All animals were held in the Monash Medical Centre Animal Facility and housed on a 12-hour light/day cycle with access to normal chow and water ab libitum. Approval for all procedures described was obtained from Monash Medical Centre Animal Ethics Committee A.
Mouse model of endometriosis
C57BL/6J background mice 8-12 weeks age underwent the mouse model of endometriosis as published previously. Briefly, donor mice were ovariectomised, then given sub-cutaneous injections of β-estradiol (1µg/ml) on days 7-9. A progesterone secreting pellet was placed sub-cutaneously from days 13-19 (500ng/day) in combination with β-estradiol sub-cutaneous injections from days 13-15 (50ng/ml). On day 15 the endometrium was artificial decidualised by injecting 20µl of oil into the uterine lumen. Progesterone support was removed on day 19 and 4 hours later the menstrual-like endometrium was removed from the outer myometrium, minced into fragments approximately 1mm3 and injected into ovariectomised estrogen primed (β-estradiol secreting pellet, 100ng/day days 7-19) recipients (approximately 20 fragments, 200mg of tissue in 200µl of PBS). Lesions were allowed to develop over 21 days (days 19-40) before collection on day 40. During this 21-day period mice were randomly allocated to one of two groups and treated twice weekly with either 250µg of anti-SHH antibody 5E1 (n=15, DSHB) or an isotype matched control antibody (n=18, BioXCell, MOPC-21 IgG1) in 200µl of sterile PBS. The 5E1 dose administered was based on previous reports that used this antibody to block hedgehog signalling in mouse in vivo tumour models.
Lesion collection and fixation
Recipient mice were euthanized using rising concentrations of carbon dioxide and cervical dislocation. Upon dissection all body cavities were photographed, the number of lesions found recorded and their size measured. Lesions were carefully removed from peritoneal organs and immersed in 4% w/v paraformaldehyde in PBS overnight at 4°C and cryoprotected in 30% w/v sucrose in PBS overnight at 4°C. Tissues were frozen in optimal cutting temperature medium and cryo-sectioned at 8µm thick.
Unless otherwise stated all sections underwent the following staining protocol. Sections were permeabilised in 0.2% Triton X-100 in PBS for 15 minutes, blocked in DAKO blocking solution for 1 hour and then stained for hedgehog activation, (GLI1 Rabbit monoclonal Thermo MA5-32553 10µg/ml), epithelial (EpCAM-PE rat anti mouse eBioscience 12-5791-81 2µg/ml), proliferation (Ki67-EF450 rat anti mouse eBioscience 50-5698-80 2µg/ml) and apoptosis (Caspase 3 Rabbit polyclonal R&D AF835 5µg/ml) markers for 1 hour at room temperature in 1% bovine serum albumin in PBS. For unconjugated primary antibodies, sections were incubated with secondary antibodies for 1 hour at room temperature (Donkey anti rabbit Alexafluor 568 LifeTech A10042 4µg/ml). Nuclei were counterstained with 5µg/ml Hoescht 33258 in PBS for 3 mins. Images were captured on an Olympus FV1200 confocal microscope using a 20x objective lens and adjusted for brightness and contrast in a linear manner using FIJI software.
Analysis of lesions
Haematoxylin and eosin staining was performed as per standard protocols on the 15th slide of each lesion (2 sections/slide x 8µm x 15 slides= 240µm deep). Lesion cross-sectional area was measured using FIIJ, using the trace outline and measure area tools.
Prior to the analysis of epithelial, proliferation and apoptosis markers, slides were blinded to reduce any bias during analysis. Markers were counted manually in FIJI. Total cell nuclei were calculated by counting the number of nuclei using the “threshold”, “watershed” and “analyse particles” functions. A minimum of 6 lesions were analysed per treatment group. A minimum of 3 fields of view per section were imaged and the average percentage of positive cell types in the total cell population was calculated for each lesion. Results were then unblinded and each lesion was then plotted as a single data point.
Group sizes were determined using G*Power software. For lesion number, we assumed an observed effect size of 0.5, alpha of 0.05 and Power of 0.8, the total sample size required was 26. For changes in lesion characteristics we assumed an effect size of 0.65, alpha of 0.05 and Power of 0.8, the total sample size required was 13.
All statistical analyses were performed in Graphpad Prism 8.0. Raw data was subjected to D’Agostino-Pearson normality testing prior to statistical analyses. Non-parametric analysis was performed when one of the sets of data failed to pass normality testing. All lesion analyses were subjected to unpaired, two-tailed Mann-Whitney testing. Significance was accepted where P≤0.05 and data represented graphically as individual data points and median.