Establishment of a novel model of endometriosis-associated ovarian cancer by transplanting uterine tissue from Arid1a/Pten knockout mice – Scientific Reports

Mouse strains including transgenic mice

C57BL/6 mice were purchased from The Jackson Laboratory Japan (Yokohama, Japan). All of the genetically engineered mouse strains used in the present study were previously established by and obtained from Johns Hopkins University²⁵, and were kindly gifted to our laboratory at Shinshu University (see Supplementary Methods in Supplementary materials). The KO of Pten and/or Arid1a in Pax8-positive cells, including endometrial epithelial cells, may be induced in these transgenic mice by the administration of DOX through the Tet-on and Cre-loxP systems. Briefly, these mice have the reverse tetracycline transactivator (rtTA) gene after the promoter sequence of Pax8, a Müllerian duct epithelial marker including an endometrial epithelium, and the Cre recombinase (Cre) gene downstream of a tetracycline response element (TRE). Therefore, Pax8-positive cells express rtTA, which binds to TRE in the presence of DOX to induce the expression of Cre. Cre cuts and removes the target “floxed” gene region flanked by loxP sequences. Among transgenic mouse strains, the “control” has wild-type, not floxed Pten and Arid1a (Pten^WT/WT, Arid1a^WT/WT). “iAD (inducible Arid1a deletion)” has the Arid1a gene floxed in both alleles (Pten^WT/WT, Arid1a^flox/flox). “iPD (inducible Pten deletion)” has the Pten gene floxed in both alleles (Pten^flox/flox, Arid1a^WT/WT). “iPAD (inducible Pten and Arid1a deletion)” has the Pten and Arid1a genes floxed in both alleles (Pten^flox/flox, Arid1a^flox/flox).

Creating peritoneal endometriosis

To create endometriotic lesions in the mouse abdominal cavity, we modified the method devised by Vernon and Wilson²¹. Ten-week-old donor mice were euthanized and their uteri were immediately removed. Small uterine pieces were prepared from the removed uteri using a 5-mm Derma punch^® (Maruho, Osaka, Japan) and surgically transplanted by silk suturing onto the peritoneum of 10-week-old recipient mice. Two weeks after transplantation, recipient mice were euthanized and their intraperitoneal lesions were observed and removed for a histological examination.

We performed all transplantation surgeries on recipient mice under appropriate anesthesia with isoflurane. We euthanized experimental mice by cervical dislocation under isoflurane anesthesia.

Induction of Arid1a and Pten KO in peritoneal endometriosis

We used the 10-week-old transgenic mouse strains, including Control, iAD, iPD, and iPAD, as donors to create carcinoma in endometriotic cysts. Uterine pieces were transplanted into the peritoneum of recipient Control mice using the same method described above for the induction of peritoneal endometriosis. Two weeks after transplantation, we started the oral administration of 6 mg/body/day DOX, which was continued for 4 weeks to induce the KO of Arid1a and Pten in Pax8-positive cells derived from donors. Recipient mice were euthanized and their intraperitoneal lesions were removed for a histological examination.

Induction of Arid1a and Pten KO in ovarian endometriosis

To create ovarian endometriosis, we transplanted small uterine pieces from donors onto the ovarian surface of recipients with silk suturing after removal of the ovarian bursa. We used 10-week-old Control, iAD, iPD, and iPAD mice as donors for uterine transplantation. Two weeks after transplantation, we started the oral administration of 6 mg/body/day DOX, which was continued for 4 weeks, to induce the KO of Arid1a and Pten in Pax8-positive cells derived from the donors. Recipient mice were euthanized, and their ovaries were removed for a histological examination.

Pathological diagnosis and immunohistochemistry (IHC)

Removed tissues were processed to 4-µm-thick formalin-fixed and paraffin-embedded sections and utilized for hematoxylin and eosin staining or IHC for a histological diagnosis or the evaluation of KO, respectively. An ectopic endometrium (endometriosis) may be identified as Pax8-positive glandular epithelial cells in the cystic lesions that formed at the transplantation site of the donor’s uterine fragment. We diagnosed the histology of the mouse ectopic endometrium based on that of humans. Endometriosis without atypia was defined as a single layer of epithelial cells without stratification, a papillary structure, or nuclear atypia. On the other hand, epithelial cells in “atypical endometriosis” are characterized by various degrees of stratification, papillary changes, and nuclear atypia³³. When ectopic endometrioid tissue shows a cribriform pattern or stromal invasion with prominent nuclear atypia, we diagnosed it as carcinoma.

We performed IHC as previously described^25,34. Briefly, indirect immunostaining for Pax8, Arid1a, and Pten was conducted using an anti-Pax8 antibody (rabbit polyclonal, 1:1000 dilution, Thermo Fisher Scientific, Waltham, MA), anti-Arid1a antibody (rabbit polyclonal, 1:800 dilution, Sigma-Aldrich, Saint Louis, MO), and anti-Pten antibody (rabbit monoclonal, 1:100 dilution, Cell Signaling Technology, Danvers, MA), respectively, as primary antibodies. Tissue sections were deparaffinized in Hemo-De (FALMA, Tokyo, Japan) and rehydrated in a graded alcohol series. Antigens were retrieved by a microwave pretreatment in 0.01 M citrate buffer (pH 6.0) for 30 min. Sections were then treated with 0.3% hydrogen peroxide for 15 min to block endogenous peroxidase activity and incubated with the primary antibody at 4 °C overnight. After washing with phosphate-buffered saline, sections were incubated with a horseradish peroxidase-conjugated secondary antibody using Histofine MAX-PO (Nichirei, Tokyo, Japan) at room temperature for 60 min and stained with diaminobenzidine in 0.15% hydrogen peroxide. Counterstaining with hematoxylin was then performed.

Ethics approval

In compliance with national regulations and guidelines, all mouse experiments were reviewed and approved by the Shinshu University Animal Experiment Committee (Approval No. 021094) and Shinshu University Genetic Recombination Experiment Committee (Approval No. 21-043) and were conducted in accordance with institutional guidelines and the Regulations for Shinshu University Animal Experimentation and Safety Management Regulations for Genetic Recombination Experiments at Shinshu University. The authors confirm that all animal experiments in the present study have been performed in accordance with ARRIVE guidelines.