Researchers Generate Stable Mouse Endometrial Tumor Cell Line Using Conditional Reprogramming

Researchers have successfully established a stable, tumorigenic mouse endometrial cancer epithelial cell line, designated PSC-01, from spontaneous uterine tumors in genetically engineered mice by utilizing conditional reprogramming technology. Endometrial cancer remains the most prevalent gynecological malignancy globally, yet preclinical exploration into disease progression and targeted interventions has been constrained by a scarcity of syngeneic murine models capable of growing in immunocompetent environments. By harvesting uterine adenocarcinoma tissue from mice bearing specific genetic modifications and cultivating the isolated epithelial cells alongside irradiated feeder cells and a Rho kinase inhibitor, the investigators created an immortalized line that mirrors the primary disease.

The experimental strategy relied on a genetically engineered mouse model characterized as Pten flox/+; Stk11 flox/flox; LTF-Cre, designated PSC. To produce these animals, researchers initially crossed Pten flox/flox mice with Stk11 flox/flox mice to establish a double-floxed PS strain, which was subsequent crossed with LTF-Cre transgenic mice. Within this genetic framework, conditional tissue-specific recombination drives the concurrent loss of STK11 protein expression and heterozygous mutation of PTEN within the endometrial epithelium. In humans, PTEN mutations occur in more than 70 to 80 percent of endometrial carcinomas, while loss of STK11 is observed in approximately 21 percent of primary endometrial tumors and is linked with hereditary Peutz–Jeghers syndrome. In the engineered mice, this dual alteration faithfully induced aggressive endometrial adenocarcinoma displaying classic pathological features of irregular glandular architecture and poorly differentiated epithelial morphology.

Primary uterine tumors were harvested from female PSC mice at approximately 17 weeks of age following euthanasia by cervical dislocation under pentobarbital sodium anesthesia. Diseased uterine tissues were washed thoroughly in sterile phosphate-buffered saline, finely minced with ophthalmic scissors, and subjected to enzymatic dissociation with 0.1 percent collagenase IV at 37 degrees Celsius for 15 minutes. Following filtration through a 70-micrometer cell sieve and centrifugation, the isolated single-cell suspension was seeded onto an irradiated feeder layer consisting of Swiss-3T3-J2 mouse fibroblasts. The primary culture relied on Complete F Medium containing DMEM/F-12 supplemented with 10 micromolar of the ROCK inhibitor Y-27,632, along with insulin, epidermal growth factor, hydrocortisone, cholera toxin, amphotericin B, gentamicin, penicillin-streptomycin, and 10 percent fetal bovine serum.

Under these conditional reprogramming parameters, multiple epithelial colonies emerged within two to three days post-seeding. The cultured cells were progressively transitioned into standard complete medium devoid of feeder cells and maintained robust proliferation without undergoing replicative senescence. Morphological assessment through light microscopy and Giemsa staining revealed classic cobblestone-like epithelial arrangements concentrated in central colonies alongside peripheral spindle-shaped cells. Cytological examination verified marked neoplastic features, including enlarged hyperchromatic nuclei, irregular chromatin clumping, and an elevated nuclear-to-cytoplasmic ratio. Flow cytometric profiling confirmed that roughly 99 percent of the cultivated PSC-01 cells expressed the epithelial-specific surface marker EpCAM, while multiplex real-time quantitative PCR authenticated that the line was purely murine, devoid of human, rat, or hamster contamination.

Biological and cytogenetic analyses confirmed that the PSC-01 cell line faithfully preserved the molecular lesions present in the donor mouse model. Genomic PCR and Western blot investigations demonstrated that PSC-01 cells completely lacked STK11 protein expression following biallelic deletion of the floxed Stk11 alleles, while maintaining wild-type PTEN protein expression from the remaining intact Pten allele. Cytogenetic G-banding demonstrated pronounced intracellular diversity and aneuploidy, revealing chromosome counts ranging from 61 to 80 across evaluated metaphase spreads. In vitro proliferation assessments documented stable growth kinetics across early and late generations, with cells successfully passaged continuously for more than 40 to 70 passages without exhibiting signs of cellular senescence or beta-galactosidase activation.

Whole-exome sequencing was performed on genomic DNA extracted from PSC-01 cells and matched primary tumor tissue to quantify genomic fidelity, achieving high sequencing quality with average GC contents near 47 percent and Q30 scores exceeding 93 percent. Circos plotting and comparative variant analysis revealed that PSC-01 cells largely recapitulated the somatic landscape of the parental primary malignancy. The cell line retained the primary tumor’s predominant point mutation spectra, single nucleotide polymorphism profiles, and small insertion-deletion distributions. Notably, high-frequency alterations in genes such as Jak3, Sfi1, Stx11, and Muc19 were conserved between the cultured cells and primary tissue, alongside highly concordant copy number variation profiles in established endometrial cancer driver genes including FBXW7, PIK3CA, NF1, and KRAS.

Functional phenotyping verified that PSC-01 cells possess strong migratory, invasive, and oncogenic capabilities. In Transwell chamber assays, the cells actively migrated through porous membranes toward a 20 percent fetal bovine serum chemoattractant gradient and invaded through reconstituted Matrigel matrices over 6, 12, and 24 hours. Immunofluorescence and cellular immunohistochemistry demonstrated robust expression of the epithelial marker E-cadherin and the broad tumor marker pan-CK, alongside positive expression for the mesenchymal markers Vimentin and Snail, reflecting an active epithelial-mesenchymal plasticity. Colony formation assays demonstrated that PSC-01 cells generated cohesive multicellular colonies within 14 days of plating at densities ranging from 500 to 2,000 cells per well, confirming cell-autonomous clonogenic potential.

