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. 2012 Jan 25;14(1):R18.
doi: 10.1186/bcr3102.

Metastasis is an early event in mouse mammary carcinomas and is associated with cells bearing stem cell markers

Affiliations

Metastasis is an early event in mouse mammary carcinomas and is associated with cells bearing stem cell markers

Desheng Weng et al. Breast Cancer Res. .

Abstract

Introduction: It is still uncertain whether metastasis is predominantly an early or late event in tumor progression. The detection of early metastases and cells responsible for the dissemination may therefore have significant clinical implications.

Methods: Lung dissemination and/or metastasis were investigated in mice carrying the polyomavirus middle-T oncogene (PyMT) during different stages of mammary tumorigenesis using the colony forming assay. Immunocytochemical or immunohistochemical staining was used to identify subpopulations of cells responsible for lung dissemination and metastasis. Histological examination was used to show primary and metastatic tumors. The tumor-initiating and metastatic capacity of cells expressing stem cell markers was assessed in syngeneic wild-type (WT) mice whose mammary fat pads were injected with these cells.

Results: Metastatic mammary epithelial cells were detected in the lungs of mice carrying the PyMT oncogene (MMT mice). These cells were observed early in breast tumorigenesis when the mammary tree appeared by histological inspection to be normal (or at a premalignant stage), suggesting the possession of disseminating and metastatic capacity even before full malignant transformation. Some of the disseminated cells and lung metastases displayed surface stem cell markers. These findings suggest that stem cells from apparently precancerous primary lesions could be a source of metastasis. Indeed, injection of lung tissue cells from MMT mice into syngeneic WT mice resulted in the formation of mammary tumors. These tumors resembled their parent mammary tumors in the MMT donors as well as grafted tumors derived from mammary tumor cells. Furthermore, when we injected lung tissue cells from GFP MMT mice into the fat pads of recipient WT mice, disseminated or metastatic GFP-expressing cells were detected in the lungs, lymph nodes and blood of the recipient WT mice. We finally identified a subpopulation of mammary epithelial/tumor cells expressing CD44 and Sca1 that was largely responsible for dissemination and metastasis in MMT mice.

Conclusions: The tumorigenic and metastatic potential of a subpopulation of mammary epithelial/tumor cells in MMT mice is endowed relatively early in mammary neoplasms and suggests a potential role for cancer stem cell sub-populations in metastasis.

