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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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 <it>in vivo</it>. Tumors and metastatic lesions were analyzed by hematoxylin and eosin (H+E) staining and immunohistochemistry (IHC).</p> <p>Results</p> <p>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 × 10³cells) 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.</p> <p>Conclusions</p> <p>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.</p

Subcellular Localization of Total and Activated Src Kinase in African American and Caucasian Breast Cancer

Background: Src, a non-receptor tyrosine kinase is elevated in cancer with expression and activity correlated with cell proliferation, adhesion, survival, motility, metastasis and angiogenesis. There is limited data on Src expression and subcellular localization in breast cancer and no information about expression in racial/ethnic groups. Methodology/Principal Findings: The present study evaluated Src expression, activity, and subcellular localization in triple negative breast cancer (TNBC) and ERa positive breast cancer (ER+BC), cancer tissue and adjacent normal epithelial ducts, and Caucasian and African American cases. 79 paraffin embedded breast carcinoma cases were obtained from Tulane University Hospital between 2007–2009. 39 cases represented TNBC (33-African Americans, 4-Caucasians, 2-unknowns) and 40 cases represented ER+BC (21-African Americans, 16-Caucasians, 3-unknowns). Immunohistochemistry was used to measure staining distribution and intensity of total Src and activated phospho-SrcY416 (p-Y416Src) in carcinoma tissue and adjacent normal mammary ducts. In TNBC and ER+BC, total Src was significantly higher in cancer compared to adjacent normal ducts (P,0.0001) in both cell membrane and cytoplasm. In membranes, p-Y416Src was elevated in cancer compared to normal ducts. Total Src in the tumor cytoplasm was significantly higher in TNBC compared to ER+BC (P = 0.0028); conversely, p-Y416Src in the tumor cell membranes was higher in TNBC compared to ER+BC (P = 0.0106). Comparison between African American (n = 21) and Caucasian ER+BC (n = 16) revealed no significant difference in expression an

Disseminated Breast Cancer Cells Acquire a Highly Malignant and Aggressive Metastatic Phenotype during Metastatic Latency in the Bone

<div><h3>Background</h3><p>Disseminated tumor cells (DTCs) in the bone marrow may exist in a dormant state for extended periods of time, maintaining the ability to proliferate upon activation, engraft at new sites, and form detectable metastases. However, understanding of the behavior and biology of dormant breast cancer cells in the bone marrow niche remains limited, as well as their potential involvement in tumor recurrence and metastasis. Therefore, the purpose of this study was to investigate the tumorigenicity and metastatic potential of dormant disseminated breast cancer cells (prior to activation) in the bone marrow.</p> <h3>Methodology/Principal Findings</h3><p>Total bone marrow, isolated from mice previously injected with tumorspheres into the mammary fat pad, was injected into the mammary fat pad of NUDE mice. As a negative control, bone marrow isolated from non-injected mice was injected into the mammary fat pad of NUDE mice. The resultant tumors were analyzed by immunohistochemistry for expression of epithelial and mesenchymal markers. Mouse lungs, livers, and kidneys were analyzed by H+E staining to detect metastases. The injection of bone marrow isolated from mice previously injected with tumorspheres into the mammary fat pad, resulted in large tumor formation in the mammary fat pad 2 months post-injection. However, the injection of bone marrow isolated from non-injected mice did not result in tumor formation in the mammary fat pad. The DTC-derived tumors exhibited accelerated development of metastatic lesions within the lung, liver and kidney. The resultant tumors and the majority of metastatic lesions within the lung and liver exhibited a mesenchymal-like phenotype.</p> <h3>Conclusions/Significance</h3><p>Dormant DTCs within the bone marrow are highly malignant upon injection into the mammary fat pad, with the accelerated development of metastatic lesions within the lung, liver and kidney. These results suggest the acquisition of a more aggressive phenotype of DTCs during metastatic latency within the bone marrow microenvironment.</p> </div

Tumor formation in the mammary fat pad upon injection of total bone marrow aspirates isolated from femurs containing metastatic tumor cells.

