Fig 1.
Fibronectin expression is upregulated in cav-1 -/- mammary glands.
(A) Immunohistochemistry of fibronectin expression in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results demonstrated an increased abundance fibronectin fibers surrounding the ducts and in the adipose stroma of cav-1 -/- glands. Fibronectin expression was also localized to the epithelial cells (red arrows) of the ducts in both cav-1 +/+ and cav-1 -/- glands. (B) The percent expression of fibronectin was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. (C) The intensity of fibronectin staining was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. Scale bars are 50 μM and 20 μM in low and high magnification images, respectively. *p≤0.05. Fn: fibronectin. Results are reported as the mean ± the SEM.
Fig 2.
Tenascin-C expression is upregulated in cav-1 -/- mammary glands.
(A) Immunohistochemistry of tenascin-C expression in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results demonstrated an increased expression of tenascin-C in the peri-ductal and adipose stroma in cav-1 -/- glands. This is in contrast to cav-1 +/+ glands where tenascin-C expression is expressed in the peri-ductal stroma and to a lesser extent in the adipose stroma. Tenascin-C was also expressed in the stromal cells in cav- 1-/- and cav-1 +/+ glands (red arrows), albeit with a greater proportion of positive cells in cav-1 -/- glands. (B) The percent expression of tenascin-C was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. (C) The intensity of tenascin-C staining was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. Scale bars are 50 μM and 20 μM in low and high magnification images, respectively. *p≤0.05, **p≤0.01. Tn-C: tenascin-C. Results are reported as the mean ± the SEM.
Fig 3.
Collagen I expression and organization are upregulated in cav-1 -/- mammary glands.
(A) PSR staining for collagen I in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Imaging demonstrates extensive expression of collagen I in the adipose stroma and in the peri-ductal region of cav-1 -/- glands. The collagen I fibers appear dense and tightly packed in cav-1 -/- glands. In contrast, overall collagen I expression is considerably lower in cav-1 +/+ glands with fibers being predominantly located to the peri-ductal region. Here, fibers are less packed and distinct fibrils are apparent. (B) The percent expression of PSR stained collagen I was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. Scale bars are 50 μM and 20 μM in low and high magnification images, respectively. (C) The intensity of PSR stained collagen I was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. (D) Representative SHG images show an abundance of dense, thick collagen fibers in cav-1 -/- glands. Collagen fibers and fiber bundles also appear more organized and aligned (red arrows). Cav-1 +/+ glands express comparably less collagen which presents as thin, unorganized fibers. Scale bars are 25 μM. Col: collagen. *p≤0.05, **p≤0.01. Results are reported as the mean ± the SEM.
Fig 4.
αSMA expression is upregulated in cav-1 -/- mammary glands.
(A) Immunohistochemistry of αSMA in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results demonstrated that αSMA expressing cells are predominantly localized to the myoepithelial compartments in cav-1 +/+ and cav-1 -/- glands. αSMA expressing cells are also observed in adipose stromal cells (arrows) in cav-1 +/+ and cav-1 -/- glands (B) The percent expression of αSMA was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. (C) The intensity of αSMA staining was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. Scale bars are 50 μM and 20 μM in low and high magnification images, respectively. *p≤0.05. Results are reported as the mean ± the SEM.
Fig 5.
Microvessel density is similar in cav-1 -/- and cav-1 +/+ glands.
(A) Immunohistochemistry of CD31 expressing endothelial cells in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results demonstrated that vessels are apparent in both cav-1 +/+ and cav-1 -/- glands. (B) Quantification of microvessels show no differences in the abundance of vessels in cav-1 +/+ and cav-1 -/- glands. Scale bars are 50μM and 20μM for low and high magnification images, respectively. Results are reported as the mean ± the SD.
Fig 6.
Ductal architecture is disrupted in cav-1 -/- glands.
(A) The number of ducts were enumerated in αSMA stained sections from cav-1 -/- and cav-1 +/+ glands. Results demonstrated a highly significant average number of ducts per image in cav-1 -/- glands in comparison to cav-1 +/+ glands. (B) Images of fibronectin stained sections illustrate the abundance of large, elongated, branched ducts (red arrows) in cav-1 -/- glands. Ducts in cav-1 +/+ glands appear small and relatively round (red arrows). Scale bars are 100μM. (C) H&E stained images of glands demonstrate the presence of stacked ductal epithelial cells in cav-1 -/- sections, indicative of ductal hyperplasia. Cav-1 +/+ glands exhibit a continuous monolayer of ductal epithelial cells. Scale bars are 20μM. (D) The ductal circumference, measured as the region immediately outside the myoepithelial compartment, was analyzed in αSMA stained sections in cav-1 -/- and cav-1 +/+ glands. The ductal circumference was significantly greater in cav-1 -/- as opposed to cav-1 +/+ glands. (E) The area, measured as the region between the myoepithelial and luminal compartment, was analyzed in αSMA stained sections in cav-1 -/- and cav-1 +/+ glands. The ductal area was significantly greater in cav-1 -/- as opposed to cav-1 +/+ glands. (F) This image of an αSMA stained cav-1 +/+ duct illustrates the vectors used to assign stromal and lumen outlines used to analyze ductal measurements. The red line shows the stromal border while the blue line shows the lumen border. Scale bar is 50uM. *p≤0.05, **p≤0.01, ***p≤0.001. Results are reported as the mean ± the SEM.
Fig 7.
Proliferation of ductal epithelial cells is increased in cav-1 -/- glands.
(A) Immunohistochemistry of ki67 cells in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results from representative images demonstrated that ki67 expressing ductal epithelial cells (arrows) are markedly increased in cav-1 -/- glands in comparison to cav-1 +/+ glands. (B) The average number of ki67 positive cells was significantly higher in cav-1 -/- glands in comparison to cav-1 +/+ glands. Scale bars are 20uM. **p≤0.01. Results are reported as the mean ± the SD.