP cells and divided into three groups: ENG-knockdown, ENG-knockdown + 8 µg/mL rINHBA, and control. During cisplatin treatment, (A) tumor growth, (B) volume, and (C) weight were monitored. (D) Tumor lysates from each group were subjected to WB to quantify apoptosis- and EMT-related proteins. (E) IHC was performed on tumor sections to assess ENG and Ki67 expression and localization(Scale bar: 100 μm, 50 μm).Data are presented as mean ± SD from 3 independent experiments ( n  = 3–5 per group). Exact p-values are provided in the figure, and a p-value < 0.05 is considered statistically significant. Corresponding normal/negative/blank controls were set for all experimental groups

Reagents and antibodies used in this study are listed as follows: cisplatin (HY-17394, MCE), collagenase IV (2091GR001, Biofroxx), amphotericin B (BL140A, Anhui Baisha). Culture media included fetal bovine serum (FBS, AUS-01 S, CELL-BOX), penicillin–streptomycin (G4003, ServiceBio), and DMEM/F12 or DMEM high glucose (Gibco); puromycin (ST551, Beyotime). Apoptosis was detected using Solarbio Annexin V-AF488/PI (CA1040) and Annexin V-PE/7-AAD (CA1030) kits; cell viability was measured with Seven Biotech CCK-8 (SC-119). Matrigel was purchased from BD Biosciences (Bedford, MA, USA). A human INHBA ELISA kit (YX-091408 H, Shanghai Youxuan), recombinant human INHBA-N-His (YB33102, YBio), and Abbkine ultrasensitive ECL substrate (BMU102-CN) were used according to the manufacturers’ instructions. Total RNA Extraction Kit (SM132) was obtained from Beijing Seven Biotech Co., Ltd.; the first-strand cDNA synthesis kit (g592) and BlasTaq 2x qPCR MasterMix (g891) were purchased from ABM (Applied Biological Materials). Triton X-100 (9002-93-1) was purchased from Beijing Solarbio Science & Technology Co., Ltd. DAPI (BL105A, Anhui Baisha) and DAB substrate (DS9800, Leica) were used for staining experiments. RIPA lysis buffer (P0013B), PMSF(ST506), and phosphatase inhibitor (P1005) were all from Beyotime; BCA kit (ZJ102) was from EPIZYME BIOTECH. Primary antibodies included rabbit anti-FAP (ab314456, Abcam); anti-INHBA (bs-1774R, Bioss; CY8360, Abways;17524-1-AP, Proteintech); anti-ENG/CD105 (bs-34063R, Bioss; CY8850, Abways;10862-1-AP, Proteintech); anti-Cleaved caspase-3 (9661, CST); anti-β-actin (60008-1-Ig, Proteintech); and α-SMA (TA1032), Vimentin ( T55134 ), E-cadherin (TA0131), N-cadherin ( T55015 ), Bax ( T40051 ), Bcl-2 ( T40056 ), and Ki-67 (TW0001) from Abmart. Secondary antibodies were HRP-conjugated goat anti-rabbit/mouse IgG ( M21003 , Abmart) and CoraLite Plus 488 goat anti-rabbit IgG (RGAR002, Proteintech). For Co-IP assays, cells were lysed with RIPA lysis buffer (HYP103, HYCEZMBIO) supplemented with protease/phosphatase inhibitors, using normal rabbit IgG ( B30011 , Abmart) as a negative control and Protein A/G Plus agarose beads (Santa Cruz Biotechnology) for immunocomplex capture. Public single-cell RNA sequencing (scRNA-seq) datasets were downloaded from the Gene Expression Omnibus (GEO) database( GSE154600 ),which was generated on the 10X Genomics platform and contained single-cell RNA sequencing data of high-grade serous ovarian cancer (HGSOC), including 2 platinum-resistant, 2 platinum-sensitive and 1 refractory tissue samples. The raw data were preprocessed using the Seurat R package, with low-quality cells filtered out. After normalization, 2000 highly variable genes were screened, and batch effects were corrected by Harmony. Cell clustering was performed following principal component analysis (PCA) dimensionality reduction, and the results were visualized via uniform manifold approximation and projection(UMAP) and t-distributed stochastic neighbor embedding(t-SNE). Cell types were annotated by the marker gene method combined with differential expression analysis; fibroblast subsets were then extracted and further clustered into four CAF subtypes (iCAF, myoCAF1, myoCAF2, myoCAF3). After grouping the samples by treatment response, we analyzed the proportion of each CAF subtype and the differential expression of the INHBA gene across groups. Intercellular communication was deciphered using the NicheNet tool, with a focus on ligand-receptor interactions between CAFs and epithelial cells. In particular, we investigated the expression correlation between INHBA and ENG in platinum-sensitive and platinum-resistant groups. Patients with OC who were admitted to the Shanxi Provincial People’s Hospital Affiliated to Shanxi Medical University from January 2020 to December 2025 were enrolled in this study. Written informed consent was obtained from all participants and the protocol for specimen use in this study was reviewed and approved by the Ethics Committee of the Fifth Clinical Medical College of Shanxi Medical University (Shanxi Provincial People’s Hospital) with the approval number 591. Inclusion criteria: The patients underwent ovarian cancer staging surgery or cytoreductive surgery at our hospital and all ovarian tissue