Contraceptive progestins with androgenic properties stimulate breast epithelial cell proliferation (2021)
Figure 5

Figure 5- Effects of PR agonists on human and mouse epithelial cells growth.

A-C Line charts showing in vivo growth of HBECs as measured by radiance after implantation of either control or different progestogen-containing pellets. Points show means of radiance in individual glands ± SEM. Statistical significance was tested by fitting a mixed effect linear model with random effects to the log10-transformed data; n=87 and 64 respectively for control and progesterone, n=34 respectively for both control and DSG, n=59 and 39 respectively for control and GSN, n=70 and 79 respectively for control and LNG, n=33 and 20 respectively for control and CMA, n=28 and 30 respectively for both control and CPA, n=48 and 35 respectively for control and DSP and shown by asterisks next to legend . To test differences at endpoint Wilcoxon rank-sum test was applied on log-transformed fold change values, asterisks at endpoints of graphs.

D Graph showing in vivo growth of HBECs from mammoplasty M354 as measured by radiance upon either control (n=9) or LNG (n=8) treatment. Points show means of radiance in individual glands ± SEM. Statistical significance of difference at endpoint was tested by Student's t-test.

E Representative micrographs showing co-IF with anti-KI67 (red), and anti-hECAD (green) of xenografted milk ducts after 21 days treatment with vehicle or LNG. Scale bar, 50 μm.

F Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ cells, dots represent individual sectors counted in 3 different glands, control (n=30) or LNG (n=28), median (red), statistical significance was assessed by non-parametric Mann-Whitney test.

G Bar plot showing relative transcript levels of MKI67 in xenografted glands from control (n=6) and LNG-treated (n=3) mice, Student's t-test.

H Graphs showing threshold cycle number of KLK3 plotted over housekeeping genes, semiquantitative RT-PCR performed with human-specific primers on RNA extracted from control (n=6) and LNG-treated (n=3) xenografted glands.

I Representative micrographs showing co-IF with anti-KI67 (red) and anti-hECAD (green) of histological sections from glands xenografted with HBECs from mammoplasty M310 exposed to vehicle or progestins. Scale bar, 50 μm.

J Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ cells in M310 derived HBECs, dots represent individual sectors counted in different glands, median (red), CTRL (n=180), P4 (n=134), DSG (n=52), GSN (n=161), LNG (n=32), CMA (n=56), CPA (n=42), DSP (n=100). Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using the CTRL group as reference.

K Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ HBECs derived from 11 different patients upon 21 days of CTRL (n=360), P4 (n=180), DSG (n=147), GSN (n=298), LNG (n=284), CMA (n=115), CPA (n=156), DSP (n=123) treatment. Red lines show medians.

L Violin plot showing percentage of AR+ intraductally-engrafted HBECs derived from 11 different patients upon 21 days of CTRL (n=258), P4 (n=97), DSG (n=173), GSN (n=159), LNG (n=168), CMA (n=68), CPA (n=76), DSP (n=135) pellets. Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions, with batches and patients as random variables and CTRL as reference, median in red. J-L dashed line shows 0%.

M Violin plot showing cycle threshold (CT) of KLK3 transcripts upon progestin treatment, as assessed by qRT-PCR, in CTRL (n=13), P4 (n=10), DSG (n=7), GSN (n=8), LNG (n=15), CMA (n=12), CPA (n=12), DSP (n=12) conditions. Statistical analysis performed by non-parametric Kruskal-Wallis test followed by Dunn's test with CTRL as reference. Red dashed line corresponds to CT40 considered detection limit.

Data information: * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 n.s. not significant.