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Homotypic or entotic cell-in-cell invasion is an integrin-independent process observed in carcinoma cells exposed during conditions of low adhesion such as in exudates of malignant disease. Although active cell-in-cell invasion depends on RhoA and actin, the precise mechanism as well as the underlying actin structures and assembly factors driving the process are unknown. Furthermore, whether specific cell surface receptors trigger entotic invasion in a signal-dependent fashion has not been investigated. In this study, we identify the G-protein-coupled LPA receptor 2 (LPAR2) as a signal transducer specifically required for the actively invading cell during entosis. We find that G12/13 and PDZ-RhoGEF are required for entotic invasion, which is driven by blebbing and a uropod-like actin structure at the rear of the invading cell. Finally, we provide evidence for an involvement of the RhoA-regulated formin Dia1 for entosis downstream of LPAR2. Thus, we delineate a signaling process that regulates actin dynamics during cell-in-cell invasion.DOI: http://dx.doi.org/10.7554/eLife.02786.001

BackgroundPancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells.Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.Methodology/Principal FindingsThe nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.ConclusionOur results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates.

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BackgroundPancreatic cancer is one of the deadliest of all human malignancies with limited options for therapy. Here, we report the development of an optimized targeted drug delivery system to inhibit advanced stage pancreatic tumor growth in an orthotopic mouse model.Method/Principal FindingsTargeting specificity in vitro was confirmed by preincubation of the pancreatic cancer cells with C225 as well as Nitrobenzylthioinosine (NBMPR - nucleoside transporter (NT) inhibitor). Upon nanoconjugation functional activity of gemcitabine was retained as tested using a thymidine incorporation assay. Significant stability of the nanoconjugates was maintained, with only 12% release of gemcitabine over a 24-hour period in mouse plasma. Finally, an in vivo study demonstrated the inhibition of tumor growth through targeted delivery of a low dose of gemcitabine in an orthotopic model of pancreatic cancer, mimicking an advanced stage of the disease.ConclusionWe demonstrated in this study that the gold nanoparticle-based therapeutic containing gemcitabine inhibited tumor growth in an advanced stage of the disease in an orthotopic model of pancreatic cancer. Future work would focus on understanding the pharmacokinetics and combining active targeting with passive targeting to further improve the therapeutic efficacy and increase survival.