The tumorigenicity of PSC-01 was corroborated in vivo through subcutaneous injection of 5 million cells into both immunodeficient BALB/c nude mice and immunocompetent C57BL/6 mice. The implanted cells rapidly initiated tumor formation within two weeks, producing expanding palpable masses that reached approximately one cubic centimeter within one month to six weeks. Histopathological evaluation of the excised allografts via hematoxylin and eosin staining demonstrated abundant cytoplasm, enlarged pleomorphic nuclei, prominent nucleoli, and poor differentiation, matching the microscopic architecture of the original parental uterine adenocarcinoma. Notably, while GEMM primary tumors demonstrated elevated CD8-positive cell infiltration relative to normal uterine tissue, the subcutaneous PSC-01 allografts exhibited modest overall infiltration by macrophages and neutrophils, while consistently retaining complete loss of STK11 expression.

The researchers also established the pharmacological baseline of the PSC-01 line against common therapeutic compounds using in vitro Cell Counting Kit-8 cytotoxicity assays over 24- to 48-hour incubation windows. The cultured endometrial tumor cells demonstrated concentration-dependent sensitivity to standard clinical chemotherapeutic agents, yielding half-maximal inhibitory concentration values of 1,370 micromolar for carboplatin, 73.1 micromolar for cisplatin, 344.2 to 614.7 nanomolar for paclitaxel, and 62.8 micromolar for azithromycin. These measured drug responses provide quantitative reference points for testing future pharmacological compounds, combinatorial chemotherapy regimens, and novel molecular therapeutics.

In their concluding evaluation, the authors noted that while genetically engineered mouse models offer indispensable biological validity, their extensive breeding times and high logistical costs restrict high-throughput testing. The establishment of PSC-01 provides an accessible, immortalized, syngeneic cellular resource that couples the speed and consistency of in vitro screening with the ability to form histologically faithful tumors in mice harboring intact immune systems. Although further transcriptomic profiling will be required to clarify why genomic Stk11 mutations were not directly picked up despite complete deficiency of the STK11 protein, the PSC-01 platform represents a robust tool for investigating endometrial cancer pathogenesis and evaluating candidate therapies.

Global epidemiological patterns underscore the pressing clinical need for tractable endometrial cancer models. Approximately 417,000 new diagnoses and more than 90,000 disease-related deaths were documented worldwide by 2020, following an overall incidence surge of 132 percent across recent decades. Although surgical hysterectomy effectively manages most localized malignancies, outcomes for recurrent, advanced, and treatment-refractory endometrial tumors remain unfavorable. To confirm active malignancy within the genetically modified donor mice prior to cell isolation, the investigators conducted serum enzyme-linked immunosorbent assays targeting circulating cancer antigen 125. Serum collected from cohorts of six genetically engineered mice demonstrated significantly elevated CA125 concentrations compared to baseline measurements in control animals, providing serological confirmation of invasive adenocarcinoma burden alongside histopathological evidence.

The laboratory protocol incorporated strict isolation, cytogenetic, and morphological benchmarks to authenticate the epithelial culture. Primary feeder fibroblasts were separated by brief exposure to 0.25 percent trypsin before the epithelial cells underwent enzymatic dissociation with 0.05 percent trypsin and subsequent staining with phycoerythrin-conjugated anti-CD326 for flow cytometric validation. For chromosomal analysis, logarithmic-phase PSC-01 cells were treated with colcemid, swelled in hypotonic potassium chloride solution, and fixed in an acetic acid and methanol mixture prior to Giemsa banding. Cellular proliferation kinetics were calculated from seven-day seeding assays using mathematical logarithmic doubling-time equations, demonstrating consistent growth across passage 20 and passage 40 without evidence of replicative arrest during senescence-associated beta-galactosidase staining. Two independent pathologists blinded to experimental allocations confirmed the cellular atypia and neoplastic morphology, supporting the formal archival deposit of the verified PSC-01 line in the China Typical Culture Biological Conservation Center.

Complementary in vivo transplantation trials also defined the relationship between inoculated cell numbers and tumor growth dynamics. When researchers varied the quantity of PSC-01 cells injected subcutaneously into mouse scapular regions, reductions in transplanted cell numbers correlated with extended latency windows and smaller resulting tumor volumes. Caliper measurements taken every two days tracked volumetric expansion using standardized dimensional formulas based on length and width squared. By establishing these kinetic parameters, the authors highlighted practical advantages over maintaining breeding colonies of genetically modified mice, including lower financial overhead, accelerated tumor development, reproducible outputs, and the capacity for real-time tracking of dynamic tumor progression in preclinical therapy evaluations.

Subject of Research: Generation and characterization of a stable mouse endometrial cancer cell line via conditional reprogramming

Article Title: Generation of a stable mouse endometrial tumor-derived cell line using conditional reprogramming

Article References: Zhang, T. A., Zhao, R., Zhang, Y., & Wang, H. (2026). Generation of a stable mouse endometrial tumor-derived cell line using conditional reprogramming. Biological Research, 59(1), Article 47. https://doi.org/10.1186/s40659-026-00706-w

Image Credits: AI Generated

DOI: 10.1186/s40659-026-00706-w

Keywords: endometrial cancer, conditional reprogramming, cell line establishment, PTEN, STK11, genetically engineered mice, PSC-01, ROCK inhibitor, in vivo tumorigenicity, cancer models, chemotherapy sensitivity, whole-exome sequencing, allograft model, epithelial cell culture, gynecological oncology