samples were obtained from the primary cytoreductive surgery. They were histopathologically diagnosed with epithelial ovarian cancer and had qualified pathological sections. They received preoperative imaging examinations with measurable lesions identified intraoperatively. Postoperative pathology confirmed epithelial ovarian cancer and they received subsequent platinum-based chemotherapy. They had no history of other malignant tumors and complete clinical data were available. Exclusion criteria: The patients had poor quality of pathological sections. Postoperative routine pathology confirmed non-epithelial ovarian malignancies. They failed to receive regular postoperative chemotherapy. They were complicated with other underlying diseases that may affect sample quality. They had received neoadjuvant chemotherapy before surgery. They were in pregnancy or lactation status. Patients were stratified into two groups according to the platinum-free interval (PFI), which is defined as the time from the last administration of platinum-based chemotherapy to disease recurrence. Specifically, there were 34 cases in the platinum-sensitive group with PFI ≥ 6 months and 16 cases in the platinum-resistant group with PFI < 6 months. All patients in the platinum-resistant group were primary platinum-resistant cases with no response to first-line platinum-based chemotherapy. Additionally, 20 patients who underwent bilateral salpingo-oophorectomy for non-ovarian diseases including adenomyosis and uterine fibroids during the same period were enrolled as the normal control group. Their ovarian tissues were confirmed to be free of pathological abnormalities by histopathology.Formalin-fixed paraffin-embedded (FFPE) ovarian tissue specimens from all participants were collected for subsequent assays. IHC analysis was performed on FFPE tissue samples from normal ovarian tissues and patients with OC who met the inclusion criteria. The detailed procedure was as follows. FFPE sections with a thickness of 6 μm were dewaxed in xylene and rehydrated in gradient ethanol. Endogenous peroxidase activity was then quenched. Two consecutive paraffin-embedded sections were incubated with anti-INHBA antibody (1:200) and anti-ENG antibody (1:200) at 4 °C for 12 h respectively. Secondary antibody was added and incubated at 25 °C for 45 min. After incubation, the sections were developed with DAB, counterstained with hematoxylin, dehydrated and mounted. The protein expression levels were evaluated by semi-quantitative analysis. The percentage of positive cells and staining intensity of INHBA and ENG in all ovarian and ovarian cancer cells were counted respectively. The percentage of positive cells was scored on a 1–4 scale with the criteria as follows. A score of 1 was assigned for 0–25% positive cells, 2 for 26–50%, 3 for 51–75% and 4 for 76–100%. The staining intensity was scored on a 0–3 scale with the criteria as follows. A score of 0 was given for negative staining, 1 for weak positive staining, 2 for moderate positive staining and 3 for strong positive staining. The total score (ranging from 0 to 12) was calculated as the product of the percentage score and the intensity score. A total score of 5–12 indicated high expression and a score of 0–4 indicated low expression. All pathological diagnoses and staining results were independently evaluated and recorded by two professional pathologists who were double-blinded to the clinical data of the patients. Sections of normal ovarian tissue, platinum-sensitive OC tissue, and platinum-resistant OC tissue were taken. After dewaxing, hydration, PBS rinsing, antigen retrieval, and cell membrane permeabilization, the sections were blocked with 5% BSA, and then the mixed working solution of anti-INHBA and anti-ENG primary antibodies was added, followed by incubation overnight at 4℃. On the next day, after PBS rinsing, fluorescent secondary antibody incubation in the dark, and TSA signal amplification, the sections were counterstained with fluorescence anti-quencher containing DAPI and mounted. Fluorescent images were collected under a laser confocal microscope to analyze the cellular localization and co-localization relationship of INHBA (excitation wavelength 555 nm, red fluorescence) and ENG protein (excitation wavelength 488 nm, green fluorescence) in the three groups of tissue sections, and the fluorescence intensity of the fluorescent images was quantitatively detected. All operating conditions must be strictly controlled throughout the experiment to effectively avoid interfering factors such as non-specific staining and fluorescence quenching, so as to ensure the accuracy and reproducibility of the experimental results. Ovarian tissue specimens for primary fibroblast isolation were collected in strict accordance with the aforementioned enrollment, exclusion and primary intraoperative sampling criteria for study participants. Epithelial ovarian cancer tissues were used for the isolation