Establishment of pathology-validated immortalized patient-derived ovarian endometriosis epithelial cell line

Endometriosis is a heterogeneous gynecological disorder for which experimental model development is often limited by diagnostic ambiguity and the restricted lifespan of primary epithelial cultures. Although immortalization strategies enable long-term expansion of patient-derived cells, concerns remain regarding the preservation of disease-specific biological characteristics and the risk of model misclassification in the absence of definitive pathological confirmation. In the present study, immortalized ovarian epithelial cell lines from clinically suspected ovarian cystic lesions representing endometriosis were established and it was evaluated whether disease-intrinsic biological features were preserved following immortalization, as determined by definitive histopathological stratification. Primary epithelial cells were isolated from two ovarian cystic lesions and immortalized via lentiviral co-expression of constitutively active cyclin-dependent kinase 4 (CDK4^R24C), cyclin D1 and human telomerase reverse transcriptase. One lesion was subsequently identified as an ovarian endometrioma, and the other as a mature cystic teratoma, in postoperative histopathology. The endometriosis-derived immortalized epithelial line iEn1 exhibited sustained proliferative capacity (Cell Counting Kit-8 assay), maintained epithelial lineage characteristics (western blotting for pan-cytokeratin, β-catenin and claudin-1), and retained a stable 46,XX karyotype. These cells demonstrated epithelial-mesenchymal plasticity evidenced by co-expression of epithelial markers with vimentin and N-cadherin, and formed microscopic glandular structures resembling endometriosis-like lesions in an estrogen-supplemented xenograft model. However, immunohistochemistry revealed that estrogen receptor β and interleukin-6, both frequently implicated in endometriosis-associated inflammation, were not detected in either the original tissue or the xenografted lesions, indicating that this particular inflammatory phenotype is not intrinsic to this model. By contrast, the teratoma-derived line iEn2 displayed marked chromosomal instability and phenotypic heterogeneity, demonstrated by karyotyping, consistent with its germ cell origin. Thus, immortalization alone does not determine the biological validity of patient-derived cellular models; rather, accurate interpretation requires integration with definitive histopathological classification. The present study established a pathology-validated immortalized epithelial model of ovarian endometriosis and highlighted the importance of pathology-integrated frameworks for improving experimental rigor in disease modeling.