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Additional Supporting Information may be found in the online version of this article.

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sc-12-0098_sm_SupplFigure1.pdf210KSupplemental Figure S1. Experimental set up for in vivo imaging (A) Low magnification picture of experimental set up for intravital imaging. Shown is microscope stage, microscopic objective, illumination, and an anesthetized live mouse with mammary gland exposed. Bar = 5cm. (B) Higher magnification image of (A). Exposed mammary gland is visible, Head of mouse is to the left, and tail of mouse is to the right, underneath surgical drape. Bar = 2cm. (C) Cross-sectional schematic view of the exposed mammary gland indicating skin in black, the mammary gland in green, the peritoneum as blue, and the mouse abdominal cavity as yellow. Supporting the mammary gland (support), rotating the mouse (long circular counterclockwise right arrow, below) and placing suture through peritoneum (short circular arrow, above) allowed improved access to the mammary gland.
sc-12-0098_sm_SupplFigure2.pdf174KSupplemental Figure S2. Fluorescent glands can be visualized in other transgenic mice with same promoter (A) Low magnification view of fluorescent mammary glands in the female, 3 month old, BL6,CAG- DsRED mouse. Red bright structures represent mammary glands. CAG promoter is Chicken Beta Actin and CMV enhancer combined in one promoter. Image demonstrates normal branching pattern. Bar = 500μm. (B) Low magnification view of fluorescent mammary glands in female, 3 month old, FvB CAG-GFP mouse. Green bright structures represent mammary glands. CAG promoter is Chicken Beta Actin and CMV enhancer combined in one promoter. Bar = 500μm.
sc-12-0098_sm_SupplFigure3.pdf271KSupplemental Figure S3. Two-photon microscopy demonstrates that ductal epithelium expresses high levels of GFP (A) High magnification view (10x) of fluorescent mammary glands in a sacrificed, female, 3 month old, pcx-GFP female mouse using two-photon microscopy. Mammary ducts and buds appear to express fluorescent protein strongly (lower arrow). Between mammary glands, endothelial cells of capillaries which surround adipocytes in the stroma also appear green, but are much thinner (upper arrow). Bar = 200μm. (B) Higher magnification view (20x) of fluorescent mammary glands in a sacrificed, female, 3 month old, pcx-GFP female mouse using two-photon microscopy. Cross-sectional view of a mammary duct is seen, and structures which appear as columnar epithelium lining lumen, strongly express fluorescent protein (bottom arrow). Additionally, and the lumen, labeled with arrow, is visualized (top arrow). This image demonstrates that fluorescent signal emanates from mammary epithelium. Bar = 200μm.
sc-12-0098_sm_SupplFigure4.pdf104KSupplemental Figure S4. Comparison of total length of ductal elements between normal and regenerating glands Bar graph demonstrating mean total length (voxels) per field of view per mouse for host mammary ducts in both the CSC and non-CSC conditions. For each field of view, mammary ducts were outlined, skeletonized, and analyzed (see Methods). Three fields of view per mouse for N=3 mice, and data is presented as mean ±SD. Length of each ductal element is shown.
sc-12-0098_sm_SupplFigure5.pdf217KSupplemental Figure S5. High magnification in vivo images of part 1 of developing mouse glands at day 3 and 4.5 (A) Schematic of primary branch, part 1 (labeled in Figure 2F) overlayed. Schematic represents growth at 3 and 4.5 weeks, and demonstrates the direction of growth over time. (B) Overlay of in vivo images (based on schematic in (A)) of part 1 at 3 and 4.5 weeks. Images taken of same in vivo location at 3 weeks (green) and at 4.5 weeks at high magnification. Images from 3 week old, BL6, GFP mice. Images co-registered using several features of image, without altering scales of either image, including two smaller buds beneath branch 1. Bar = 100μm. (C) Bar graph demonstrating quantitation of growth at 3 weeks and 4.5 weeks of part 1. Total number of pixels was used as arbitrary units, and plot demonstrates a 0.54 fold increase in growth. Growth measurement was performed by outlining structures from same anatomical location in both images.
sc-12-0098_sm_SupplFigure6.pdf229KSupplemental Figure S6. Endogenous mammary duct remodeling 2 weeks after CSC transplantation (A) In vivo image of endogenous fluorescent mammary gland in female, 5 week old FvB- GFP mouse, 2 weeks after transplantation of 2500 Non-Cancer Stem Cells (non-CSC) (non-Thy1+ CD24+) cells. Site of tumor implantation is labeled (T). Terminal end buds (TEB) are present near tumor and ducts (D) display increased lateral budding (LB). Bar = 500μm. (B) Same experiment as above, but different field of view. Site of tumor implantation is labeled (T). Ducts (D) display increased lateral budding (LB). Bar = 500μm.
sc-12-0098_sm_SupplFigure7.pdf236KSupplemental Figure S7. Endogenous mammary duct remodeling 2 weeks after CSC transplantation Mosaic, In vivo image of endogenous fluorescent mammary gland in female, 5 week old FvB- GFP mouse, 2 weeks after transplantation of 2500 Non-Cancer Stem Cells (non- CSC) (non-Thy1+ CD24+) cells. Site of tumor implantation is labeled (T). Terminal end buds (TEB) are present near tumor and ducts (D) display increased lateral budding (LB). Bar = 500μm.
