Ir053

Tissue slices were stained with hematoxilyn and eosin (HE) and tumor tissue vitality was confirmed. Slices with necrosis >50% were excluded from further immunohistochemical stainings. Slices were stained with antibodies against Ki67 (MIB-1, Dako, Glostrup, Denmark), PD-L1 (ab213524, Abcam, Cambridge, UK) or double stained using the Envision G/2 Doublestain System or Envision Flex Doublestain System (Dako). The antibody combinations were CD3 (IR503, Dako) + Ki67 (MIB-1, Dako), CD3 (IR503, Dako) + CK AE1/3 (IR053, Dako), CD8 (IR623, Dako) + Ki67 (MIB-1, Dako), PD1 (ab52587, Abcam) + Ki67 (MIB-1, Dako), and PD1 (ab52587, Abcam) + CK AE1/3 (IR053, Dako). All slides were stained with automatized immunostainers (Autostainer Plus, Dako).

The absence of bone metastasis was confirmed by X-ray and bone scintigraphy. Neoplastic cells infiltration in BM aspirates was ruled out by immunocytochemistry staining (Universal LSAB System, K0673, Dako) using monoclonal antibodies (Abs) against epithelial membrane antigen (IS629, Dako) and cytokeratin AE1–AE3 (IR053, Dako). Cell morphology analysis was performed by the Pappenheim technique. Patient´s BM were considered positive for metastasis if cells expressed epithelial membrane antigen and cytokeratins AE1–AE3 and were morphologically malignant.

Primary endometrial epithelial cells and stromal cells were seeded onto 48-well plates and grown until 90% confluency. The cells were washed with 1X PBS before being fixed with 10% neutral formalin for 20 min at room temperature. Cells were permeated with 0.1% Triton for 15 min and non-specific binding sites were blocked with 10% normal goat serum for 1 h. After washing twice with PBST, cells were incubated with primary antibodies against cytokeratin (AE1/AE3, 1:100, IR053, DAKO, Glostrup, Denmark) and vimentin (1:100, M0725, DAKO, Glostrup, Denmark) in the blocking medium at 4 °C overnight. The cells were washed with PBST four times before incubating with a secondary goat anti-mouse antibody with Alexa Fluor 488 (1:500, Abcam, MA, USA) in a blocking solution at room temperature for 1 h. After washing, the nuclei were stained with DAPI (20 µg/mL, Invitrogen, Carlsbad, CA, USA) in PBST for 3 min. Fluorescence images were captured under a fluorescence microscope (Nikon, Tokyo, Japan).