Figure 1.
Transplanted bMECs generate developing spherical outgrowths in the mouse mammary fat pad with location-dependent morphology.
A: Timeline of basic transplantation procedure. B–D: Carmine-stained wholemounts depicting typical outgrowth development at, respectively, 3, 6 and 8.5 weeks after cell transplantation. Bar = 1 mm. E: Carmine-stained wholemount of biopsy from the parenchymatic region of the heifer’s mammary gland. Bar = 1 mm. F: H&E-stained paraffin section from a Carmine-stained wholemount of 6-week-developed outgrowth. Bar = 50 µm. G: H&E-stained paraffin section from the parenchymatic region of a heifer’s mammary gland. Bar = 25 µm. H: H&E-stained paraffin section from a region containing outgrowths. A fibrous stromal area (dashed red line) encloses a solid, multilayered spherical outgrowth (red arrowhead) and partially encloses a hollow single-layered outgrowth (black arrowhead). Arrows indicate disorganized epithelial cells embedded in the adipose stroma. Bar = 100 µm.
Figure 2.
Bovine mammary outgrowths developed in the mouse, resemble the immunohistochemical characteristics of the donor tissue.
Immunohistochemical analyses of selected markers on paraffin sections from bovine outgrowths (left panels) and mammary epithelium from heifer’s mammary gland (right panels). The outgrowths and the heifer’s tissue were subjected to similar histological procedures, including Carmine staining, prior to immunohistochemistry and hematoxylin counterstaining. Bar = 50 µm.
Figure 3.
A TDLU-like structure develops from transplanted bovine cells in mice treated with estrogen and progesterone.
A: Timeline of hormone treatment and bMEC transplantation. Hormone pellets were inserted 2 days prior to bMEC transplantation. B, C: Carmine-stained endogenous mammary epithelium from recipient mice carrying placebo (B) or hormone pellets (C). Enhanced branching of the endogenous ductal network is noted in (C). D, E: Carmine-stained wholemount (D) and H&E-stained paraffin section (E) depicting typical morphology of bovine outgrowth in the cleared mammary fat pad of hormone-treated mice. F–I: Carmine staining of mammary wholemounts (F, G) and H&E staining of paraffin sections (H, I), depicting a particular case of an outgrowth exhibiting significant growth and TDLU-like morphology under continuous systemic treatment of estrogen and progesterone. J: Frequency and morphology of outgrowths developed from transplanted bMECs in the cleared mammary fat pad of recipient mice that received, or did not receive, hormone treatment.
Figure 4.
Co-transplantation of mouse and bovine epithelial cells rarely produces a chimeric outgrowth in the mouse.
A: Frequency of outgrowths with mouse and bovine characteristics developed in the cleared mouse mammary fat pad. Outgrowths with ductal elongations and outgrowths with detectable DNA levels of the bovine-specific BLG sequences were monitored in fat pads transplanted with a range of bovine-to-mouse cell ratios. B, C: Low and high magnification, respectively, of Carmine-stained outgrowths developed from mouse and bovine MECs, co-transplanted at a ratio of 10:10. Region of spherical outgrowths is outlined in dashed line. Ductal protrusion is marked with arrowhead. Bar = 1 mm. D: Number of BLG-positive outgrowths developed in distinctly spherical and ductal outgrowth regions (n = 14 for each group).
Figure 5.
Differential effects for pre- and co-transplantation of fibroblasts on bMEC intake.
A: Timeline of 10T1/2 fibroblast pre- and co-transplantation with bMECs. B, C: Representative Sirius red-stained tissue sections from cleared mammary fat pads, 2 weeks after fibroblast transplantation (B), or without intervention (C). D: The stained fibrotic area is significantly larger in fat pads transplanted with fibroblasts than in intact controls (p = 0.02, n = 5, error bars represent SEM). Percentage of red pixels was calculated using the Adobe Photoshop software. E: Effects of pre- and co-transplantation of fibroblasts on outgrowth frequency. F: Representative Carmine-stained wholemount of bovine outgrowth that developed within the cleared mammary fat pad 2 weeks after fibroblast transplantation. Bar = 1 mm.
Figure 6.
Growth of bovine epithelial mammospheres is suppressed by adipose-conditioned medium and rescued by FGF supplementation.
A: Mammospheres formed by bMECs cultured for 3 days within Matrigel are smaller when supplemented with adipose-conditioned medium as compared to bMEC-conditioned or fresh medium. Columns represent average size ± SEM of 28 mammospheres measured in each group. Results of one representative experiment out of three biological replicates are presented. Inset: ammonia levels accumulated in adipose-conditioned, bMEC-conditioned and fresh media. Columns represent average size ± SEM from four analyses of each medium. **Statistically significant difference (p ≤ 0.01) compared to other columns. B: Light microscopy demonstrating the effect of the culture medium on the size of the developing mammospheres. Bar = 50 µm. C: The effect of the culture medium (fold change) on the size of 3-day-developed mammospheres. Columns represent average size ± SEM of 60 mammospheres measured in each group. Different letters above the columns represent statistically significant differences (p ≤ 0.05). D: Immunofluorescent staining of PCNA in representative mammospheres formed by bMECs in fresh and conditioned media. PCNA-positive cells are encircled by a white dotted line. Bar = 50 µm. E: Light microscopy demonstrating no effect of adipose-conditioned medium on the size of mammospheres formed by mMECs. Absence of fibroblasts in cultures supplemented with adipose-conditioned medium is noted. Bar = 100 µm. F: FGF7 and FGF10 supplementation to the adipose-conditioned medium induces mammosphere size increase in a dose-dependent manner. Columns represent average size ± SEM of 60 mammospheres measured in each group. Different letters above the columns represent statistically significant differences (p ≤ 0.05).
Figure 7.
Adipose-conditioned medium suppresses the development of human breast mammospheres in culture.
A: Average size of mammospheres formed by hMECs from the breast tissue of two donors after 3 days of culture in fresh or adipocyte-conditioned medium. Columns represent average size ± SEM of 60 mammospheres measured in each group. ***Statistically significant difference (p ≤ 0.001). B: Light microscopy of representative mammospheres formed by hMECs in 3D Matrigel culture with fresh or adipose-conditioned medium. Bar = 100 µm.