Paracrine-Rescued Lobulogenesis in Chimeric Outgrowths Comprising Progesterone-Receptor-Null Mammary Epithelium and Redirected Wild-Type Testicular Cells

We have previously shown that non-mammary and tumorigenic cells can respond to the signals of the mammary niche and alter their cell fate to that of mammary epithelial progenitor cells. Here we tested the hypothesis that paracrine signals from mammary epithelial cells expressing progesterone receptor (PR) are dispensable for redirection of testicular cells, and that re-directed wild-type testicular-derived mammary cells can rescue lobulogenesis of PR-null mammary epithelium by paracrine signaling during pregnancy. We injected PR-null epithelial cells mixed with testicular cells from wild-type adult male mice into cleared fat-pads of recipient mice. The testicular cells were redirected in vivo to mammary epithelial cell fate during regeneration of the mammary epithelium, and persisted in second-generation outgrowths. In the process, the redirected testicular cells rescued the developmentally deficient PR-null cells, signaling them through the paracrine factor RANKL to produce alveolar secretory structures during pregnancy. This is the first demonstration that paracrine signaling required for alveolar development is not required for cellular reprogramming in the mammary gland, and that reprogrammed testicular cells can provide paracrine signals to the surrounding mammary epithelium

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

<div><p>Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In <em>vivo</em>, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.</p> </div

PTN and ALK mRNA expression in mouse mammary glands during pregnancy.

<p>Mammary glands from mice that were virgin, pseudopregnant (PsPreg), at different days of pregnancy or after weaning ( = involution). Quantitative, real-time PCR was used. Pseudopregnants denotes 12 days since plug date. Pups were removed from the mother at 21 days of age and involution followed thereafter. Expression is given relative to keratin 18. Mean ± SE (n = 4; PsPreg n = 2). **P<0.01; ***P<0.001, by ANOVA.</p

PTN protein expression and function in mammary epithelial cells (MECs).

<p>(A) Immunofluorescence (IF) staining of MECs grown on poly-D-lysine coated slides. PTN (green), DAPI (blue), Phalloidin (red). Images are shown as merged images of anti-PTN with DAPI and anti-PTN with Phalloidin staining. Scale Bar  = 20 μm. (B, C) Real-time impedance sensing of MECs growth relative to stimulation time (20 hours) with anti-PTN blocking antibody (anti-PTN), recombinant PTN (PTN), or antibody elution buffer (Control) (B), bFGF or resuspension buffer (C). (D) Real-time impedance sensing of MECs migration relative to 30 hours from plating and four hours from scratching. MECs were treated with anti-PTN blocking antibody (anti-PTN), recombinant PTN (PTN), alone or in combination or antibody elution buffer (Control). (E) Real-time impedance sensing of MECs invasion relative to 30 minutes from plating. MECs were treated with anti-PTN blocking antibody (anti-PTN), or antibody elution buffer (Control) (E), recombinant PTN (PTN) or PTN resuspension buffer (Control) (F). The data are representative of at least three independent experiments done in duplicates. ***P<0.001 versus control by ANOVA.</p

PTN effect on MAP kinase pathway activity in developing mammary glands.

<p>Phospho-ERK1/2 (A), phospho-p38 (B), and phospho-JNK (C) in whole mammary gland extracts from anti-PTN treated mice (n = 10) versus controls (n = 9). The ratio of phospho- to total protein was obtained from multiplex assays. Ns, not significant; *P<0.05. (D, E) Western blot analysis for pERK1/2. Representative Western blots (mouse m1 to m6) and quantitation of phospho ERK1/2 relative to total ERK1/2 in 3 independent experiments (n = 9 to 10 per group). (F) Representative Western blots of whole cell lysates from MECs treated for 24 hours with either UO126 (500 nM) alone or in combination with anti-PTN blocking antibody and quantitation of phospho ERK1/2 relative to total ERK1/2. Numbers represent the ratio of the quantification of phospho ERK1/2 to total ERK1/2. Data are means ± SE; *P<0.05.</p

PTN effect on cultured mammary epithelial cell (MEC) differentiation.

<p>(A, B) Effect of treatment of MECs with a blocking anti-PTN antibody. (A) Progenitor marker mRNA, (B) Keratin 18 (K18), Vimentin (VM) and PTN mRNA expression were measured by quantitative, real-time PCR. The effect of continuous passaging of MECs ± anti-PTN is shown relative to passage #1 MECs. Data are means ± SE; n = 4 and 2 independent experiments for VM and K18 respectively; **P <0.01; ***P <0.001 by ANOVA. MECs passage#1 expression relative to Actin: CD29, 3.5; CD49f, 5.6; CD10, 6.6; SCA-1, 2.3; K18, 2.9; VM, 5.3; PTN, 12.2. Control MECs passage#3 expression relative to Actin: CD29, 2.9; CD49f, 5.1; CD10, 9; SCA-1, 3.8; K18, 2.9; VM, 5.5; PTN, 10.3. Treated MECs passage#3 expression relative to Actin: CD29, 3.5; CD49f, 5.4; CD10, 7.5; SCA-1, 3.2; K18, 2.9; VM, 5.9; PTN, 10.9. (C) Expression of K18, VM and PTN in mammary fibroblasts relative to MECs passage #1. Expression of K18, VM and PTN in mammary fibroblasts relative to Actin: PTN, 15.7; K18, 15.9; VM, 3.</p

PTN effect on ductal outgrowth and branching in developing mammary glands.

<p>(A) Whole mounts of representative mammary glands from mice treated with anti-PTN blocking antibody versus control. White arrows: distance of the outgrowth of a representative duct measured from the midpoint of the lymph node. Black arrowheads: branch points. Scale bar  = 1.5 mm. (B to F) Controls (n = 9 mice, white), anti-PTN (n = 10 mice, grey). (B, C) Ductal length and number of branch points with the ductal length expressed in mm and the number of branch points per mm of duct. Data are means ± SE; *P = 0.04; ** P = 0.006; n = 3 ducts per mouse. (D to F) Quantitation of Terminal Ends (TE) and Terminal End Buds (TEBs) of mammary ducts; (D) TEBs per duct, ns =  not significant; (E) TEs per duct, * P = 0.03; (F) TEBs per total number of duct ends ( = TEBs + TEs), *P = 0.027. Data are means ± SE; n = 6 ducts per mouse.</p

PTN protein expression in mammary glands and mammary epithelial cells (MECs).

<p>(A) Immunohistochemistry of virgin mouse mammary glands with a polyclonal anti-PTN antibody or secondary antibody only (sec. only) as a negative control. The dark brown staining shows PTN crossreactivity. Scale Bar  = 50 μm. Magnified ductal areas are indicated by the red squares (Scale Bar  = 25 μm) and individually magnified duct epithelial cells by the black squares (Scale Bar  = 10 μm).</p