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. 2012 Jan 10:12:10.
doi: 10.1186/1471-2407-12-10.

"A novel in vivo model for the study of human breast cancer metastasis using primary breast tumor-initiating cells from patient biopsies"

Carolyn G Marsden  1 , Mary Jo Wright, Latonya Carrier, Krzysztof Moroz, Radhika Pochampally, Brian G Rowan
Affiliations

"A novel in vivo model for the study of human breast cancer metastasis using primary breast tumor-initiating cells from patient biopsies"

Carolyn G Marsden et al. BMC Cancer. .

Abstract

Background: The study of breast cancer metastasis depends on the use of established breast cancer cell lines that do not accurately represent the heterogeneity and complexity of human breast tumors. A tumor model was developed using primary breast tumor-initiating cells isolated from patient core biopsies that would more accurately reflect human breast cancer metastasis.

Methods: Tumorspheres were isolated under serum-free culture conditions from core biopsies collected from five patients with clinical diagnosis of invasive ductal carcinoma (IDC). Isolated tumorspheres were transplanted into the mammary fat pad of NUDE mice to establish tumorigenicity in vivo. Tumors and metastatic lesions were analyzed by hematoxylin and eosin (H+E) staining and immunohistochemistry (IHC).

Results: Tumorspheres were successfully isolated from all patient core biopsies, independent of the estrogen receptor α (ERα)/progesterone receptor (PR)/Her2/neu status or tumor grade. Each tumorsphere was estimated to contain 50-100 cells. Transplantation of 50 tumorspheres (1-5 × 103 cells) in combination with Matrigel into the mammary fat pad of NUDE mice resulted in small, palpable tumors that were sustained up to 12 months post-injection. Tumors were serially transplanted three times by re-isolation of tumorspheres from the tumors and injection into the mammary fat pad of NUDE mice. At 3 months post-injection, micrometastases to the lung, liver, kidneys, brain and femur were detected by measuring content of human chromosome 17. Visible macrometastases were detected in the lung, liver and kidneys by 6 months post-injection. Primary tumors variably expressed cytokeratins, Her2/neu, cytoplasmic E-cadherin, nuclear β catenin and fibronectin but were negative for ERα and vimentin. In lung and liver metastases, variable redistribution of E-cadherin and β catenin to the membrane of tumor cells was observed. ERα was re-expressed in lung metastatic cells in two of five samples.

Conclusions: Tumorspheres isolated under defined culture conditions from patient core biopsies were tumorigenic when transplanted into the mammary fat pad of NUDE mice, and metastasized to multiple mouse organs. Micrometastases in mouse organs demonstrated a dormancy period prior to outgrowth of macrometastases. The development of macrometastases with organ-specific phenotypic distinctions provides a superior model for the investigation of organ-specific effects on metastatic cancer cell survival and growth.