and culture of CAFs, while normal ovarian tissues were for NFs. Fresh intraoperative specimens were processed immediately after resection, rinsed with PBS containing penicillin-streptomycin-amphotericin B, and epithelial and adipose tissues were carefully removed. The remaining connective tissues were minced into 1 mm³ tissue fragments, which were then seeded in DMEM/F12 medium supplemented with 5% FBS and 1% antibiotic-antimycotic agent. The cultures were maintained in a humidified incubator at 37 °C with 5% CO₂, and the medium was refreshed every 2–3 days. When the cells reached confluence, they were passaged at a ratio of 1:2, and the 3rd passage cells were used for subsequent experiments. Conditioned media were collected after 48 h of monolayer culture of NFs and CAFs at passages 3–10, then centrifuged at 800 × g for 10 min, and labeled as NF-CM and CAF-CM respectively. All fibroblasts were used within passages 3–10 to preserve their original phenotypic characteristics. Adherent cell slides were placed in 24-well plates and fixed with 4% paraformaldehyde solution for 20 min at room temperature in the dark. After permeabilization with PBS containing 0.1% Triton X-100 for 30 min at room temperature, the slides were completely covered with blocking buffer (PBS containing 5% bovine serum albumin, BSA) and blocked for 1 h at room temperature, followed by aspiration of the buffer. The diluted primary antibody working solutions against α-SMA (1:200), FAP (1:100), vimentin (1:200) or INHBA (1:100) were added dropwise to the slides separately, which were then incubated overnight at 4 °C in a humidified chamber. The primary antibodies were recovered the next day, and secondary antibody incubation was performed according to the experimental type: for immunocytochemistry (ICC) assays, HRP-conjugated species-specific secondary antibody (1:500) was added and incubated for 1 h at room temperature; for immunofluorescence (IF) assays, FITC-conjugated species-specific secondary antibody (1:500) was added and incubated for 1 h at room temperature in the dark. For ICC, DAB chromogenic solution was added for color development for 3–5 min at room temperature in the dark, and the reaction was terminated with deionized water when distinct positive signals were observed under a microscope. For IF, cell nuclei were counterstained with DAPI solution at a final concentration of 5 µg/mL for 5 min at room temperature in the dark. Finally, the slides were mounted: for ICC, slides were inverted and mounted on glass slides with neutral balsam, then air-dried at room temperature; for IF, slides were inverted and mounted on glass slides with anti-fade mounting medium and stored in the dark. All samples were examined under an Olympus or Leica upright microscope, and images were acquired from 5 randomly selected non-overlapping fields of view (10× objective lens). Human epithelial OC cell lines SKOV3 and A2780, along with their cisplatin-resistant derivatives SKOV3/DDP and A2780/DDP, were purchased from Shanghai Jinyuan Biotechnology and authenticated by STR profiling. All cell lines were cultured in DMEM supplemented with 10% FBS and maintained at 37 °C in a humidified incubator with 5% CO 2 . To maintain the resistant phenotype, the culture medium for SKOV3/DDP and A2780/DDP was continuously supplemented with 0.5 µg/mL cisplatin. INHBA knockout plasmids were constructed based on the CRISPR/Cas9 system and purchased from abm Biosciences (Shanghai). CAF cells were seeded one day prior to transfection to ensure 70%-90% confluency at the time of transfection. The transient transfection procedure was as follows: in each well of a 6-well plate, 5 µL of Lipofectamine™ 3000 reagent (Cat. No. L3000015, Thermo Fisher Scientific) was used to transfect control plasmids and sgRNA plasmids targeting INHBA (5’-GCACTCACATCGCTACATCA-3’). The sgRNA sequences were sgRNA-A1 (5’-GCTTCATGTGGGCAAAGTCG-3’) and sgRNA-A2 (5’-GGCAAACGTGATGATCTCCG-3’). Following 4–6 h of incubation at 37 °C, the medium was replaced with complete medium, and culturing was continued for 48 h. Conditioned media (CM) collected using the above method were designated as INHBA NC-CM and INHBA KO-CM. For stable transduction of cisplatin-resistant cells, packaged negative control (NC) vectors and GV493 lentiviral vectors carrying shRNA-ENG were utilized; these were purchased from Haixing Biotechnology Co., Ltd. (Suzhou). The shRNA sequences were shENG#1 (5’-CCCTGTCATTTGAACCTGGAT-3’) and shENG#2 (5’-GCGAGGTGACATATACCACTA-3’). One week before transduction, the cell culture medium was replaced with cisplatin-free medium. SKOV3/DDP and A2780/DDP cells were seeded in 6-well plates and cultured at 37 °C for 24 h, after which NC groups and shRNA groups were established, with the working concentration of shRNA being 50 nM. The volume of NC and shRNA lentivirus to be added was calculated according to cell number, with a multiplicity of infection (MOI) of 10 for both SKOV3/DDP and A2780/DDP