Humanization of the mouse mammary gland by replacement of the luminal layer with genetically engineered preneoplastic human cells

Abstract

Introduction: The cell of origin for estrogen receptor α-positive (ERα+) breast cancer is probably a luminal stem cell in the terminal duct lobular units. To model these cells, we have used the murine myoepithelial layer in the mouse mammary ducts as a scaffold upon which to build a human luminal layer. To prevent squamous metaplasia, a common artifact in genetically-engineered breast cancer models, we sought to limit activation of the epidermal growth factor receptor (EGFR) during in vitro cell culture before grafting the cells.

Methods: Human reduction mammoplasty cells were grown in vitro in WIT medium. Epidermal growth factor in the medium was replaced with amphiregulin and neuregulin to decrease activation of EGFR and increase activation of EGFR homologs 3 and 4 (ERBB3 and ERBB4). Lentiviral vectors were used to express oncogenic transgenes and fluorescent proteins. Human mammary epithelial cells were mixed with irradiated mouse fibroblasts and Matrigel, then injected through the nipple into the mammary ducts of immunodeficient mice. Engrafted cells were visualized by stereomicroscopy for fluorescent proteins and characterized by histology and immunohistochemistry.

Results: Growth of normal mammary epithelial cells in conditions favoring ERBB3/4 signaling prevented squamous metaplasia in vitro. Normal human cells were quickly lost after intraductal injection, but cells infected with lentiviruses expressing CCND1, MYC, TERT, BMI1 and a short-hairpin RNA targeting TP53 were able to engraft and progressively replace the luminal layer in the mouse mammary ducts, resulting in the formation of an extensive network of humanized ducts. Despite expressing multiple oncogenes, the human cells formed a morphologically normal luminal layer. Expression of a single additional oncogene, PIK3CA-H1047R, converted the cells into invasive cancer cells. The resulting tumors were ERα+, Ki67+ luminal B adenocarcinomas that were resistant to treatment with fulvestrant.

Conclusions: Injection of preneoplastic human mammary epithelial cells into the mammary ducts of immunodeficient mice leads to replacement of the murine luminal layer with morphologically normal human cells. Genetic manipulation of the injected cells makes it possible to study defined steps in the transformation of human mammary epithelial cells in a more physiological environment than has hitherto been possible.

Figure 1

EGFR family gene expression in normal and tumor cells. A. Illumina gene expression data from normal human reduction mammoplasty cells (mean + SEM). MaSC, mammary stem cell; LP, luminal progenitor; ML, mature luminal. Error bars sem; † p < 0.01; * p < 0.05; n = 3; the p values are for comparisons of ML with the other groups. B&C. Affymetrix gene expression data for large operable or locally advanced breast cancer. The scatterplot in B shows how the tumors were classified into luminal (ER+/FOXA1+, 43 samples); molecular apocrine (ER-/FOXA1+, 49 samples); and basal-like (ER-/FOXA1-, 69 samples). The plots in C show the expression profile of EGFR family members in each tumor class. The distributions are strikingly bimodal (see text for details). The maximum density for each distribution is scaled to 1 to facilitate comparison of the different tumor classes.

Figure 2

Silencing of p53 and activation of PI3K in 4G cells. A. Western blots showing that the shp53 vector silences p53 expression, and the PIK3CA vector increases PI3K expression and AKT phosphorylation on T308. B. Growth curves comparing parental 4G cells with cells infected with the shp53 and PIK3CA vectors. Error bars sem; † p < 0.001; * p < 0.05; n = 5 biological replicates.