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Figures

Figure 1
Figure 1
Tumor formation in the mammary fat pad upon injection of human tumorspheres. A. Light microscopy of a representative tumorsphere isolated from a patient core biopsy following in vitro culture for 10 days. B. Injection of Matrigel alone into the 3rd mammary fat pad of female NUDE mice. C, D. ≤5 × 103 cells derived from the original patient biopsy were injected into the 3rd mammary fat pad in the form of 'tumorspheres' (with Matrigel) and resulted in formation of small, palpable tumors within 3 months injection with an approximate, sustainable tumor volume of 100 mm3
Figure 2
Figure 2
Morphology, proliferation and apoptosis within primary tumors formed upon injection of tumorspheres. A, B. H+E staining of 5 μm paraffin-embedded tumor sections derived from patient Sample 5 at 40× and 200×, respectively. C, E. IHC and quantitation for Ki67 were performed on 5 μm paraffin-embedded tumor sections derived from patients Samples 5-9. Ki67 positive cells and total number of cells were counted using Image J software in five randomly microphotographed images from each tumor sample and represented as the mean % positive cells with SD. D, F. TUNEL staining and quantitation (as described above) for apoptosis. MDA-MB-231 human breast cancer xenograft incubated with 4 U/ml DNase I was used as a positive control for TUNEL. Values are reported as mean +/- SD.
Figure 3
Figure 3
Expression of markers for epithelial and mesenchymal lineages in tumor samples. A-G. Representative IHC demonstrating patterns of expression of E-cadherin, fibronectin, Her2/neu, cytokeratin 8, cytokeratin 14, and aldehyde dehydrogenase 1 (ALDH1) in tumors formed upon the injection tumorspheres into the mammary fat pad of female NUDE mice. 200× magnification in all panels. H. Quantitation of positive staining and total number of cells were counted using Image J software in five randomly microphotographed images from each tumor sample and represented as the mean % positive cells with SD. I. Histological scoring of Her 2 expression within tumor samples. Values are reported as mean +/- SD.
Figure 4
Figure 4
Detection of micrometastasis of human cancer cells in mouse tissues. A. PCR for a centromeric region in human chromosome 17 was used to detect human cells in mouse organs isolated from mice injected with tumorspheres into the mammary fat pad 3 months post-injection. Human DNA was detected in the lungs, kidneys, brain, bone marrow, and liver. DNA isolated from MCF-7 cells and mouse tail was used as a positive and negative control, respectively. B. PCR for DNA isolated from organs collected from a mouse injected with Matrigel alone was also used as a negative control. C-F. Micrographs representing micrometastasis in the lung, liver, brain and kidney, respectively, collected from mice previously injected with tumorspheres. 100× magnification in all panels. Arrows indicate micrometastases surrounded by normal mouse tissue.
Figure 5
Figure 5
Macrometastasis in the organs of mice injected with tumorspheres into the mammary fat pad. A-C. Representative visual macrometastatic lesions detected in the lung, liver and kidney, respectively, 10 months post-injection of tumorspheres into the mammary fat pad of NUDE mice. D-F. H+E staining performed on 5 μm paraffin-embedded section of a lung, liver and kidney, respectively, illustrates macrometastatic lesions in the organs. Metastatic lesion indicated by £; normal mouse tissue indicated by §. 100× magnification in all panels.
Figure 6
Figure 6
Quantitation of the organ tropism of the metastatic cells and the metastatic burden within the mouse organs. A. Comparison of the percentage of total organs that contained metastatic cells (Overall Metastatic Spread) for samples 5-9 as assessed by morphological analysis of H+E stained sections. The total number of mouse organs analyzed is indicated by n above each bar. B. Comparison of the percent of lungs, kidneys, brains and livers that contained metastatic cells (Organ Tropism) for samples 5-9 as assessed by morphological analysis of H+E stained sections. C-E. Graphical representation of the metastatic burden calculated for the total number of lungs, kidneys, brains and livers that were analyzed from mice injected with sample 5-9. The number of mouse organs analyzed is indicated by n in each graph. Each bar represents the calculated metastatic burden for one organ. The metastatic burden in each organ was calculated by dividing the pixels present in the metastatic lesion/s (x pixels) by the total pixels comprising the field of view (y pixels) then multiplying by 100 [(x pixels/y pixels)*100], resulting in a percent value. The percent metastatic burden was calculated from the means from 5 fields of view (100× magnification) per organ analyzed. Values are reported as mean +/- SD.
Figure 7
Figure 7
Expression of markers for epithelial and mesenchymal lineages in metastatic lesions of lung and liver. A-B. IHC performed on 5 μm paraffin-embedded sections of a lung and liver using a monoclonal anti-human antibody to E-cadherin. C-D. IHC performed on 5 μm paraffin-embedded sections of a lung and liver using a polyclonal anti-human antibody to β-catenin. E-F. IHC performed on 5 μm paraffin-embedded sections of a lung and liver using a polyclonal anti-human antibody to fibronectin. G-H. IHC performed on 5 μm paraffin-embedded sections of a lung and liver using a monoclonal anti-human antibody to ERα. G. Inset panel (bottom left) demonstrates negative staining in normal lung cells in proximity to the metastatic lesion in the lung. 200× magnification in all panels.
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