cells. After addition to the cells, cultures were incubated at 37 °C for 24 h, followed by replacement with fresh complete medium. At 48–72 h post-transduction, transduction efficiency was evaluated by observing green fluorescent signals under a fluorescence microscope, and stable transduced cell lines were selected using 2 µg/mL puromycin. Finally, gene knockdown efficiency was verified by quantitative real-time PCR(qRT-PCR) and Western blot (WB) analyses. Total RNA was extracted from cells using the SevenFast Total RNA Extraction Kit. After quantification, the RNA was used for complementary DNA (cDNA) synthesis and subsequent PCR amplification. The reaction conditions for qRT-PCR were as follows: pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s. GAPDH was used as the internal reference gene, and the relative expression of the target gene was calculated using the 2 −ΔΔCT method. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific sequences are provided in Supplementary Table 1 . Cells were lysed on ice with RIPA lysis buffer containing PMSF and phosphatase inhibitor. Protein concentration was quantified with a BCA kit.Twenty micrograms of protein were separated on 10% SDS-PAGE gels and transferred to 0.45 μm PVDF membranes (Millipore). After blocking with 5% non-fat milk for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies, followed by 1 h at room temperature with HRP-conjugated secondary antibodies (Supplementary Table 2 ). Signals were visualised using an ECL chemiluminescent reagent. Cells were seeded in 6-well plates at a density of 2 × 10⁵ cells/mL and cultured at 37 °C with 5% CO₂ for 48 h. The supernatants of NFs and CAFs were then collected and centrifuged at 3000 r/min for 10 min at 4 °C to remove cell debris. The ELISA kit was equilibrated at room temperature for 20 min, after which standard wells and sample wells were set up. 50 µL of standard solution with the corresponding concentration was added to each standard well, while 10 µL of the supernatant to be tested and 40 µL of sample diluent were added to each sample well. Except for the blank wells, 100 µL of HRP-conjugated detection antibody was added to all other wells. The plate was sealed and incubated at 37 °C for 60 min, followed by five washing cycles. 50 µL each of Substrate A and Substrate B were added to every well, and the plate was incubated at 37 °C for 15 min in the dark. Subsequently, 50 µL of stop solution was added to each well, and the OD values were measured at a wavelength of 450 nm within 15 min. A standard curve was plotted to calculate the INHBA concentration in the samples, and the final results were multiplied by 5 to obtain the actual concentrations. All protein sequences used in this study were retrieved from the UniProt database (INHBA and ENG sequences are listed in Supplementary Table 3 ). Using these sequences, the three-dimensional structures of the two target proteins were predicted using AlphaFold3. Previous studies have shown that AlphaFold3 achieves prediction accuracy comparable to or even higher than that of X-ray crystallography, eliminating the need for further validation of these predictions [ 27 ]. For each protein, five parallel structural models were generated. The optimal structure was selected based on the predicted template modeling (pTM) score, and the conformation with the highest pTM score was used for subsequent molecular docking analyses. Protein docking analyses were performed using these optimized structures, and PyMOL 2.3.1 was used to visualize the final protein structures, docking complexes and generate the corresponding figures. Co‑IP was performed to detect the endogenous interaction between INHBA and ENG in CAF–OC cell co‑cultures [ 28 ]. CAFs and SKOV3 or A2780 cells were seeded at a 1:1 ratio and cultured until 80%–90% confluence. Cells were washed with ice‑cold PBS and lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates were pre‑cleared and immunoprecipitated overnight at 4 °C with anti‑ENG antibody, anti‑INHBA antibody, or normal rabbit IgG as a negative control. Protein A/G Plus agarose beads were added for 4 h at 4 °C. After washing three times, bound complexes were eluted by boiling in 1× SDS loading buffer and analyzed by WB using antibodies against the partner protein. Cells in logarithmic growth phase were seeded into 96-well plates at a density of 8 × 10³ cells per well in 100µL of medium. After overnight incubation to ensure complete adhesion, the following protocols were applied: Cytotoxicity assay: The culture medium was replaced with fresh medium containing 0, 1, 2, 4, 8–10 µg/mL cisplatin, and cells were incubated for 48 h ( n  = 3 replicates per concentration). Proliferation assay: The culture medium was replaced with medium containing 0, 1, 2, 4, 8–10 µg/mL recombinant INHBA (rINHBA), and cells were incubated for 48 h ( n  = 3 replicates per concentration). Combination assay: Cells were pretreated with the optimal concentration