Abstract P2-19-02: mTOR Pathway Inhibitor Nanoparticles Beneficially Modulate Autophagy in Triple Negative Breast Cancer Cells

Background: Autophagy, a cellular degradation pathway, has shown promise as a therapeutic target for many cancers. Depending on the cell type, induction of autophagy has been reported as a cell survival mechanism, making the cells more resistant to common therapies or else a cell death mechanism, synergizing with standard therapeutic agents. We hypothesized that certain nanoparticle-drug combinations could modulate autophagy beneficially for cancer treatment, as they can be targeted to the cells of interest to decrease off-target effects in normal cells. In this study, we propose that nanoparticles containing rapamycin, would induce autophagy in a human triple negative ductal breast cancer cell line, HIM-3 (“human-in-mouse”). Materials and Methods: HIM3 cells were cultured in McCoys 5A supplemented with 10% FBS. HIM3 is a human ductal breast cancer cell line derived from a patient failing chemotherapy that can be grown in vivo in mice. Rapamycin nanoparticles were formulated with a lipid/surfactant coating and a perfluorocarbon (PFC) core. The surfactant layer composition comprised 97.7%phosphatidylcholine, 2mol % DPPE, and 0.28mol% rapamycin. After blending, the slurry was emulsified in one step at 20,000 PSI for 4 min in an ice bath (S110 Microfluidics emulsifier, Microfluidics, Newton, MA). Particle size was measured using dynamic laser light scattering (Brookhaven Instruments Corp., Holtsville, NY). Cell viability assays were performed after 6 days of treatment with Alamar Blue reagent (Invitrogen, Carlsbad, CA). Autophagy flux assays were performed in 6 hour incubations with the nanoparticles and bafilomycin (Sigma, St. Louis, MO) and western blot analysis of LC3B expression for autophagy detection. Western blot analysis was also done to assess phosphorylation of S6 ribosomal protein as evidence for mTOR inhibition. Results: In this study, cell viability decreased (LC50=5nM) in HIM3 when treated with rapamycin nanoparticles. As expected these particles inhibit phosphorylation of ribosomal protein S6, a downstream target of mTOR, by 96% after half an hour of treatment. In order to assess autophagy, an autophagy flux assay was performed over the course of six hours under blockade with bafilomycin treatment to accumulate the marker LC3B. The results from this assay demonstrates a comparable increase in autophagy by the rapamycin nanoparticles as is achieved with a one hundred fold more potent dose of free rapamycin. Discussion: We have demonstrated a novel method for delivering an autophagy modulator to a new line of triple negative breast cancer cells. By increasing direct delivery of the payload to a specific cell type and enhancing trafficking to appropriate intracellular targets, a nanoparticle carrier system appears to be more effective than the free drug in causing a decrease in cell viability through upregulation of autophagic processes. The potential for enhanced therapeutic efficacy with reduced off target effects could emerge as a benefit to this strategy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-19-02.