Fig 1.
(A) Example of a uterus used in the study, photographed from i) anterior, ii) posterior, and iii) open anterior view. Scale bars represent 5 cm. (B) Cell culture methodology. i) Explant tissue size. ii) Example of plate containing 2x explants per well. Black arrows point to individual explants sitting on top of a gelatin sponge. iii) Schematic of cell culture technique.
Fig 2.
Representative images from Patient 1 of H&E and IHC from Day 0 to Day 21 in ex vivo culture.
H&E and IHC for PR expression, proliferation (Ki67, PHH3) and apoptosis (CC3, TUNEL). In the TUNEL row, blue staining reflects DAPI indicating intact DNA and green reflects TUNEL, indicating apoptosis. H&E, haematoxylin and eosin; IHC, immunohistochemistry; PR, Progesterone Receptor; PHH3, Phosphohistone H3; CC3, Cleaved Caspase 3; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labelling; DAPI, 4′,6-diamidino-2-phenylindole.
Fig 3.
Representative images from three patients (P5, P6, P7) on untreated explants after 21 days in ex vivo culture with the addition of BrdU (BrdU was added to culture medium 24 h before fixing explants). H&E, Haematoxylin and Eosin; BrdU, Bromodeoxyuridine / 5-bromo-2’-deoxyuridine.
Table 1.
Patient characteristics.
Fig 4.
Variability in viability of the tumour tissue within explant replicates after 21 days in ex vivo culture.
Explants from six patients were treated with LNG for 21 days in the ex vivo culture model. A histopathologist assessed the viability of the tumour tissue within each explant based on H&E stains. Patients 2 (P2) and 4 (P4) were excluded from downstream analysis due to low viability of 4 μg/mL LNG explants, and Patient 3 (P3) was excluded due to having a small epithelial tumour compartment. Red circles indicate the explants selected for downstream assessment. Explants were selected based on high viability and large epithelial tumour compartment. Neg, negative control (culture media alone); Vhc, vehicle control (culture media plus 0.08% DMSO); LNG, levonorgestrel (4 ng/mL or 4 μg/mL).
Fig 5.
Assessment of apoptosis in LNG quasi-resistant explants.
Visualised using TUNEL, in endometrial cancer explants after 21 days in ex vivo culture. (A) Representative images of TUNEL. Explants from Patient 5 which were treated with either vehicle control (culture media plus 0.08% DMSO) or 4 μg/mL LNG for 21 days and stained with TUNEL (green) and DAPI (blue). (B) TUNEL positive cells as a percentage of all cells from explants treated with either culture media (Negative), DMSO (Vehicle), 4 ng/mL LNG or 4 μg/mL LNG. Plot shows mean and standard error of the mean (SEM) bars. A mixed effects analysis showed no significant difference in TUNEL positivity between treatment groups. LNG, Levonorgestrel; H&E, Haematoxylin and Eosin; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labelling; DAPI, 4′,6-diamidino-2-phenylindole.
Fig 6.
Candidate biomarker immunohistochemical analysis.
Day 0 tumour tissue samples were taken on the day of surgery and tumour explants were fixed after 21 days of culture in culture media alone (Negative), culture media plus 0.08% DMSO (Vehicle), or LNG (4 ng/mL, 4 μg/mL). IHC stains were scored by three independent authors (H.V, C.H, D.K) on a four-point scale of 0–3 based on the proportion of positive tumour cells within the explant and the staining intensity. (A) Representative images of the 0–3 point scale for all markers. (B) Heatmap of the median stain score for each biomarker against treatment for three patients. LNG, Levonorgestrel; IHC, Immunohistochemistry.