of rINHBA, conditioned medium (CM), or vehicle control, then exposed to the half-maximal inhibitory concentration (IC 50 ) of cisplatin previously determined in cisplatin-sensitive OC cells, and incubated for 24–48 h ( n  = 3 replicates per time point). At the end of the indicated incubation periods, 10 µL of CCK-8 reagent was added to each well, the plates were gently mixed, and cells were incubated for 1 h. The absorbance values were measured at 450 nm using a microplate reader, and half-maximal inhibitory concentration (IC 50 ) values were calculated based on the cytotoxicity data. For cell seeding, 700 cells were plated per well in 6-well plates. After attachment, cells were divided into different treatment groups and cultured for 48 h in CM supplemented with cisplatin and 10% FBS or medium containing rINHBA. After 48 h of treatment, cisplatin was removed, and the medium was replaced with appropriate fresh medium for an additional culture period of approximately 10 days. At the end of the culture period, cells were fixed and stained as follows: Cells were first fixed with 4% paraformaldehyde for 30 min. After fixation, cells were stained with 0.1% crystal violet for 30 min. Colonies were then imaged using a high-resolution digital camera. Three independent experiments were performed, and ImageJ was used to quantify colony number and analyze colony morphology. Cells in each group were treated with CM or medium containing rINHBA, both supplemented with cisplatin for 48 h. Then, cells were stained with Annexin V-FITC (or PE) and PI (or 7-AAD) according to the manufacturer’s instructions. Flow cytometry was performed using a BD Biosciences flow cytometer to detect fluorescence signals. Three cell populations were distinguished: viable cells (Annexin V⁻/PI⁻), early apoptotic cells (Annexin V⁺/PI⁻), and late apoptotic/necrotic cells (Annexin V⁺/PI⁺). All flow cytometry data were analyzed using FlowJo (BD Biosciences), and each experiment was repeated at least three times independently. Migration assays were conducted using Transwell 24-well chemotaxis chambers (BD Biosciences, Franklin Lakes, NJ, USA). A total of 2 × 10⁴ cells suspended in 200 µL serum-free CM containing cisplatin were seeded into the upper chamber. For invasion assays, the upper surface of the filter membrane was pre-coated with 50 µL diluted Matrigel. After incubation for 48 h, cells that had migrated or invaded to the lower surface of the membrane were fixed and stained with 0.1% crystal violet. Stained cells were counted in 5 randomly selected fields under an optical microscope at ×10 magnification.Each experiment included 3 replicate wells per group and was repeated independently at least 3 times. The experimental procedures strictly adhered to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences and approved by the National Institutes of Health (NIH) to ensure the health and welfare of the experimental animals. The experimental protocol and animal care plan were reviewed and approved by the Ethics Committee of the Fifth Clinical Medical College of Shanxi Medical University (Shanxi Provincial People’s Hospital) with approval number: 591. Five-week-old female BALB/c nude mice were used to establish subcutaneous xenograft tumor models, and the experimental mice were provided by Saibeifu (Suzhou) Biotechnology Co., Ltd.To minimize selection bias and ensure consistent baseline characteristics across experimental groups, all mice were randomly numbered and assigned to six groups before modeling via a completely random grouping method. The experimental groups included the SKOV3 group, SKOV3 + NC-CAFs group, SKOV3 + INHBA KO-CAFs group, shNC SKOV3/DDP group, shENG SKOV3/DDP group, and rINHBA+shENG SKOV3/DDP group, with 5 mice per group [ 29 ]. For xenograft experiments including the SKOV3 alone, SKOV3 + INHBA NC-CAFs, and SKOV3 + INHBA KO-CAFs groups, CAFs were first stably transfected with INHBA sgRNA. Subsequently, SKOV3 cells alone or mixed with CAFs at a 1:1 ratio (5 × 10⁵ cells per mouse) were subcutaneously injected into the right axilla of the mice [ 30 ].In the shNC SKOV3/DDP group, shENG SKOV3/DDP group, and rINHBA+shENG SKOV3/DDP group, 1 × 10⁶ SKOV3-DDP cells were suspended in 100 µl PBS. Among them, shENG SKOV3/DDP cells were incubated with rINHBA (8 µg/ml) at 37 °C for 2 h prior to injection [ 31 ], after which cells from all groups were subcutaneously injected into the right axilla of mice. All mice bearing xenografts were administered cisplatin (4 mg/kg) via intraperitoneal injection every 6 days [ 32 ]. Tumor volume was calculated according to the formula: V = (L × W × H)/2, where L, W and H represent the longest, shortest and vertical diameters of the tumor, respectively, for dynamic monitoring of tumor growth. Tumor dimensions were measured every 2 days throughout the experiment to ensure no tumor exceeded 15 mm in maximum diameter. On day 28, mice were euthanized by carbon dioxide asphyxiation. Briefly, CO₂ was delivered at a flow rate of 30%-70% of the chamber volume per minute. Once mice reached a surgical plane of anesthesia (confirmed by loss of pedal withdrawal reflex, decreased muscle tone, and absence of corneal reflex), the CO₂ flow was increased to 100% and maintained for 2 min to ensure death. CO₂ euthanasia was selected for its rapid onset, reliable analgesia, and clearly definable anesthesia endpoints. Immediately following euthanasia, tumor tissues were harvested and photographed. The tissue samples were then snap-frozen in liquid nitrogen or fixed in 4% paraformaldehyde solution for subsequent WB and IHC analyses. The IHC protocol for animal tissues was performed following that for clinical samples, with the only modification being the addition of anti-Ki-67 antibody (1:200) during primary antibody incubation. Anti-Ki-67 antibody was co-incubated with anti-INHBA and anti-ENG/CD105 antibodies (both 1:200) at 4 °C for 12 h, and all subsequent steps were carried out identically as described previously. GraphPad Prism 9.5 software was utilized for all statistical analyses. Prior to hypothesis testing, all quantitative datasets were assessed for normal distribution and homogeneity of variance to select suitable statistical approaches. The Kruskal-Wallis H test, followed by Bonferroni post-hoc pairwise comparison, was employed to evaluate differences in IHC staining scores across normal control, platinum-sensitive, and platinum-resistant groups. Normally distributed quantitative data with homogeneous variance were presented as mean ± standard error of the mean (SEM). For parametric comparisons, Student’s t-test was adopted for two-group analyses, while one-way or two-way ANOVA was performed for multigroup comparisons. All exact p-values were reported in the respective figures, and statistical significance was defined as p  < 0.05.

OC exhibits the highest fatality rate among gynecologic malignancies, with high-grade serous carcinoma (HGSC) as the leading histologic subtype [ 1 ].Approximately 70% of patients with HGSC acquire resistance to platinum-based combination chemotherapy, which represents a major clinical bottleneck. Such drug resistance further promotes tumor metastasis and recurrence, ultimately leading to adverse patient prognosis [ 2 , 3 ].Accordingly, identifying specific therapeutic targets for platinum resistance is an urgent unmet demand for basic research and clinical transformation of OC treatment. OC mainly metastasizes within the peritoneal cavity [ 4 ]. EMT drives this process by converting quiescent epithelial cells into motile mesenchymal cells, remodeling the extracellular matrix (ECM) and adjusting adhesion molecules. All these alterations boost chemoresistance and help cancer cells cross tissue barriers, which contributes to peritoneal metastasis [ 5 , 6 ].Tumor-stromal interactions in the tumor microenvironment (TME) further amplify cisplatin resistance and metastasis [ 6 – 8 ].As a critical modulator of OC onset and progression, the TME enables cancer cells to recruit and remodel stromal cells, thereby forming a protective niche that sustains tumor cell survival, proliferation and dissemination under cisplatin treatment stress [ 9 ]. Fibroblasts within the stromal compartment remodel the extracellular matrix, amplifying metastatic signaling and interacting with EMT pathways, thereby aggravating cisplatin resistance and driving tumor progression [ 10 , 11 ]. Notably, CAFs, a key stromal subset within the TME, have attracted increasing attention [ 12 , 13 ]. By secreting proteases and extracellular matrix components, CAFs help construct metastatic niches that facilitate tumor cell colonization. At the same time, these cells drive disease progression and treatment resistance. They do so through several mechanisms, including chronic inflammation, angiogenesis, immunosuppression, drug tolerance, and stromal remodeling [ 14 – 16 ]. INHBA encodes inhibin βA, a key ligand of the transforming growth factor-β (TGF-β) superfamily, which can form activin A via homodimerization or inhibin via heterodimerization with inhibin βB [ 17 , 18 ].Previous studies have confirmed that INHBA⁺ CAFs are key drivers of tumorigenesis and immune evasion [ 19 ], while the INHBA they secrete regulates stromal activation, immunosuppression, and tumor proliferation in OC [ 20 , 21 ]. As a transmembrane co-receptor of the TGF-β receptor complex, ENG is indispensable for TGF-β signal transduction and serves as a core regulator of tumor angiogenesis. Aberrant activation of this pathway in cancer cells drives uncontrolled cell proliferation, invasion, metastasis and EMT [ 22 ].Elevated ENG expression predicts poor prognosis in multiple cancers including OC [ 23 ]. In patients with advanced serous ovarian cancer, significantly higher ENG levels are detected in tumor cells from post-chemotherapy effusions and solid metastases than in pre-chemotherapy samples [ 24 ].While INHBA and ENG are known