Influence of stromal-epithelial interactions on breast cancer in vitro and in vivo.

Stromal cell-secreted chemokines including CCL2 have been implicated in the primary tumor microenvironment, as mediators of tumor cell migration, proliferation, and angiogenesis. Expression of CCL2 and its principal receptor CCR2 was analyzed by RQ-PCR in primary tumor cells and breast cancer cell lines. Breast cancer cell lines (MDA-MB-231, T47D) were co-cultured directly on a monolayer of primary breast tumor and normal stromal cells, retrieved using EpCAM+ magnetic beads, and changes in expression of CCL2, CCR2, MMP11, ELK1, VIL2, and Ki67 detected by RQ-PCR. Epithelial cell migration and proliferation in response to stromal cell-secreted factors was also analyzed. In vivo, tumor xenografts were formed by co-injecting T47D cells with primary tumor stromal cells. Following establishment, tumors were harvested and digested, epithelial cells retrieved and analyzed by RQ-PCR. Whole tumor tissue was also analyzed by immunohistochemistry for CD31 and the VIL2 encoded protein Ezrin. Tumor stromal cells expressed significantly higher levels of CCL2 than normal cells, with no CCR2 expression detected. Primary epithelial cells and breast cancer cell lines expressed elevated CCL2, with relative expression of CCR2 found to be higher than the ligand. Interaction of breast cancer epithelial cells with primary tumor, but not normal stromal cells, stimulated increased expression of CCL2 (8-fold), ELK1 (6-fold), VIL2 (6-fold), and MMP11 (17-fold). Factors secreted by stromal cells, including CCL2, stimulated a significant increase in epithelial cell migration, with no effect on cell proliferation in vitro observed. In vivo, the presence of stromal cells resulted in tumors of increased volume, mediated at least in part through neoangiogenesis demonstrated by immunohistochemistry (CD31). Admixed tumor xenografts exhibited increased expression of Ki67, MMP11, VIL2, and ELK1. Elevated Ezrin protein was also detected, with increased cytoplasmic localization. The results presented highlight mechanisms through which breast cancer epithelial cells can harness stromal cell biology to support tumor progression.

The sodium iodide symporter (NIS) and potential regulators in normal, benign and malignant human breast tissue.

INTRODUCTION: The presence, relevance and regulation of the Sodium Iodide Symporter (NIS) in human mammary tissue remains poorly understood. This study aimed to quantify relative expression of NIS and putative regulators in human breast tissue, with relationships observed further investigated in vitro. METHODS: Human breast tissue specimens (malignant n = 75, normal n = 15, fibroadenoma n = 10) were analysed by RQ-PCR targeting NIS, receptors for retinoic acid (RARα, RARß), oestrogen (ERα), thyroid hormones (THRα, THRß), and also phosphoinositide-3-kinase (PI3K). Breast cancer cells were treated with Retinoic acid (ATRA), Estradiol and Thyroxine individually and in combination followed by analysis of changes in NIS expression. RESULTS: The lowest levels of NIS were detected in normal tissue (Mean(SEM) 0.70(0.12) Log(10) Relative Quantity (RQ)) with significantly higher levels observed in fibroadenoma (1.69(0.21) Log(10)RQ, p<0.005) and malignant breast tissue (1.18(0.07) Log(10)RQ, p<0.05). Significant positive correlations were observed between human NIS and ERα (r = 0.22, p<0.05) and RARα (r = 0.29, p<0.005), with the strongest relationship observed between NIS and RARß (r = 0.38, p<0.0001). An inverse relationship between NIS and PI3K expression was also observed (r =  0.21, p<0.05). In vitro, ATRA, Estradiol and Thyroxine individually stimulated significant increases in NIS expression (range 6-16 fold), while ATRA and Thyroxine combined caused the greatest increase (range 16-26 fold). CONCLUSION: Although NIS expression is significantly higher in malignant compared to normal breast tissue, the highest level was detected in fibroadenoma. The data presented supports a role for retinoic acid and estradiol in mammary NIS regulation in vivo, and also highlights potential thyroidal regulation of mammary NIS mediated by thyroid hormones.

Systemic chemokine levels in breast cancer patients and their relationship with circulating menstrual hormones.

INTRODUCTION: The chemokines Stromal Cell-Derived Factor-1alpha (SDF-1alpha/CXCL12) and Monocyte Chemotactic Protein-1 (MCP-1/CCL2) have been implicated in breast cancer progression. We recently reported elevated systemic MCP-1 in breast cancer patients. This study investigated circulating levels of SDF-1alpha in breast cancer patients, and addressed potential hormonal regulation of these two potent chemokines. METHODS: SDF-1alpha levels were determined by ELISA in 114 breast cancer patients and 85 controls, and correlated with clinical data. Blood samples were collected from 36 healthy premenopausal volunteers weekly for four weeks to measure Luteinising Hormone (LH), Follicular Stimulating Hormone (FSH), Oestradiol and Progesterone using a Bayer ADVIA Centaur Immunoassay system, in parallel with SDF-1alpha and MCP-1. CXCL12 expression was determined using RQ-PCR in primary tumour stromal cells (n = 16) harvested at surgery. RESULTS: Plasma SDF-1alpha was significantly higher in breast cancer patients than age-matched controls and had a significant correlation with tumour grade and epithelial subtype. Investigation of menstrual variations of these chemokines revealed lower SDF-1alpha levels in the mid-luteal phase of the menstrual cycle and a significant positive correlation with circulating Oestradiol. MCP-1 levels showed no correlation with menstrual hormones. There was a trend towards increased CXCL12 expression in tumour compared to normal stromal cells. CONCLUSIONS: The elevated level of SDF-1alpha detected in breast cancer patients, and it's correlation with prognostic indicators, highlights the importance of this chemokine in disease progression. Elucidation of factors influencing chemokine secretion supports clarification of their role in tumourigenesis.