One of the key challenges in anticancer therapy is the toxicity and poor bioavailability of the anticancer drugs. Nanotechnology can play a pivotal role by delivering drugs in a targeted fashion to the malignant cells that will reduce the systemic toxicity of the anticancer drug. In this report, we show a stepwise development of a nanoparticle-based targeted delivery system for in vitro and in vivo therapeutic application in pancreatic cancer. In the first part of the study, we have shown the fabrication and characterization of the delivery system containing gold nanoparticle as a delivery vehicle, cetuximab as a targeting agent, and gemcitabine as an anticancer drug for in vitro application. Nanoconjugate was first characterized physico-chemically. In vitro targeting efficacy, tested against three pancreatic cancer cell lines (PANC-1, AsPC-1, and MIA Paca2) with variable epidermal growth factor receptor (EGFR) expression, showed that gold uptake correlated with EGFR expression. In the second part, we showed the in vivo therapeutic efficacy of the targeted delivery system. Administration of this targeted delivery system resulted in significant inhibition of pancreatic tumor cell proliferation in vitro and orthotopic pancreatic tumor growth in vivo. Tumor progression was monitored noninvasively by measuring bioluminescence of the implanted tumor cells. Pharmacokinetic experiments along with the quantitation of gold both in vitro and in vivo further confirmed that the inhibition of tumor growth was due to targeted delivery. This strategy could be used as a generalized approach for the treatment of a variety of cancers characterized by overexpression of EGFR.

Background and Objectives New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumor-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesize that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC exosomes as the mediator of this lipolysis. Design Exosomes from patient derived cell lines and from plasma of PC patients and non-PC controls were isolated and characterized. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink assay was used to study AM and AM receptor (ADMR) interaction in adipocytes treated with exosomes. Results In murine and human adipocytes we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by AM receptor blockade. AM interacted with its receptor on the adipocytes, activated p38 and ERK1/2 MAPKs and promoted lipolysis by phosphorylating hormone sensitive lipase. PKH67 labeled PC-exosomes were readily internalized into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. Conclusions Pancreatic cancer secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal adrenomedullin is a candidate mediator of this effect.

Background & Aims New-onset diabetes in patients with pancreatic cancer is likely to be a paraneoplastic phenomenon caused by tumor-secreted products. We aimed to identify the diabetogenic secretory product(s) of pancreatic cancer Methods Using microarray analysis, we identified adrenomedullin as a potential mediator of diabetes in patients with pancreatic cancer. Adrenomedullin was up-regulated in pancreatic cancer cell lines, in which supernatants reduced insulin signaling in beta cell lines. We performed quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry on human pancreatic cancer and healthy pancreatic tissues (controls) to determine expression of adrenomedullin messenger RNA and protein, respectively. We studied the effects of adrenomedullin on insulin secretion by beta cell lines and whole islets from mice and on glucose tolerance in pancreatic xenografts in mice. We measured plasma levels of adrenomedullin in patients with pancreatic cancer, patients with type 2 diabetes mellitus, and individuals with normal fasting glucose levels (controls) Results Levels of adrenomedullin messenger RNA and protein were increased in human pancreatic cancer samples compared with controls. Adrenomedullin and conditioned media from pancreatic cell lines inhibited glucose-stimulated insulin secretion from beta cell lines and islets isolated from mice; the effects of conditioned media from pancreatic cancer cells were reduced by small hairpin RNA-mediated knockdown of adrenomedullin. Conversely, overexpression of adrenomedullin in mice with pancreatic cancer led to glucose intolerance. Mean plasma levels of adrenomedullin (femtomoles per liter) were higher in patients with pancreatic cancer compared with patients with diabetes or controls. Levels of adrenomedullin were higher in patients with pancreatic cancer who developed diabetes compared those who did not. Conclusions Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in β cells and mice.

Background & Aims Pancreatic cancer (PC) frequently causes diabetes. We recently proposed adrenomedullin (AM) as a candidate mediator of pancreatic β-cell dysfunction in PC. How PC-derived AM reaches β-cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that PC sheds AM-containing exosomes into circulation which are transported to β-cells and impair insulin secretion. Methods We characterized exosomes from conditioned media of PC-cell lines (n=5) and portal/peripheral venous blood of PC patients (n=20). Western blot analysis showed the presence of AM in PC-Exosomes. We determined the effect of AM-containing PC-Exosomes on insulin secretion from INS-1 β-cells and human islets, and showed how exosomes internalize into β-cells. We studied the interaction between β-cell AM receptors and AM present in PC-Exosomes. In addition, we studied the effect of AM on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β-cells. Results Exosomes were found to be the predominant extracellular vesicles secreted by PC into culture media and human plasma. PC-Exosomes contained AM and CA19-9, readily entered β-cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. AM in PC-Exosomes interacted with its receptor on β-cells. AM receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β-cells exposed to AM or PC-Exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Conclusions Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding AM+/CA19-9+ exosomes into circulation that inhibit insulin secretion, likely through AM-induced ER stress and failure of the UPR.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF or VEGF-A) is a pivotal driver of cancer angiogenesis that is a central therapeutic target in treatment of malignancy. However, little work has been devoted to investigating functions of VEGF that are independent of its pro-angiogenic activity. Here we report that VEGF produced by tumor cells acts in an autocrine manner to promote cell growth through interaction with the VEGF receptor neuropilin-1 (NRP-1). Reducing VEGF expression by tumor cells induced a differentiated phenotype in vitro and inhibited tumor-forming capacity in vivo independent of effects on angiogenesis. Autocrine activation of tumor cell growth was dependent on signaling through NRP-1 and Ras was determined to a critical effector signaling molecule downstream of NRP-1. Our findings define a novel function for VEGF in de-differentiation of tumor cells, expanding its role in cancer beyond its known pro-angiogenic function.