to regulate CAF activation and TGF-β signaling individually, the nature of their functional interplay, downstream crosstalk, and translational relevance in CAF-driven cisplatin resistance and metastasis remains unclear. Breakthrough advances in scRNA-seq have revolutionized our understanding of CAF heterogeneity in the tumor microenvironment, overcoming the limitations of traditional bulk sequencing that mask cellular diversity. This high-resolution technology enables dissection of tumor cell heterogeneity, identification of rare subsets, tracking of cell lineages, localization of key genes and mutation sites, and discovery of novel biomarkers. It can also accurately identify unrecognized CAF subtypes, clarify their transcriptional profiles and specialized functions, and localize key gene expression in specific cell subsets [ 25 , 26 ]. In this study, we combined scRNA-seq with clinical sample validation and complementary in vitro and in vivo experiments to elucidate the mechanisms by which CAFs drive malignant progression in OC. Single‑cell profiling enabled us to delineate CAF subtypes and characterize the expression patterns of INHBA and ENG. Functional analyses further revealed how CAF‑derived INHBA interacts with ENG to modulate cisplatin sensitivity and epithelial–mesenchymal transition. These insights may inform novel therapeutic strategies against cisplatin‑resistant ovarian carcinoma.

Tumor heterogeneity drives malignant progression through dynamic crosstalk between tumor cells, ECM and stromal components in the TME [ 37 , 38 ]. CAFs, as the most abundant stromal cells in solid tumors, actively remodel the TME and mediate chemoresistance by secreting cytokines, chemokines and growth factors [ 10 , 39 , 40 ]. Among these factors, TGF-β superfamily members play central roles in ECM remodeling and malignant phenotype regulation, and their contributions to ovarian cancer platinum resistance remain a major research focus [ 19 , 41 – 44 ]. INHBA, a TGF-β superfamily member, is aberrantly overexpressed in multiple malignancies and correlates with poor prognosis [ 45 – 47 ].Recent single-cell studies have identified INHBA+ CAFs as a key functional subset driving tumor progression and immune suppression across cancer types [ 19 ]. For example, in oral squamous cell carcinoma, INHBA triggers the INHBA-ACVR1/ACVR2A/ACVR2B axis to modulate INHBA-positive macrophages and inflammatory CAFs, ultimately steering regulatory T-cell (Treg) differentiation [ 48 ].In OC specifically, CAF-derived activin A (an INHBA homodimer) induces PD-L1 expression via SMAD2 signaling and promotes Treg expansion [ 19 ].Beyond these contexts, INHBA—also recognized as a CAF-associated signature gene—predicts poor colorectal cancer outcome and correlates with macrophage polarization [ 49 ].However, the primary cellular source of INHBA, its specific functional receptor, and the precise mechanisms underlying its role in platinum resistance remain incompletely elucidated in OC. Single-cell dissection of the TME identified myoCAF1 as the predominant source of INHBA, exhibiting strict CAF subtype specificity. This strict compartmentalization of INHBA within the myoCAF1 subtype, rather than iCAFs, suggests a specialized functional division within the stromal compartment. Unlike iCAFs that primarily regulate immunity via IL-6 and CXCL12 [ 50 ], myoCAF1 appears dedicated to remodeling the epithelial phenotype through the INHBA-ENG axis. Importantly, its putative receptor ENG was predominantly localized to tumor epithelial cells, and the spatial colocalization of this ligand-receptor pair in clinical tissues underscores the clinical relevance of this axis in platinum-resistant tumors [ 51 ].Clinical validation using 50 epithelial ovarian cancer and 20 normal tissues confirmed that elevated INHBA–ENG expression and their spatial co‑localization are associated with platinum resistance, supporting the clinical relevance of this axis in situ. To elucidate the INHBA-ENG interaction, we employed AlphaFold3-based molecular docking and functional assays. As a key component of the TGF-β receptor complex, ENG is essential for signal transduction [ 25 ].Previous studies have confirmed that in breast cancer, ovarian cancer, and lung adenocarcinoma harboring p53 mutations, INHBA can influence patient prognosis and tumor cell sensitivity to chemotherapeutic agents via an ENG/CD105-dependent signaling pathway [ 52 ]. However, the structural basis remains unclear. Molecular docking predicted a high-affinity interaction (ΔG = − 12.3 kcal/mol), which was empirically validated by Co-IP, confirming a direct physical interaction between INHBA and ENG at the cellular level. The regulatory mechanisms underlying tumor chemoresistance are highly complex, and accumulating evidence has identified EMT, high expression of ABC transporters, and enhanced DNA damage repair capacity as core mechanisms mediating tumor chemoresistance [ 53 ].This indicates that EMT