P-040Phosphatidylserine Targeted Therapy of Pancreatic Cancer Using SapC-DOPS Nanovesicles

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer (∼ 85% cases). PDAC has an extremely poor prognosis with a 5-year relative survival rate < 6%. There is thus an urgent, unmet clinical need to develop truly effective therapeutics. We have created novel protein (saposin C, SapC)-lipid (dioleoylphosphatidylserine, DOPS) nanovesicles, which selectively target a newly identified biomarker (phosphatidylserine, PS) on PDAC tumor cell surfaces. This interaction induces apoptotic cancer cell death in cell cultures and xenografted tumors (Chu et al. PLOS ONE, 8(10), e75507. 2013). We now show significantly enhanced targeting and anticancer activity of SapC-DOPS nanovesicles after induction of increased surface PS on PDAC cells in vitro and in vivo. Methods: Various cell types were analyzed for surface PS exposure by flow cytometry with annexin V-FTIC. Involvement of flippases in the regulation of surface PS exposure was analyzed by flippase activity assay. Total cellular PS was quantified by TLC separation of phospholipids and estimation of PS phosphorous. Orthotopic PDAC tumors were established by injecting human or murine PDAC cells into animal pancreas. In vivo bioluminescence and fluorescence imaging were performed to monitor tumor targeting and growth. The molecular mechanisms underlying the induction of apoptosis by SapC-DOPS were evaluated in cultured PDAC cells through measurements of cell viability, TUNEL, and flow cytometric DNA fragmentation. Results: We demonstrated in a live animal imaging system that fluorescently labeled SapC-DOPS nanovesicles accumulate in orthotopic PDAC tumors via a PS-selective mechanism. We determined that PS exposure on the surface of PDAC cells was variable. Cancer cells exhibited elevated surface PS and fell into low and high surface PS groups. Our results identify differential flippase activity as one of the major regulators of surface PS exposure among cancer cells. We also observed a correlation between total cellular PS and surface PS exposure, with high surface PS cancer cells having relatively high intracellular calcium and total cellular PS compared to low surface PS cells. Chemotherapy (i.e. gemcitabine) was found to increase surface PS levels on PDAC cells. Enhancement of SapC-DOPS anticancer effect was determined by combining chemotherapy in PDAC cells and tumors. Combination treatment with SapC-DOPS and gemcitabine showed a synergistic effect on PDAC cells and tumors, presumably by enhancing SapC-DOPS anticancer potency. Conclusion: SapC-DOPS nanovesicles have PS-specific targeting activity on PDAC cells in orthotopic tumors. PS is heterogeneously exposed on the surface of PDAC cell lines. PS low and high cancer cell lines exhibit differential flippase activity and differ in total cellular PS. Chemodrug treatments elevated surface PS levels on pancreatic cancer cells. Such surface PS increase led to enhancement of SapC-DOPS efficacy in vitro and in vivo.