Fig 2: (A) Mesothelial displacement assays. Ovarian cancer spheroids were generated from wild-type (WT), KRT14KO and KRT14OE lines and overlaid onto a confluent layer of mesothelial cells and imaged by light microscope. Representative images following overnight and 48-h co-culture are presented where dotted lines indicate spheroid outgrowth and mesothelial displacement. (B,C) Embedding and outgrowth assays. OVCAR4 ovarian cancer spheroids were formed and overlaid into (B) Collagen I (24 h) or (C) Matrigel (48 h) samples, and subsequently fixed and stained for KRT14 expression. (D) Wound-healing assays OVCAR4 cells were grown as a monolayer, cells were wounded, fixed, and stained for KRT14 expression at time-points preceding total wound closure. Images from a representative well are shown with scale bars = 100 µm.

Fig 3: (A) Representative qRT-PCR of KRT14 expression in LP9 cells, whole normal ovary, benign fibroma, primary ovarian tumour, or ascites-derived cells; and migratory leader cell populations (n = 3 separate patient isolations where individual data points represent individual patient samples). (B) KRT14 expression in cultured cells by immunofluorescence. KRT14 expression was detected as a granular cytoplasmic stain expressed by a small proportion of ovarian cancer cells in the OVCAR4 line cultured as a two-dimensional (2D) monolayer. Scale bar = 100 µm. (C) KRT14 expression was detected at the periphery of OVCAR4 ovarian cancer cells cultured as three-dimensional (3D) spheroids with no signal detected in the core of the spheroid. Scale bar = 100 µm. (D) Localisation of KRT14 at the periphery of ovarian cancer spheroids by comparison to the intracellular staining observed for N-cadherin. Scale bar = 100 µm. (E) Vector control (NT), KRT14KO, and KRT14OE cells were suspended in SFM/0.25% methylcellulose in U-bottom 96-well plates and imaged at regular intervals (0–48 h) to observe spheroid aggregation. Images are representative of multiple wells observed at the 13-hour and 48-hour time points. Scale bar = 100 µm. (F) Spheroid attachment to the target mesothelium. Vector control (NT) and KRT14KO spheroids were co-cultured with the target mesothelium, and attachment was measured 6 h post-addition. The results presented are from one representative assay where data points are the attachment counts of three individual wells per cell line.

Fig 4: (A) Parallel endpoint Boyden chamber assays. Boyden chamber assays using labelled mesothelial cells overlaid with individual patient-derived ovarian cancer spheroids; we observe no invasion of the mesothelial cells at MALDI imaging collection points; n = 3 wells/sample of one representative experiment. (B) Haemotoxylin and Eosin (H&E) staining of the invasive interface. H&E staining identifying the invading interface of ovarian cancer spheroid mesothelial co-cultures and the interface used for MALDI imaging mass spectrometry (IMS). (C) MALDI IMS of the invading interface. MALDI IMS identifies: CDCA8, HNRN, keratin-14 (KRT14) and FNDC3B expressed at the invading interface of ovarian/mesothelial co-cultures. (D) Representative qRT-PCR of MALDI identified candidates using fresh-frozen confirmed primary high-grade serous ovarian tumours or normal whole ovary (n = 3/group) where individual data points represent individual patient samples. (E) Representative IHC of individual MALDI identified candidates in HGSC primary ovarian samples.

Fig 5: Genetic ablation of keratinocyte-specific Cyp11b1 abrogates de novo GC synthesis in the skin.(A) Scheme of Cre/LoxP strategy for Cyp11b1 exons 3 to 5 excision. (B) Generation of mice with inducible Cyp11b1 deletion in keratinocytes (K14-CreERTamCyp11b1L2/L2) by breeding Cyp11b1L2/L2 mice with K14-CreERTam mice. (C) Experimental protocol for in vivo Cyp11b1 deletion in the skin. (D) Agarose gel electrophoresis of the PCR analysis of genomic Cyp11b1 excision from dorsal skin of control (L2/L2) and KO animals. Deletion fragment (349 base pairs) indicates successful Cyp11b1 in vivo deletion. bp, base pairs. (E) Cyp11b1 and Hsd11b1 expression in ear skin. Expression was normalized to Actb. Data are presented as 2(-?Ct) and shown as fold change over L2/L2 controls. Dots represent individual animals (n = 10 to 13 per group), pooled from three independent experiments. (F and G) Corticosterone radioimmunoassay of blood serum (F) and supernatant of untreated (G, left) or adrenocorticotropin (ACTH), forskolin, or pregnenolone-treated dorsal skin ex vivo cultures (G, right). Dots represent individual animals (n = 6 to 12 per group), pooled from three (F), four (G, left), or two (G, right) independent experiments. (H) GRE luciferase (Luc) reporter assay with HEK 293T cells using supernatant of untreated or metyrapone-treated ex vivo skin culture from L2/L2 or KO animals. Empty luciferase vector–transfected cells served as controls. Normalized relative luciferase activity was depicted as fold change over the mean of untreated L2/L2 controls (dashed line). Paired dots represent skin biopsies from one individual animal (n = 10 to 13 per group). Data are pooled from two to three independent experiments. Box plots (E to G) show the 25th to 75th percentiles with whiskers indicating minimum to maximum values. Statistical significance was determined using unpaired two-tailed t test (F), two-tailed Mann-Whitney test (E and G, left) and ordinary two-way ANOVA with Sidak’s multiple comparisons test (G, right and H). (A) and (B) were created with biorender.com.