not only drives tumor metastasis but may also play a pivotal role in chemoresistance-associated metastasis. Consistent with this notion, previous studies have demonstrated that INHBA can promote proliferation, invasion, migration and EMT in osteosarcoma, breast cancer and cervical cancer cells via the TGF-β signaling pathway [ 54 – 56 ].In the subsequent functional experiments of our study, two complementary approaches, namely conditioned co-culture of ovarian cancer cells with CAFs and exogenous supplementation of rINHBA, yielded consistent results in both in vitro and in vivo models: INHBA significantly upregulated ENG expression in ovarian cancer cells. Furthermore, analysis of TCGA database revealed that ENG exerts a pro-EMT effect during the EMT process of OC.The molecular mechanism by which INHBA upregulates ENG expression remains to be fully elucidated. Previous studies have demonstrated that ENG transcription is regulated by a complex network of transcription factors, including not only SMAD proteins (the canonical mediators of TGF-β signaling) but also EMT-associated transcription factors such as Snail and Twist [ 57 – 59 ]. Notably, TGF-β superfamily ligands can activate both canonical SMAD-dependent pathways and non-canonical non-SMAD pathways, such as PI3K/AKT and MAPK/ERK signaling. Therefore, INHBA-induced ENG upregulation may be mediated by SMAD-dependent transcriptional activation, or alternatively, through non-SMAD signaling cascades. This potential dual regulatory mode, together with the possible existence of a positive feedback loop, may amplify the pro-tumorigenic and chemoresistant effects of the INHBA-ENG axis, representing a critical direction for future mechanistic investigations. Functional intervention experiments provided further mechanistic clues: knocking down ENG effectively reversed the cisplatin-resistant phenotype of drug-resistant ovarian cancer cells, while exogenous rINHBA supplementation markedly attenuated this reversal effect. Although rINHBA partially restored ENG expression and the chemoresistant phenotype, it failed to recover them to the levels observed in control cells, suggesting that INHBA may exert partial biological functions through minor ENG-independent bypass mechanisms.This finding highlights the critical role of INHBA-mediated ENG regulation in maintaining cisplatin resistance. Mechanistic investigations further showed that both CAF culture supernatants and rINHBA significantly downregulated the expression of pro-apoptotic proteins and upregulated anti-apoptotic proteins in ovarian cancer cells. Additionally, they increased the expression of mesenchymal markers and decreased the expression of epithelial markers, which serves as clear evidence of enhanced EMT progression.Flow cytometry experiments further validated that INHBA inhibits cisplatin-induced apoptosis in ovarian cancer cells.Notably, when INHBA was knocked out in CAFs or ENG was knocked down in cisplatin-resistant OC cells, the aforementioned changes in the expression of apoptosis-related proteins and EMT markers were significantly attenuated. Collectively, these results suggest that EMT may mediate cisplatin resistance in epithelial ovarian cancer cells induced by CAFs, with the underlying mechanism potentially involving the induction of apoptotic resistance. This study has several limitations. First, the isolation of primary CAFs and NFs relied on a limited number of clinical surgical samples, which may constrain the statistical power of primary cell-based findings. Future studies will expand the sample size and incorporate multicenter cohorts to validate these conclusions. Second, the SKOV3 and A2780 cell models used in this study have limited representativeness and cannot fully recapitulate the complex biological characteristics of OC. Future investigations will include more types of OC cell lines as well as patient-derived clinical samples to verify and extend our findings.

Our data unveil a novel mechanism whereby CAF-derived INHBA acts in a paracrine manner on the transmembrane receptor ENG of OC cells, thereby inducing EMT and enhancing apoptotic resistance (Figure 8 ). This study provides a systematic evaluation of how CAFs govern EMT and cisplatin resistance in OC, offering not only experimental evidence for the molecular mechanisms by which the tumor microenvironment drives malignant progression in platinum-resistant OC, but also a theoretical basis for future microenvironment-targeted therapies. Fig. 8 Schematic diagram of the molecular mechanism by which CAF-derived INHBA regulates platinum resistance in OC via paracrine action on ENG receptor to induce EMT and enhance apoptosis resistance. (by figdraw.com) Schematic diagram of the molecular mechanism by which CAF-derived INHBA regulates platinum resistance in OC via paracrine action on ENG receptor to induce EMT and enhance apoptosis resistance. (by figdraw.com)

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