The normal mammary microenvironment suppresses the tumorigenic phenotype of mouse mammary tumor virus-neu-transformed mammary tumor cells - PubMed (original) (raw)
The normal mammary microenvironment suppresses the tumorigenic phenotype of mouse mammary tumor virus-neu-transformed mammary tumor cells
B W Booth et al. Oncogene. 2011.
Abstract
The microenvironment of the mammary gland has been shown to exert a deterministic control over cells from different normal organs during murine mammary gland regeneration in transplantation studies. When mouse mammary tumor virus (MMTV)-neu-induced tumor cells were mixed with normal mammary epithelial cells (MECs) in a dilution series and inoculated into epithelium-free mammary fat pads, they were redirected to non-carcinogenic cell fates by interaction with untransformed MECs during regenerative growth. In the presence of non-transformed MECs (50:1), tumor cells interacted with MECs to generate functional chimeric outgrowths. When injected alone, tumor cells invariably produced tumors. Here, the normal microenvironment redirects MMTV-neu-transformed tumorigenic cells to participate in the regeneration of a normal, functional mammary gland. In addition, the redirected tumor cells show the capacity to differentiate into normal mammary cell types, including luminal, myoepithelial and secretory. The results indicate that signals emanating from a normal mammary microenvironment, comprised of stromal, epithelial and host-mediated signals, combine to suppress the cancer phenotype during glandular regeneration. Clarification of these signals offers improved therapeutic possibilities for the control of mammary cancer growth.
Conflict of interest statement
Conflict of interest
The authors declare no conflict of interest.
Figures
Figure 1
Tumor-derived cells give rise to normal mammary structures. (a) Schematic outlining the experimental protocol used (modified from Bissell and Inman, 2008). Cells were isolated from mammary tumors from WAP-Cre/Rosa26/MMTV-neu female mice. These were transplanted into the cleared mammary fat pads of 3-week-old virgin Nu/Nu female mice alone (100, 10 or 1.0 K) or mixed with 50K normal FVB/N mammary epithelial cells in ratios of 2:1, 1:5 or 1:50. These transplants were allowed to grow for 6–8 weeks or until tumors arose. The mature transplanted gland was removed and 80% was used for tissue analyses and the remaining 20% was transplanted into new 3-week-old virgin Nu/Nu female mice. After 8 weeks, these female mice were placed with male mice and allowed to complete a full-term pregnancy, after which the transplanted mammary glands were removed and analyzed or viably frozen as fragments. (b, b1) Tumor-derived cells (50 K) were transplanted and the resulting focal tumors were whole mounted and X-gal stained. (b2) Cross-section of the portion of the tumor outlined in (b1), indicating that the entire tumor is comprised of lacZ+ cells. (b3) Tumor-derived cells (1.0 K) were mixed with 50K normal FVB/N mammary epithelial cells and transplanted. Resulting mammary glands were whole mounted and stained with X-gal. Normal ductal tree and duct termini are evident. (b4) Higher power magnification of whole mount in (b3) showing X-gal-stained endbuds. (b5) Cross-section of chimeric mammary gland illustrating mosaic-staining pattern of β-gal+ cells. (b6) Cross-section of chimeric mammary gland showing, in some instances, normal development is independent of tumor-derived cells. (b7) Second transplant generation mammary outgrowths displaying similar distribution of lacZ+ tumor-derived cells as seen in (b3) and (b4). (b8) Cross-section of (b7). Nuclei in (b2), (b5), (b6) and (b8) were counterstained pink with Nuclear Fast Red. Scale bars, 2.5mm (b1) and (b3); 50 μm (b2), (b5), (b6) and (b8); and 500 μm (b4) and (b7).
Figure 2
Tumor-derived cells differentiate into different cell lineages and contribute to the formation of a functional mammary gland. Tumor-derived cells (1.0K) were mixed with 50K normal FVB/N mammary epithelial cells and injected into the cleared fat pad of Nu/Nu female hosts. Tissue sections of chimeric glands and mammary tumors were stained for β-gal (green) and ERα or PR (red). (a, a1) Chimeric mammary outgrowth showing ERα expression (red) indicated by arrows only in β-gal− cells. (a2) Cross-section of a β-gal+ mammary hyperplasia that is ERα−. (b, b1) Chimeric mammary outgrowth showing PR expression (arrows) only in β-gal− cells. (b, b2) Cross-section of a β-gal+ mammary hyperplasia that is PR−. (c) Cross-section of a chimeric gland at day 2 post-partum showing that tumor-derived cells, determined by β-gal (green) expression, become functional secretory epithelial cells and produce the milk protein β-casein (red). Arrows indicate tumor-derived cells that are producing β-casein. L – lumen. Sections (a2), (b2) and c) were counterstained with 4′,6-diamidino-2-phenylindole. Scale bars, 20 μm (a) and (b) and 10 μm (c).
Figure 3
Tumor-derived cells maintain their erbB2+ status following transplantation. Immunofluorescent staining of chimeric cross-section showing (a) erbB2+ expression (red) and β-gal expression (green). (b) Tumor that developed following transplantation that is completely erbB2+ and β-gal+. (c) Section of wild-type FVB/N mammary gland showing non-existent erbB2 and β-gal expression. Far right image is section stained with nuclear fast red. Scale bars, 20 μm.
Figure 4
Tumor-derived cell receptor phosphorylation status following transplantation. (a) Immunofluorescent staining of chimeric cross-section showing that within the chimera, there is no detectable phospho-erbB2 (red). Tumor-derived cells identified by β-gal expression (green). (b) Cross-section showing that within the chimera, tumor-derived cells continue to express phospho-EGFR (phospho-EGFR, red; β-gal, green). (c) Immunofluorescent image of a chimeric cross-section showing that within the chimera, tumor-derived cells continue to express EGFR (EGFR, red; β-gal, green). All sections counterstained with 4′,6-diamidino-2-phenylindole. Scale bars, 30 μm. (d) Section of a normal FVB/N mammary gland stained for P-EGFR and β-galactosidase (phospho-EGFR, red; β-gal, green.
Figure 5
Recovered erbB2+ cells form mammary tumors. Dissociated cells from chimeric outgrowths were magnetically sorted for erbB2 before transplantation. (a) ErbB2+ fraction formed mammary tumors when 1000 cells were transplanted even when fat pad contained host outgrowth (unstained ducts–add arrows). Tumors from two separate mammary recipients are shown. (b) Cross-sections analyses of mammary outgrowths shown in (a) showing entire mammary tumors consist of lacZ+ cells. Sections counterstained with nuclear fast red. Scale bars, 100 μm.
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