Reconstruction of functionally normal and malignant human breast tissues in mice

Abstract

The study of normal breast epithelial morphogenesis and carcinogenesis in vivo has largely used rodent models. Efforts at studying mammary morphogenesis and cancer with xenotransplanted human epithelial cells have failed to recapitulate the full extent of development seen in the human breast. We have developed an orthotopic xenograft model in which both the stromal and epithelial components of the reconstructed mammary gland are of human origin. Genetic modification of human stromal cells before the implantation of ostensibly normal human mammary epithelial cells resulted in the outgrowth of benign and malignant lesions. This experimental model allows for studies of human epithelial morphogenesis and differentiation in vivo and underscores the critical role of heterotypic interactions in human breast development and carcinogenesis.

Fig. 1.

GFP whole-mount and histological analysis of human-murine chimeric fat pads. (A and B) GFP whole-mount examination of cleared mouse mammary fat pads injected with immortalized human breast fibroblasts 4 weeks postinjection (×10). Single GFP-positive cells can be seen infiltrating the adipose stroma (B Inset). (C) Hematoxylin/eosin-stained section of an area of fibroblastic cord growth within the cleared fat pad. (D and E) Serial sections of this region were examined by immunohistochemistry by using antibodies against human-specific vimentin (purple, D) or PCNA (brown, E). Genomic FISH was performed by using specific probes for mouse Cot I gene (red) and human genomic DNA (green) (F).

Fig. 2.

Whole-mount analysis of breast tissue of human breast and xenografted outgrowths. Normal human breast tissue isolated from reduction mammoplasty was stained with hematoxylin and whole-mounted exhibits both ductal and TDLU (in brackets) structures (A). Carmine-stained whole-mount examination of outgrowth from xenografts in humanized fat pads contain both ductal (B) and TDLU structures (C and D). Arrows point out commonly observed acini structures with hollow lumina. TDLU formation occurred during murine puberty (C) or pregnancy (D). Some outgrowth displayed evidence of both ductal and TDLU-like growths (E).

Fig. 3.

Epithelial and stromal cell contribution in xenograft breast tissue. Genomic FISH was performed on sections from mice in which human breast epithelial cells were injected into the humanized fat pads along with normal primary breast fibroblasts (A). Human cells (green) comprise all of the epithelial cells in the xenografts, but the stroma is comprised of both human and mouse cells (red). A significant amount of angiogenesis is observed within the xenograft (B). White-light photomicrograph of the xenografted implanted region 8 weeks after surgery displays significant infiltration of blood vessels. (C) Hematoxylin/eosin-stained section of a region of the xenograft reveals many capillaries (arrows) within the stroma indicative of neovascularization.

Fig. 4.

Histological and molecular analysis of xenograft breast tissues. Paraffin sections of breast xenograft ductal outgrowths were stained with hematoxylin/eosin (A and B). Double labeling of breast markers ductal structures (C). Myoepithelial cell-specific expression of smooth-muscle actin (purple) is not detected in cells expressing the estrogen receptor (brown). Immunohistochemistry was performed on serial sections with antibodies specific for PCNA (D) to demonstrate the proliferative state of the outgrowth. Brown nuclei can be detected in both epithelial and stromal cells. Luminal-specific expression of cytokeratin 19 is detected in ER expressing cells (E), and E-cadherin expression is detected on the membrane of luminal epithelial cells (F). Paraffin sections of fully differentiated breast xenograft ductal outgrowths in pregnant mice were stained with hematoxylin and eosin (G). Milk protein and fat droplets are seen (arrows) in the differentiated epithelial cells under the control of the hormones of pregnancy. Immunohistochemistry was performed on these sections with antibodies against human-specific β-casein milk protein (H). β-Casein expression is detected in secretory epithelial cells as well as in the luminal space of the alveoli.

Fig. 5.

Hyperplastic and tumorigenic outgrowths derived from reduction mammoplasty epithelium. Hematoxylin/eosin-stained sections of hyperplasias that developed from normal organoids in the absence of normal admixed fibroblasts (A and B). (C) Frequency of tumor formation when reduction mammoplasty organoids were introduced directly into humanized stroma overexpressing TGF-β or HGF (O) or when the organoids were coinjected with normal primary human breast fibroblasts (O + F). Histology of the tumors arising in glands humanized with growth factor expressing stroma: stroma that overexpresses either HGF (D) or TGF-β1 (E). Histology of outgrowths of various presumed stages of human breast cancer detected in the same field. Normal ductal tissue (normal), benign hyperplastic ducts (hyperplasia), in situ ductal cancer (DCIS), and invasive carcinoma (invasive) (F).