sc-12-0098_sm_SupplFigure8.pdf260KSupplemental Figure S8. Imaging and quantitation of lateral branching after CSC transplantation and relative size comparison of CSC and non-CSC tumors (A) Image from same field of view as Figure 5C demonstrating lateral branching (LB), 2 weeks after transplantation of CSC into FvB-GFP mice. Bar = 500μm. (B) Skeletonized view of branched portion of image as in Figure 5C. Bar = 500μm. (C) Bar graph demonstrating total voxels per field of view per mouse for host mammary ducts in both the CSC and non-CSC conditions. For each field of view, mammary ducts were outlined, skeletonized, and analyzed (see Methods). Three fields of view per mouse for N=3 mice, and data is presented as mean ±SD. (D) Hematoxylin and Eosin (H & E) stain of mammary fat pad of non-CSC (left panel) and CSC (right panel) transplanted mice. Image is at two weeks after transplantation of 2500 cells into 3 week old FvB-GFP mice. “T” labels tumor in both panels. Bar = 500μm.
sc-12-0098_sm_SupplFigure9.pdf217KSupplemental Figure S9. Flow Cytometry of the MMTV-Wnt1 mouse Flow cytometric plot after isolation and staining of the CSC tumor. Flow cytometry panel is on left, and population analysis of each gate shown on right. Cells are stained for Lin- ,Thy1+,CD24+. Gating based on the previous results of MMTV-Wnt 1transplant experiments. Top right gate on the flow cytometer panel is the P6 population and represents the “CSC” population (Thy1+,CD24+), while the rest of the cells represent the non-CSC population. Cells are sorted a second time as a purity check and typically 99% of P5 cells from the initial sort also are present in the P5 gate in the second sort.
sc-12-0098_sm_SupplFigure10.pdf241KSupplemental Figure S10. Flow Cytometry of the DB7 cell line (A) Flow cytometric plot after isolation and staining of the DB7 cell line after culturing. Flow cytometry panel is on left, and population analysis of each gate shown on right. Cells are stained for Lin-,Thy1+,CD24+. Gating is based on previous results of MMTV-Wnt1 transplant experiments. Top right gate on the flow cytometric panel is the P5 population (Thy1+,CD24+), and represents the “CSC” population, while the rest of the cells represent the non-CSC population. (B) Flow cytometric plot the same cells in (A) in gate P5 that are resorted using the same gates. Normally, 99% of the same cells from the P5 (Lin-,Thy1+,CD24+) population are expected to be present in the same P5 gate upon resorting. In this case of the DB7 cells, only 20.2 % of the cells that were previously gated positive actually sort into the same, P5 gate.
sc-12-0098_sm_Supplmovie1.avi247KSupplemental Online Video S1 High magnification intravital depth stack movie of normal mammary gland in a female, 3 month old, pcx-GFP adult mouse. Bright green tube-like structures represent mammary glands. Movie is motion corrected, and then a maximum intensity projection image is generated and viewed individually or as part of a mosaic image.
sc-12-0098_sm_Supplmovie2.avi197KSupplemental Online Video S2 Same as above, except another field of view shown.
sc-12-0098_sm_Supplmovie3.avi311KSupplemental Online Video S3 Higher magnification (10x objective) of intravital depth stack movie of normal mammary gland in a female, 3 month old, pcx-GFP adult mouse. Bright green tube-like structures represent mammary glands.
sc-12-0098_sm_Supplmovie4.avi493KSupplemental Online Video S4 High magnification intravital depth stack movie of Lin- GFP expressing mammary epithelial cells, 16 weeks after transplantation into the cleared mammary fat pad of a wild type BL6, female, 3 week old mouse.
sc-12-0098_sm_Supplmovie5.avi132KSupplemental Online Video S5 High magnification intravital depth stack movie of a developing female pcx-GFP mouse at 3 weeks of age. Bright green tube-like structures represent mammary glands. Movie is motion corrected, and then a maximum intensity projection image is generated and viewed individually or as part of a mosaic image.
sc-12-0098_sm_Supplmovie6.avi172KSupplemental Online Video S6 High magnification intravital depth stack movie of a developing female pcx-GFP mouse at 4.5 weeks of age. Bright green tube-like structures represent mammary glands. Movie is motion corrected, and then a maximum intensity projection image is generated and viewed individually or as part of a mosaic image.
sc-12-0098_sm_Supplmovie7.avi262KSupplemental Online Video S7 High magnification intravital depth stack movie of endogenous fluorescent mammary gland in female, 5 week old FvB-GFP mouse, 2 weeks after transplantation of 2500 Non-Cancer Stem Cells (non-CSC) (non-Thy1+ CD24+) cells. Bright green tube-like structures represent mammary glands.
sc-12-0098_sm_Supplmovie8.avi185KSupplemental Online Video S8 High magnification intravital depth stack movie of endogenous fluorescent mammary gland in female, 5 week old FvB-GFP mouse, 2 weeks after transplantation of 2500 Cancer Stem Cells (non-CSC) (Thy1+ CD24+) cells. Bright green tube-like structures represent mammary glands. Increased branching and terminal end buds were observed in the case of CSC transplantation.
sc-12-0098_sm_Supplmovie9.avi208KSupplemental Online Video S9 High magnification intravital depth stack movie of dye-labeled, 10,000 non-CSC (non-Thy1+ CD24+), 2 weeks after transplantation in 3 month old, nude mouse. Cell implant appears yellow.
sc-12-0098_sm_Supplmovie10.avi372KSupplemental Online Video S10 High magnification intravital depth stack movie of dye-labeled, 10,000 CSC (Thy1+ CD24+), 2 weeks after transplantation in 3 month old, nude mouse. Cell implant appears yellow.
sc-12-0098_sm_SupplInfo11.docx37KSupporting Information

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