Open Access Highly Accessed Research article

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

Carolyn G Marsden1, Mary Jo Wright2, Latonya Carrier3, Krzysztof Moroz4, Radhika Pochampally5 and Brian G Rowan6*

Author Affiliations

1 Department of Structural and Cellular Biology, Tulane University Health Sciences Center, The Louisiana Cancer Research Consortium, New Orleans, LA 70112, USA

2 Department of Surgery, Tulane University School of Medicine, The Louisiana Cancer Research Consortium, New Orleans, LA 70112, USA

3 Department of Structural and Cellular Biology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA

4 Section of Surgical Pathology & Cytopathology, Tulane University School of Medicine, Louisiana Cancer Research Consortium, New Orleans, LA 70112, USA

5 Department of Pharmacology, Tulane University Health Sciences Center, Center for Gene Therapy, Louisiana Cancer Research Consortium, New Orleans, LA, 70,112, USA

6 Department of Structural and Cellular Biology, Tulane University Health Sciences Center, Louisiana Cancer Research Consortium, Center for Gene Therapy, New Orleans, LA 70112, USA

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BMC Cancer 2012, 12:10  doi:10.1186/1471-2407-12-10

Published: 10 January 2012

Additional files

Additional file 1:

Figure S1. Characterization of cell surface marker expression of tumorspheres. A. Immunocytochemistry (ICC) of tumorspheres prepared by formalin fixation and 5 μm paraffin-embedded sections using pre-conjugated antibodies against CD44-PE, and CD24-FITC. ICC for ESA-FITC was performed on tumorspheres prepared by centrifugation onto glass coverslips (cytospins). Tumorspheres demonstrate a CD44+/CD24low-med/ESA+ cell surface marker phenotype. B. Isotype matched, pre-conjugated IgG control antibody mixture (IgG1-PE/IgG2a-FITC) was used as a negative control for ICC. 200× magnification in all panels.

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Additional file 2:

Figure S2. MCF-7 and MDA-MB-231 breast tumor xenografts used as controls for IHC. A-L IHC performed on 5 μm paraffin-embedded sections of MCF-7 (A-F) and MDA-MB-231 (G-L) xenografts using rabbit monoclonal E-cadherin antibody (A+G), rabbit polyclonal β-catenin antibody (B+H), rabbit polyclonal fibronectin antibody (C+I), rabbit monoclonal Her2/ErbB2 antibody (D+J), rabbit polyclonal cytokeratin 8 antibody (E+K), and rabbit monoclonal cytokeratin 14 antibody (F+L). IHC results on MCF-7 and MDA-MB-231 xenograft sections were used as positive and negative controls for the IHC results on tumors formed after injection of tumorspheres in the mammary fat pad (Figure 3). All panels 200× magnification.

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Figure S3. Human nuclear antigen (HNA) staining detects human cells at in the primary tumor and at the metastatic sites. A. 5 μm paraffin-embedded sections of MDA-MB-231 breast tumor xenograft used as a positive control for HNA (mouse anti-human nuclei monoclonal antibody) staining. B. Tumor sample matched negative control, with the replacement of the primary antibody with 1× PBS. C. Kidney isolated from a non-injected NUDE mouse, incubated with HNA to demonstrate human specificity with the lack of nuclear staining of the mouse kidney cells. D. Cells stain positive for HNA in 5 μm paraffin-embedded sections of a tumor removed from the mammary fat pad after injection of tumorspheres. E-F. HNA staining of 5 μm paraffin-embedded sections of metastatic lesions in the liver and lung, respectively confirms the human origin of the lesion, with the majority of nuclei staining positive. All panels 200× magnification.

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Figure S4. Correlation between metastatic burden and the time after injection of tumorspheres that mouse organs were removed. Graphical representation of the percent metastatic burden, previously calculated as described in Figure 6, for each tissue for each sample as a function of the time the organs were removed after initial injection of tumorspheres into the mammary fat pad (Days post-injection). Values are reported as mean +/- SD.

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Additional file 5:

Table S1. Summary of heterogeneous marker expression between primary tumor (mammary fat pad) and metastatic lesions. Tabular representation of the expression of E-cadherin, β-catenin, fibronectin, and ERα between samples in the primary tumor (mammary fat pad), metastatic lesions in the lung and the liver. Cytoplasmic localization is expressed as 'cyto' in the table. 'Variable' indicates the variability of staining within metastatic lesions.

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