Purpose: Major barriers to effective adenovirus-based gene therapy include induction of an immune response and tumor-specific targeting of vectors. The use of mesenchymal stem cells (MSC) as systemic delivery vehicles for therapeutic genes has been proposed as a result of their combined ability to home in on the tumor site and evade the host immune response. This study is aimed at investigating factors mediating homing of human MSCs to breast cancer primary cultures and cell lines in vitro and in vivo. Experimental Design: Fluorescently labeled MSCs were given to mice bearing breast cancer xenografts, and tumor tissue was harvested to detect MSC engraftment. MSC migration in response to primary breast tumors in vitro was quantified, and chemokines secreted by tumor cells were identified. The role of monocyte chemotactic protein-1 (MCP-1) in cell migration was investigated using antibodies and standards of the chemokine. Serum MCP-1 was measured in 125 breast cancer patients and 86 healthy controls. Results: Engrafted MSCs were detected in metastatic breast tumors in mice after systemic administration. There was a significant increase in MSC migration in response to primary breast tumor cells in vitro (6-fold to 11-fold increase). Tumor explants secreted a variety of chemokines including GROa, MCP-1, and stromal cell^derived factor-1a. An MCP-1antibody caused a significant decrease (37-42%) in MSC migration to tumors. Serum MCP-1 levels were significantly higher in postmenopausal breast cancer patients than age-matched controls (P < 0.05).Conclusions: These results highlight a role for tumor-secreted MCP-1in stimulating MSC migration and support the potential of these cells as tumor-targeted delivery vehicles for therapeutic agents.

Bone marrow derived Mesenchymal Stem Cells (MSCs) are known to specifically migrate to and engraft at tumor sites. Understanding interactions between cancer cells and MSCs has become fundamental to determining whether MSC-tumour interactions should be harnessed for delivery of therapeutic agents or considered a target for intervention. Breast Cancer Cell lines (MDA-MB-231, T47D & SK-Br3) were cultured alone or on a monolayer of MSCs, and retrieved using epithelial specific magnetic beads.Alterations in expression of 90 genes associated with breast tumorgenicity were analyzed using low density array. Expression of markers of Epithelial-Mesenchymal transition and array results were validated using RQ-PCR. Co-cultured cells were analyzed for changes in protein expression, growth pattern, and morphology. Gene expression and proliferation assays were also performed on indirect co-cultures. Following direct co-culture with MSCs, breast cancer cells expressed elevated levels of oncogenes (NCOA4, FOS), protooncogenes (FYN, JUN), genes associated with invasion (MMP11), angiogenesis (VEGF)and anti-apoptosis (IGF1R, BCL2). However, universal downregulation of genes associated with proliferation was observed (Ki67, MYBL2), and reflected in reduced ATP production in response to MSC-secreted factors. Significant upregulation of Epithelial-Mesenchymal Transition specific markers (N-cadherin, Vimentin, Twist and Snail) was also observed following co-culture with MSCs, with a reciprocal downregulation in E-cadherin protein expression. These changes were predominantly cell contact mediated and appeared to be MSC specific. Breast cancer cell morphology and growth pattern also altered in response to MSCs. Mesenchymal Stem Cells may promote breast cancer metastasis through facilitation of Epithelial-Mesenchymal Transition.

Introduction

IntroductionThe 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.MethodsHuman breast tissue specimens (malignant n = 75, normal n = 15, fibroadenoma n = 10) were analysed by RQ-PCR targeting NIS, receptors for retinoic acid (RARα, RARβ), oestrogen (ERα), thyroid hormones (THRα, THRβ), 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.ResultsThe lowest levels of NIS were detected in normal tissue (Mean(SEM) 0.70(0.12) Log10 Relative Quantity (RQ)) with significantly higher levels observed in fibroadenoma (1.69(0.21) Log10RQ, p<0.005) and malignant breast tissue (1.18(0.07) Log10RQ, 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 RARβ (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).ConclusionAlthough 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.

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