<p><b>A.</b> Experimental design to determine the tumorigenicity of disseminated human cancer cells in the bone marrow of mice previously injected with tumorspheres into the mammary fat pad. Bone marrow, aspirated from femurs of mice previously injected with tumorspheres into the mammary fat pad, was injected into the mammary fat pad of NUDE mice to determine the tumor-forming ability of dormant cancer cells in the bone marrow. <b>B.</b> Injection of 12.5×10⁶ cells/pad aspirated from the femurs of mice injected with tumorspheres resulted in large tumor formation in the mammary fat pad two months post-injection. <b>C.</b> Injection of 12.5×10⁶ cells/pad aspirated from the femurs of non-injected mice resulted in no tumor formation in the mammary fat pad three months post injection. <b>D.</b> Summary of tumor formation and metastasis for sample 5–9 BM, including ER/PR/Her2 status of patients samples from which the parental tumorspheres were first derived. <b>E.</b> Representative positive HNA staining of 5 µm paraffin-embedded sections of sample 5 BM tumors demonstrated the presence of human cells. <b>F.</b> No positive nuclear HNA staining of 5 µm paraffin-embedded sections of mouse kidney from non-tumor bearing mouse (negative control). <b>G.</b> Positive HNA staining of 5 µm paraffin-embedded sections of a human MCF-7 xenograft (positive control). 200× magnification in all panels.</p

Metastatic lesions in the lungs of mice bearing mammary fat pad tumors that were derived from transplantation of bone marrow aspirate containing metastatic tumor cells.

<p><b>A–D.</b> H+E staining performed on 5 µm paraffin-embedded sections of lung from sample 5–8 BM illustrates metastatic lesions. Metastatic lesions indicated by £; normal mouse tissue indicated by §. 100× magnification. <b>E–H.</b> IHC performed on 5 µm paraffin-embedded sections of lung from sample 5–8 BM using a monoclonal antibody to E-cadherin. <b>I–L.</b> IHC performed on 5 µm paraffin-embedded sections of lung from sample 5–8 BM using a polyclonal antibody to β-catenin.</p

Expression of markers for epithelial and mesenchymal lineages in tumor samples.

<p><b>A–E.</b> H+E staining of tumors formed in the mammary fat pad upon injection of bone marrow aspirated from the femurs of mice injected with tumorspheres isolated from samples 5–9 BM, respectively. <b>F–T.</b> Representative IHC demonstrating patterns of expression of E-cadherin (<b>F–J</b>), β-catenin (<b>K–O</b>), and fibronectin (<b>P–T</b>) in sample 5–9 BM tumors. 200× magnification in all panels.</p

Metastatic lesions in the livers of mice bearing mammary fat pad tumors that were derived from transplantation of bone marrow aspirate containing metastatic tumor cells.

<p><b>A–D.</b> H+E staining performed on 5 µm paraffin-embedded sections of liver from sample 5–7, and 9 BM illustrates metastatic lesions. Lesion indicated by £; normal tissue indicated by §. 100× magnification. <b>E–H.</b> IHC performed on 5 µm paraffin-embedded sections of liver from sample 5–7, and 9 BM using a monoclonal antibody to E-cadherin. <b>I–L.</b> IHC performed on 5 µm paraffin-embedded sections of liver from sample 5–7, and 9 BM using a polyclonal antibody to β-catenin.</p

Relationship between lymph node status and Src expression/activity/localization in ER+BC.

<p>Mean histoscore values ± SEM (standard error of mean) were calculated for total Src and p-Y416Src expression in lymph node positive and negative cases of ER+BC. Statistical differences in the distribution of Src and p-Y416Src in ER+BC were calculated using the Mann-Whitney <i>U</i> test. *P<0.05 was considered statistically significant.</p