An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ

Abstract

Introduction: Human models of noninvasive breast tumors are limited, and the existing in vivo models do not mimic inter- and intratumoral heterogeneity. Ductal carcinoma in situ (DCIS) is the most common type (80%) of noninvasive breast lesions. The aim of this study was to develop an in vivo model whereby the natural progression of human DCIS might be reproduced and studied. To accomplish this goal, the intraductal human-in-mouse (HIM) transplantation model was developed. The resulting models, which mimicked some of the diversity of human noninvasive breast cancers in vivo, were used to show whether subtypes of human DCIS might contain distinct subpopulations of tumor-initiating cells.

Methods: The intraductal models were established by injection of human DCIS cell lines (MCF10DCIS.COM and SUM-225), as well as cells derived from a primary human DCIS (FSK-H7), directly into the primary mouse mammary ducts via cleaved nipple. Six to eight weeks after injections, whole-mount, hematoxylin and eosin, and immunofluorescence staining were performed to evaluate the type and extent of growth of the DCIS-like lesions. To identify tumor-initiating cells, putative human breast stem/progenitor subpopulations were sorted from MCF10DCIS.COM and SUM-225 with flow cytometry, and their in vivo growth fractions were compared with the Fisher's Exact test.

Results: Human DCIS cells initially grew within the mammary ducts, followed by progression to invasion in some cases into the stroma. The lesions were histologically almost identical to those of clinical human DCIS. This method was successful for growing DCIS cell lines (MCF10DCIS.COM and SUM-225) as well as a primary human DCIS (FSK-H7). MCF10DCIS.COM represented a basal-like DCIS model, whereas SUM-225 and FSK-H7 cells were models for HER-2+ DCIS. With this approach, we showed that various subtypes of human DCIS appeared to contain distinct subpopulations of tumor-initiating cells.

Conclusions: The intraductal HIM transplantation model provides an invaluable tool that mimics human breast heterogeneity at the noninvasive stages and allows the study of the distinct molecular and cellular mechanisms of breast cancer progression.

Figure 1

Intraductal HIM transplantation model mimics human breast cancer noninvasive-to-invasive progression. The model allows temporal analysis of many processes involved in early breast cancer invasive progression including intraductal cancer cell growth, the cell interactions with the surrounding normal epithelial and myoepithelial cells, and their escape into the surrounding stroma.

Figure 2

Hematoxylin and eosin (H&E) and whole-mount staining of intraductal xenografts. The panels show whole-mount and H&E staining of xenografts generated by intraductal transplantation of DCIS.COM (a, b), SUM-225 (c, d), and FSK-H7 (e, f). The H&E figures depict ×20 magnification, and whole mounts were captured at ×1.6 (SUM-225 and DCIS.COM) and ×0.7 (FSK-H7). Cells were injected intraductally (20,000/μl cells in 2 μl of PBS) into the primary mammary ducts (via cleaved nipple) of intact 8-week-old immunocompromised SCID-beige female mice. The human primary DCIS was enzymatically digested into single cells before injections. The pictures were taken at 6 weeks after injection.

Figure 3

iFish analysis of a DCIS.COM intraductal xenograft. Human-derived DCIS.COM cells are contained within the boundaries of mouse myoepithelial cells and basement membrane. The panels depict immunofluorescence staining of paraffin sections from a fat pad containing DCIS.COM intraductal lesions by using anti-mouse smooth muscle actin antibody (a-c), anti-human pan cytokeratin antibody (d-e), iFish with fluorescently labeled human (green) (f-j), and mouse (red) Cot-1 DNA as probes, and merged images of all panels (p-t).

Figure 4

Expression of subtype-specific markers by the intraductally generated DCIS-like lesions. Intraductal lesions were generated by injection of SUM-225 (a, d, g), DCIS.COM (b, e, h), and FSK-H7 (c, f, i). Six weeks after injection, fat pads containing intraductal lesions were removed and fixed, followed by paraffin embedding by using standard histological techniques. Paraffin sections were subjected to immunofluorescence studies with antibodies against human CK-19 (a-c), Her-2 (d-f), and CK-5 (g-i). Primary antibodies were conjugated to secondary antibodies, Alexa-594 against mouse anti-human CK-5 and CK-19 (red), and Alexa-488 against rabbit anti-human Her-2 antibody (green).

Figure 5

Flow-cytometry analysis of DCIS.COM and SUM-225. Cells grown in 2D were stained by using the indicated anti-human antibodies to the surface markers, CD44/CD24, CD49f/CD24, and CD49f/MUC-1. The antibodies were directly conjugated, except for MUC-1, which was conjugated by using an anti-mouse FITC antibody. The histograms show expression levels for the indicated surface markers. The expression level is arbitrarily designated as low (lo) if the log¹⁰ of median fluorescence intensity (FI) is between about 0 and 2, medium (med) if the FI is between about 2.1 and 3.6, and high (hi) if the FI is higher than about 3.7.