Abstract
Abstract
Increased expression of CCL2 correlates with poor patient survival and development of invasive breast cancer. CCL2 regulates mammary tumor progression by recruitment of tumor promoting macrophages. Our laboratory and others have shown that CCL2 signaling mediates stromal: tumor interactions to regulate breast cancer cell survival, invasion and metastasis. While the CCL2 signaling represents a novel and potentially effective therapeutic target in invasive breast cancer, it is necessary to develop new tools to specifically inhibit CCL2 signaling in vivo. siRNA knockdown could specifically target CCL2, but is unstable and toxic when delivered in vivo. The HIV-1 derived TAT peptide penetrates cell plasma membranes at high efficiency and contains nuclear translocation signals to enhance siRNA delivery to the nucleus. Recent studies from our research team have shown that CaCl2 condenses TAT/nucleic acids to nanoparticles facilitating cellular uptake. The goals of these studies are to design TAT nanoparticle formulas to target CCL2 protein expression in breast tumors. Using an in vitro luciferase reporter system, we identified specific N/P (TAT:siRNA) ratios and CaCl2 concentrations that would efficiently deliver siRNAs to PyVmT mammary tumor cells. Utilizing a novel ex vivo mammary tumor system, TAT/CaCl2 nanocomplexes carrying GFP plasmid or Cy3 labeled siRNAs were then injected into PyVmT mammary tumors. We observed visible GFP expression and Cy3 labeled siRNA uptake in over 50% of the PyVmT mammary tumor. Flow cytometry analysis showed that low N/P ratio resulted in efficient TAT/CaCl2/siRNA nanoparticle uptake in tumor epithelial cells. Normal mammary tissues did not show significant uptake of TAT nanoparticles. CCL2 knockdown was observed in tumor epithelial cells, but not stromal cells as determined by flow cytometry. Similar results were observed in cultured cells. At low N/P ratio, PyVmT tumor cells showed a 50% reduction in CCL2 expression upon delivery of TAT/CaCl2 nanoparticles carrying CCL2 siRNAs, while there was no significant effect on stromal cells as determined by ELISA. Downregulation of CCL2 in the tumor cells significantly decreased proliferation as determined by flow cytometry analysis. Importantly, treatment with TAT/siRNA nanoparticle did not significantly affect viability of normal cells. These data indicate this nanoparticle strategy represents a novel, feasible approach to target the CCL2 signaling in breast cancer, with important implications on therapeutic targeting.
Citation Format: Wei Bin Fang, Nabil Alhakamy, Cory Berkland, Nikki Cheng. Targeting the CCL2 chemokine pathway in breast tumors through intratumoral delivery of calcium cross linked TAT peptide: siRNA complexes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A063.