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Figures

Figure 1
Figure 1
Development of mammary tumors and detection of disseminated and/or metastatic cells in MMT mice. (a) Mammary glands were harvested from mice double transgenic for PyMT and MUC1 antigen (MMT) or MUC1 transgenic (MUC1.Tg) mice at indicated ages, processed for whole mount or sections and stained with H&E. The solid masses are indicated by circles. (b) Comparison of mammary masses in different age groups (n = three to seven per group) of MMT, MUC1.Tg and MT (mice transgenic for PyMT oncogene) mice. The whole mount was digitized and the solid masses were traced and measured using SPOT advanced software. (c) Detection of disseminated cells or metastasis in lungs. Lungs were harvested from MMT mice at the indicated ages and perfused to remove circulating cells. The lung tissue was minced and digested in a collagenase enzyme cocktail solution. The cells were then cultured on tissue culture plates for two weeks and stained with 0.5% crystal violet (upper panels). Lungs from mice at the indicated ages were processed for histological examination with H&E staining. The green square indicates the enlarged area. (d) Colonies of more than 50 cells from lung cell cultures of each mouse (n = three to seven per group) were counted. Statistical analysis was performed using one-way analysis of variance.
Figure 2
Figure 2
Detection of cells expressing stem cell markers or MUC1 in the lungs of MMT mice. (a) Immunocytochemical (ICC) staining. The colonies growing from lung tissue cells (as in Figure 1) were stained with anti-CD44 or Sca1 antibodies. The cells staining red were positive cells and those stained purple were considered double-positive cells (10x). The red square indicates the enlarged area. (b) Single-cell suspensions were made from the lungs of mice double transgenic for PyMT and MUC1 antigen (MMT) at the indicated ages and stained with monoclonal antibodies against CD44, Sca1 or MUC1 using ICC staining. Cells isolated from mammary tumors of MMT mice were used as positive control. Cells staining red were considered positive (60x). (c) Comparison of positive cells among different age groups (n = three to eight per group) of MMT and MT mice. The cells positive for the indicated antibody from each mouse were counted and are presented in the bar graph. Statistical significance between groups was determined using Chi-square test and the symbol "*" indicates P < 0.05 when 20 to 35 day group was compared with 53 to 87 day or 98 to 170 day groups. (d and e) Expression of CD24, ESA (epithelial specific antigen) or estrogen receptor (ER) on CD44/Sca1+ cells. The lung cells isolated from MMT mice at the indicated ages were processed for staining with anti-CD44-Cy, Sca1-FITC and CD24-PE, ESA-PE or ER-PE antibodies. The percentage of cells double positive for CD44 and Sca1 were gated and then further analyzed by FACS for expression of CD24, ESA or ER. The percentage of gated CD44/Scal+ cells in total lung or tumor cells and percentage of triple-positive cells are presented. (e) Mammary tumor cells isolated from 162-day-old MMT mice were used as controls.
Figure 3
Figure 3
Tumorigenic potential of lung tissue cells from MMT mice. (a) Single-cell suspensions were isolated from lungs of mice double transgenic for PyMT and MUC1 antigen (MMT) at the indicated ages, counted and then injected into the left and right mammary fat pads of syngeneic wild-type (WT) mice at various doses. Eight weeks after inoculation, the recipient mice were sacrificed and the mammary glands were harvested, processed for whole mount and photographed. WT mice injected with sorted CD44+ mammary tumor cells or single-cell suspension obtained from the lungs of MUC1.Tg mice were used as positive or negative controls, respectively. The solid masses indicated by circles are the tumors. (b) Tumor incidence and tumor size in the whole mount were summarized and compared using one-way analysis of variance. (c and d) The mammary glands as shown in (a) were processed for histology and (c) stained with H&E or anti-CD44 and Sca1 monoclonal antibodies by (d)immunohistochemical (IHC) staining.
Figure 4
Figure 4
Detection of disseminated and/or metastatic tumor cells in the new hosts. Single-cell suspensions from the lungs of mice double transgenic for PyMT and MUC1 antigen (MMT) or MUC1 transgenic (MUC1.Tg) mice at indicated ages were injected into the fat pads of mammary glands of wild type (WT) mice. Mice injected with sorted CD44+ mammary tumor cells were used as controls. Eight weeks after the inoculation, the lungs were harvested from the mice, perfused to remove the circulation cells, and then processed for clonogenic assay. (a) Colonies from lung cell cultures of each mouse were counted and are presented in the bar graph. Statistical significance among groups was compared using one-way analysis of variance. (b) Expression of estrogen receptor (ER) in some cells from colonies as shown in (a) using immunocytochemical (ICC) staining. (c) Detection of MUC1/Sca1 positive tumor cells in lung tissue cells isolated from WT mice using ICC staining. (d) Metastatic lesions in the lungs of WT mice injected with sorted CD44/Sca1+ from mammary tumors (left two panels) or lungs of MMT mice (right two panels). The lungs of recipient mice were processed for immunohistochemical (IHC) staining with anti-CD44 or anti-ER monoclonal antibodies. The red square indicates the enlarged area.
Figure 5
Figure 5
Detection of disseminated cells in WT recipient mice after inoculation with lung tissue cells from GFP MMT mice. (a) Mammary glands harvested from mice transgenic for PyMT and MUC1 antigen (MMT) that also express green fluorescent protein (GFP MMT) were processed for histological examination under light or fluorescence microscopy (60 x). (b) Colony-forming assay. WT mice were injected with lung tissue cells isolated from GFP MMT mice at the indicated ages. Lungs were then collected and perfused to remove circulating cells. Single-cell suspensions from the lungs were cultured in DMEM for two weeks. Colonies with GFP and Sca1 expression were observed under fluorescence microscopy (lower panels). (c) Detection of GFP and CD44-positive cells in blood vessels of lung from a WT recipient mouse. Samples from the recipient lungs were also processed for staining with H&E or anti-CD44 monoclonal antibodies.
Figure 6
Figure 6
Tumorigenic and metastatic potential of CD44/Sca1+ cells. CD44/Sca1+ and CD44/Sca1- cells were obtained by cell sorting from mammary epithelial cells (MECs), tumor cells or the lungs of mice transgenic for PyMT and MUC1 antigen (MMT) that also express green fluorescent protein (GFP MMT) at different stages of tumor development and injected into the mammary fat pads of wild type (WT) mice at the doses indicated (if not indicated in this way, 8 × 104 cells were injected). Tissue samples were obtained from WT mice injected with either sorted CD44/Sca1+ cells [(+)] or CD44/Sca1- cells [(-)]. (a) Comparison of tumorigenic potential between CD44/Sca1+ and CD44/Sca1- tumor cells. The tumor incidence and tumor size are presented for each group of mice inoculated with the indicated numbers of cells. (b) Mammary tumor from a WT mouse inoculated with CD44/Sca1+ tumor cells was viewed by H&E staining (left panel) and examined for GFP (middle panel) or CD44 (right panel) expression using immunohistochemical (IHC) staining. (c) Sections of mammary glands from WT mice inoculated with CD44/Sca1+ tumor cells (> 120 days, upper panels) or MECs (< 40 days, lower panels) were stained with H&E and examined for expression of GFP and ESA or CD44. (d) Quantification of disseminated cells. GFP-positive cells were gated from lung, lymph node cells (LNC) or blood cells of recipient mice inoculated with CD44/Sca1+ and CD44/Sca1- cells from tumor cells or MECs of GFP MMT mice, and stained with anti-ESA monoclonal antibodies and analyzed by FACS. (e) Tumors from the lungs of WT mice inoculated with CD44/Sca1+ lung cells from GFP MMT mice at more than 120 days were viewed by H&E staining and examined for GFP and estrogen receptor (ER) expression. (f) Quantification of disseminated cells. GFP-positive cells were gated from lung, LNC or blood cells of recipient mice inoculated with CD44/Sca1+ and CD44/Sca1- cells from GFP MMT mice at the indicated ages, stained with anti-ESA monoclonal antibodies and analyzed by FACS. (d and f) The average ± standard deviation of ESA/GFP-positive cells in total blood, LN or lung cells were presented.

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