Figure 3

Tumor growth is not inhibited by fulvestrant. A. Western blot showing that treatment of 4G-shp53-PI3K tumor cells with 1 μM fulvestrant reduces the level of ERα. B&C. Fulvestrant has no effect on proliferation of the cells measured by crystal violet staining (B) or growth curves (C; fulvestrant treatment was started at the arrow; n = 3; NS, not significant). D. 4G-shp53-PI3K cells were injected into the ducts, allowed to form tumors for 3 weeks then treated with fulvestrant or vehicle for 5 weeks. Representative immunohistochemical images show a marked reduction in ERα level in treated tumors. Each image is derived from a separate animal (n = 6 for control, n = 7 for fulvestrant). The slides were all processed together. Scale bars in F, 50 μm. E&F. 4G-shp53-PI3K cells were injected into the ducts, allowed to form tumors for 3 weeks then treated with fulvestrant or vehicle for 3 weeks. E. PhotonIMAGER scans of excised mammary glands after treatment with fulvestrant or vehicle (control) to show how the response was quantified. The scanner counts fluorescence emitted by tdTomato. F. Quantitative data from the glands in E (n = 8 for control, n = 9 for fulvestrant; NS, not significant).

Figure 4

Subcutaneous tumor formation requires activation of PI3K. A. Red fluorescence was used to measure subcutaneous tumor growth. Fluorescence in photons per second per cm² per steradian was normalized to the starting value one week after injection. n = 6; *, p < 0.05. B. Histopathology (H&E stain) and immunohistochemistry show that the tumors were ERα+, Ki67+ adenocarcinomas. The central region in the sections contains an island of mouse stroma surrounded by human tumor cells (PGR gives a non-specific cytoplasmic stain in the stroma). Scale bars 100 μm.

Figure 5

Transformed BPECs grown in pWIT medium form squamous tumors. Human mammary epithelial cells were infected with vectors expressing TERT, BMI1, MYC and ESR1 (3G-ER cells). A. Western blot showing expression of the transgenes in parental (NT) and transformed cells. B&C. Tumor formation two weeks after subcutaneous injection of 3G-ER cells into NSG mice (n = 2). B. Stereomicrograph showing tdTomato fluorescence from the tumor. C. Glandular regions express keratin 8/18; squamous regions express keratin 14. All of the cells are ERα + because one of the lentiviruses used to transform the cells expresses ESR1. D&E. At 3 weeks (D, n = 2) and 5 weeks (E, n = 5) after injection, the tumors are dominated by stratified cells undergoing terminal squamous differentiation leading to the formation of keratin pearls. Keratin pearls are marked by asterisks. Scale bars: B 1 mm; upper panels in C 500 μm; lower panels in C 200 μm; D&E 200 μm.

Figure 6

Comparison of WIT medium containing EGF or NRG1/AREG. Human mammary epithelial cells were infected with vectors expressing TERT, BMI1, CCND1 and MYC. A. Western blots showing that the transgenes are expressed. The cells in medium containing AREG and NRG1 (svWIT) have luminal characteristics. The cells in medium containing EGF (pWIT) have stronger phosphorylation of EGFR. B. Photomicrographs showing that cells in pWIT are a mixture of spindle shaped and rounder cells, whereas cells in svWIT have a more uniform cobblestone appearance. Scale bars 250 μm. C. Flow cytometry shows that cells in svWIT have a putative luminal progenitor phenotype (CD49+/EPCAM+) whereas cells in pWIT are a mixture of putative luminal progenitors and myoepithelial cells. The quadrants were defined by the isotype controls.

Figure 7

Genomic profiles at different stages in the transformation protocol. CGH plots of 4G cells and 4G cells infected with the PIK3CA vector show no copy number changes. Silencing of p53 in 4G cells leads to loss of chr X and minor changes on chromosomes 3 and 5 which do not progress after introduction of the PIK3CA vector. The spike on chromosome 3q in 4G PIK3CA and 4G shp53 PIK3CA cells is caused by four exonic probes in the PIK3CA locus. There were no additional changes after passage in mice.

