Stress-induced anoikis-resistance triggers malignant-like phenotypes in uterine leiomyoma cells

Background: Uterine leiomyomas (LM) are the most common tumors in women of reproductive age and represent a major clinical burden. Although LM are classically considered benign, a long-standing scientific question concerns whether a subset of these tumors may undergo malignant transformation into leiomyosarcomas (LMS), a rare but highly aggressive uterine sarcoma. To date, this progression has not been conclusively demonstrated, yet it also cannot be fully excluded, and the biological mechanisms that might enable such a transition remain poorly understood. Anoikis resistance - the capacity of cells to survive loss of extracellular matrix attachment - is a hallmark of malignant transformation and early metastatic behavior in several solid tumors. Therefore, experimental models that induce anoikis resistance may offer insight into whether LM cells can acquire malignant-like traits under sustained cellular stress. Methods: Established cell lines of myometrium (MM; PCS-460-011) and leiomyoma (LM; THESCs; CRL-4003) were cultured (1×10⁵ cells/mL) on plates coated with 1% sterile agarose to prevent cell-matrix adhesion and maintained for 96 hours at 37°C and 5% CO₂. Surviving non-adherent spheroids (anoikis-resistant cells) were collected and replated under adherent conditions. This spheroid-formation cycle was repeated four times, generating the derived cell populations LM1C, LM2C, LM3C, and LM4C. Subsequently, spheroids were subjected to limiting dilution to obtain clonal populations. Five clones were randomly selected for expansion and functional/molecular analyses. Results: Preliminary findings indicate that LM cells progressively lose adhesion capacity and acquire anoikis resistance, accompanied by notable phenotypic alterations. MM cells did not survive repeated adhesion-blocking cycles and were eliminated before cycle 5 (C5), whereas LM cells successfully persisted through all cycles. After clonal expansion, genetic identity was confirmed through sequencing, validating their LM origin. Clonogenic assays demonstrated significant behavioral changes: anoikis-resistant LM derivatives exhibited an increased ability to survive and proliferate independently of cell-cell contact compared with parental LM cells. Ongoing assays of proliferation, migration, and invasion further indicate important phenotypic and behavioral alterations in vitro. Conclusion: The anoikis-resistance model appears to be a valuable tool for studying uterine leiomyoma biology. Our findings suggest that LM cells may acquire malignant-like traits when subjected to repeated adhesion stress, supporting the hypothesis that anoikis resistance may contribute to malignant transformation in uterine smooth muscle tumors.