Abstract NG03: A novel endocytic and intercellular transport pathway for drug delivery across blood vessels and into nutrient-deprived tumor cells
Authors: Pang Hongbo , Braun Gary B. , Friman Tomas , Aza-Blanc Pedro , Ruidiaz Manuel E. , Sugahara Kazuki N. , Teesalu Tambet , Ruoslahti Erkki 
Published: August 2015

Abstract
A major limiting factor in the development of cancer therapy, especially for solid tumors, is the delivery of therapeutic agents into target cells in vivo to reach the site of action. Major barriers to the efficient delivery of drugs, especially macromolecules and nanomaterials, include the vascular walls, extravascular tissue, and membrane of cells and intracellular organelles. Peptides with high affinity to signature surface receptors displayed on tumor vasculature have been attractive carriers of therapeutic and diagnostic agents into tumors. Major efforts have been devoted to complexing these peptides with drug carriers of various physical and chemical properties, while little is known about either the cellular machinery that mediates the intake of these agents, or the underlying regulatory mechanism for delivery efficiency. We tackle this problem by characterizing the cell entry and tissue penetration process of a novel class of tumor-penetrating peptides. These peptides contain a carboxy (C)-terminal, basic sequence R/KXXR/K motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) bind to neuropilin-1 (NRP1) on the cell surface and initiate an endocytic process into cells. Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development, and their expression is often upregulated on tumor vasculature and tumor cells. Many growth factors and other signalling molecules bind to NRPs through the CendR motif. To achieve a tumor-specific homing in vivo, the CendR motif is rendered cryptic in the middle of the peptide. Tumor-homing CendR peptides recognize different primary receptors to achieve the initial homing to the target tissues. The notable example is iRGD (CRGD(K/R)GP(D/E)C; the terminal Cys residues form a disulfide bond; CendR motif underlined), which use the RGD motif to first recognize αv integrins highly expressed on tumor endothelium. The peptide is then proteolytically cleaved to expose the CendR motif at the C-terminus. The activated CendR peptides thus bind to NRP1, which initiate an active penetration process across the blood vessels and throughout the extravascular tumor tissue. This tumor homing and penetration effect has been shown in a wide range of tumor types, and displays a good compatibility with various drug types. Most interestingly, the cargo does not need to be chemically conjugated to the peptide; co-injection with iRGD can also enhance the transport of diverse therapeutic payloads from the circulation into the tumor parenchyma (bystander activity), leading to an improved antitumor efficacy.
To decipher the molecular machinery of CendR-mediated cell entry, we performed a genome-wide RNAi screening to identify important genes for the cellular uptake of a prototypic CendR peptide displayed on silver-based nanoparticles. Silver-based platform is advantageous here since it allows the exclusive tracing of the internalized particles while the extracellular ones are effectively removed by a mild etching procedure. The genome screen and subsequent validation studies demonstrated that NRP1-mediated endocytosis of CendR peptides is a novel cell entry mechanism distinct from known endocytic pathways. CendR endocytosis depends little on critical genes of the other pathways, and is resistance to the treatment of the established endocytic inhibitors. CendR cargo also exhibits little colocalization with structural components of classic endocytic vesicles (e.g. clathrin-coated pits and caveolae) over time. Ultrastructurally, CendR endocytosis resembles macropinocytosis. But they are mechanistically different, especially in the receptor (NRP1) dependence of CendR endocytosis.
The uniqueness of CendR pathway also lies in the regulatory mechanism of its activity. The genome screen surprisingly showed that nutrient-sensing networks, such as mTOR signaling, rank the highest among the canonical pathways regulating the activity of CendR endocytosis. Nutrient deprivation was shown to enhance the penetration of CendR cargo into cells in vitro, live tumor slices ex vivo and tumor tissue in vivo. Moreover, we developed assays to observe the intercellular transport of CendR cargo, which is also stimulated by nutrient deprivation.
We are carrying on subsequent studies to elucidate the structural and molecular basis of CendR transport pathway. A methodology has been developed to synchronize the peptide entry, and ultrastructural imaging pinpoints a step-wise roadmap for the subcellular transport of CendR cargo. We have also performed magnetic isolation of intracellular vesicles containing CendR cargo, and identified signature molecules associated with these vesicles based on proteomic analysis. Moreover, we are able to observe the engulfment of bystander cargo in vitro, and molecular and ultrastructural characterization is undergoing to elucidate this intriguing cellular process. Using zebrafish and mouse models, we are monitoring the vascular penetration in vivo and characterizing the dynamic profile. Meanwhile, we are continuing to decode how mTOR regulates the activity of CendR endocytosis. A transcription factor, Sp1, has been found to serve as a link between mTOR and cell surface NRP1 level. Overall, we have discovered a novel pathway for peptide-functionalized payloads to enter tumor cells and tissue after receptor engagement. The ultrastructural studies of CendR endocytic structures supports the notion that CendR peptides activate a bulk transport system sweeping along bystander compounds, such as drugs and imaging agents present within tumor vessels. The platform to monitor the intercellular transfer of CendR cargo in vitro, together with the in vivo models, enables us to further optimize the transport efficiency of therapeutic agents across the vascular barriers. Particularly, the nutrient regulation of CendR transport pathway is of great significance by linking cancer metabolism with drug delivery. Due to dysfunctional angiogenesis, the nutrient conditions vary significantly in solid tumors. Some regions (e.g. hypoxia) are well known to undergo molecular changes and thus adapt to the nutrient-deprived environment. As a bulk transport pathway, our results suggest a role for CendR pathway in nutrient transport. Moreover, our studies point out a new direction to apply CendR-enhanced drug delivery into nutrient-deprived pathological tissues. These results shed new light in the fields of cell biology, cancer metabolism and drug delivery, while they also highlight the importance to bridge drug delivery with mechanistic studies.
Citation Format: Hongbo Pang, Gary B. Braun, Tomas Friman, Pedro Aza-Blanc, Manuel E. Ruidiaz, Kazuki N. Sugahara, Tambet Teesalu, Erkki Ruoslahti. A novel endocytic and intercellular transport pathway for drug delivery across blood vessels and into nutrient-deprived tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG03. doi:10.1158/1538-7445.AM2015-NG03