Figure 8

DCIS formation 3 weeks after intraductal injection of 4G-shp53-PI3K tumor cells. A. Composite image showing human cells expressing CFP scattered throughout the gland. B. Enlarged images; the right panel shows alternating clusters of yellow and green cells. C. H&E stain and immunohistochemistry showing ERα+, Ki67+ DCIS (the PGR staining is negative in the tumor cell nuclei relative to controls on the slide). Scale bars A & B 1 mm, C 100 μm.

Figure 9

The myoepithelial layer in DCIS is murine in origin. Ducts containing DCIS 3 weeks after intraductal injection of 4G-shp53-PI3K tumor cells were costained for p63 (red), GFP (green) and DNA (DAPI, blue) (n = 4). The p63-positive cells do not stain for GFP, indicating that they are murine in origin. Red arrow, normal murine duct; green arrow, DCIS. Scale bars 50 μm.

Figure 10

Invasive tumor formation 6 and 8 weeks after intraductal injection of 4G-shp53-PI3K tumor cells. A. Mammary ducts dilated by human tumor cells at 6 weeks. B. Diffuse blue masses 8 weeks after intraductal injection caused by tumor cells invading the stroma. C. Mixed DCIS and invasive tumor at 8 weeks. H&E stain and immunohistochemistry show increased keratin 14 staining as cells migrate from the ducts into the stroma. Early squamous changes are marked by arrows. There are small difference in the structures in the serial sections in C because the structures gradually change as the sectioning proceeds through the paraffin block. Scale bars A 1 mm, B 2 mm, C 100 μm.

Figure 11

Foci of engrafted human cells 3 weeks after intraductal injection of 4G-shp53 cells. A. Stereomicrograph showing isolated foci of human cells (the images cover a single gland with a small overlap in the middle). B. Attachment of 4G-shp53 cells to the duct wall 3 weeks after intraductal injection. Left panels, H&E staining. Right panels, matched sections showing GFP staining to identify the human cells. The clumps of human cells correspond to the scattered foci of fluorescent cells seen by stereomicroscopy in A. Scale bars A 1 mm, B 100 μm. n = 2.

Figure 12

Replacement of the murine luminal layer 6 weeks after intraductal injection of 4G-shp53 cells. A. Stereomicrograph showing spread of human cells within the ducts. Alternating yellow and green areas in the right panel demonstrate independent engraftment of multiple clones. B&C. Histopathology (H&E stain) and immunohistochemistry show that the human cells have replaced the murine luminal layer with morphologically normal human cells. The human cells are larger, as seen at junctions between murine and human luminal cells. GFP, keratin 7 and keratin 19 staining specifically label the human cells. The cells are ERα+, PGR-, Ki67+ luminal cells. SMA staining shows that the myoepithelial layer is intact. D. GATA3 and FOXA1 are positive in murine ducts (marked by arrows) but negative in humanized ducts (marked by an arrowhead). Scale bars A 1 mm, B 100 μm, C 50 μm, D 200 μm.

Figure 13

The myoepithelial layer is murine in humanized glands. Ducts humanized with 4G-shp53 cells were costained for p63 (red), GFP (green) and DNA (DAPI, blue) (n = 3). All injected human cells express GFP strongly. Green arrow, human luminal cells in a humanized duct; red arrow, murine myoepithelial cells in a murine duct. Scale bars 50 μm.

Figure 14

4G-shp53 cells can undergo secretory differentiation but do not form normal alveoli. Mice with human cells engrafted in the ducts were sacrificed on day 1 of lactation (n = 2). Histopathology (H&E stain) and immunohistochemistry for GFP show that human cells are present in the ducts but do not spread into the lactating alveoli. Immunohistochemistry for casein shows that some of the human cells in the ducts have undergone secretory differentiation. The structures in the middle of the image are mouse alveoli (open arrowheads); they do not stain for casein because the antibody is human specific. The structure on the right is a humanized duct containing cells that do not express casein (closed arrowheads). The structure on the left is a humanized duct containing cells that do express casein (arrows). Scale bars 200 μm.