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1.
PLoS One ; 8(10): e77065, 2013.
Article in English | MEDLINE | ID: mdl-24204737

ABSTRACT

BACKGROUND: Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC. METHODS: Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression. RESULTS: Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines. CONCLUSIONS: The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy.


Subject(s)
Adenocarcinoma/genetics , Carcinoma, Pancreatic Ductal/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Pancreatic Neoplasms/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cluster Analysis , Cytokines/metabolism , ErbB Receptors/metabolism , Humans , Kaplan-Meier Estimate , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Mice , Mice, Inbred NOD , Mice, SCID , Mutation , Neoplasm Transplantation , Oligonucleotide Array Sequence Analysis , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Peritoneal Neoplasms/genetics , Peritoneal Neoplasms/metabolism , Peritoneal Neoplasms/secondary , Smad4 Protein/genetics , Transplantation, Heterologous , Tumor Suppressor Protein p53/genetics , ras Proteins/genetics
2.
Neoplasia ; 15(2): 143-55, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23441129

ABSTRACT

Mutations of the oncogene KRAS are important drivers of pancreatic cancer progression. Activation of epidermal growth factor receptor (EGFR) and human EGFR2 (HER2) is observed frequent in pancreatic adenocarcinomas. Because of co-activation of these two signaling pathways, we assessed the efficacy of inhibition of EGFR/HER2 receptors and the downstream KRAS effector, mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK) kinase 1 and 2 (MEK1/2), on pancreatic cancer proliferation in vitro and in a murine orthotopic xenograft model. Treatment of established and patient-derived pancreatic cancer cell lines with the MEK1/2 inhibitor trametinib (GSK1120212) inhibited proliferation, and addition of the EGFR/HER2 inhibitor lapatinib enhanced the inhibition elicited by trametinib in three of eight cell lines. Importantly, in the orthotopic xenograft model, treatment with lapatinib and trametinib resulted in significantly enhanced inhibition of tumor growth relative to trametinib treatment alone in four of five patient-derived tumors tested and was, in all cases, significantly more effective in reducing the size of established tumors than treatment with lapatinib or trametinib alone. Acute treatment of established tumors with trametinib resulted in an increase in AKT2 phosphorylation that was blunted in mice treated with both trametinib and lapatinib. These data indicate that inhibition of the EGFR family receptor signaling may contribute to the effectiveness of MEK1/2 inhibition of tumor growth possibly through the inhibition of feedback activation of receptor tyrosine kinases in response to inhibition of the RAS-RAF-MEK-ERK pathway. These studies provide a rationale for assessing the co-inhibition of these pathways in the treatment of pancreatic cancer patients.


Subject(s)
ErbB Receptors/metabolism , Pancreatic Neoplasms/drug therapy , Pyridones/administration & dosage , Pyrimidinones/administration & dosage , Quinazolines/administration & dosage , Animals , Antineoplastic Agents/administration & dosage , Cell Line, Tumor , ErbB Receptors/antagonists & inhibitors , Humans , Lapatinib , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Signaling System/genetics , Mice , Neoplasm Transplantation , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Phosphorylation/drug effects , Protein Kinase Inhibitors/administration & dosage , Receptor, ErbB-2/antagonists & inhibitors , Signal Transduction/drug effects
3.
Mol Cancer Ther ; 10(11): 2135-45, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21903606

ABSTRACT

Current therapies for pancreatic ductal adenocarcinoma (PDA) target individual tumor cells. Focal adhesion kinase (FAK) is activated in PDA, and levels are inversely associated with survival. We investigated the effects of PF-562,271 (a small-molecule inhibitor of FAK/PYK2) on (i) in vitro migration, invasion, and proliferation; (ii) tumor proliferation, invasion, and metastasis in a murine model; and (iii) stromal cell composition in the PDA microenvironment. Migration assays were conducted to assess tumor and stromal cell migration in response to cellular factors, collagen, and the effects of PF-562,271. An orthotopic murine model was used to assess the effects of PF-562,271 on tumor growth, invasion, and metastasis. Proliferation assays measured PF-562,271 effects on in vitro growth. Immunohistochemistry was used to examine the effects of FAK inhibition on the cellular composition of the tumor microenvironment. FAK and PYK2 were activated and expressed in patient-derived PDA tumors, stromal components, and human PDA cell lines. PF-562,271 blocked phosphorylation of FAK (phospho-FAK or Y397) in a dose-dependent manner. PF-562,271 inhibited migration of tumor cells, cancer-associated fibroblasts, and macrophages. Treatment of mice with PF-562,271 resulted in reduced tumor growth, invasion, and metastases. PF-562,271 had no effect on tumor necrosis, angiogenesis, or apoptosis, but it did decrease tumor cell proliferation and resulted in fewer tumor-associated macrophages and fibroblasts than control or gemcitabine. These data support a role for FAK in PDA and suggest that inhibitors of FAK may contribute to efficacious treatment of patients with PDA.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Pancreatic Ductal/drug therapy , Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Indoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Sulfonamides/pharmacology , Tumor Microenvironment/drug effects , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Pancreatic Ductal/enzymology , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Indoles/therapeutic use , Male , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Metastasis , Protein Kinase Inhibitors/therapeutic use , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Sulfonamides/therapeutic use
4.
Ultrasound Med Biol ; 36(11): 1907-18, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20800945

ABSTRACT

The objective of the study was to examine the role of acoustic power intensity and microbubble and plasmid concentrations on transfection efficiency in HEK-293 cells using a sonoporator with a 1-MHz transducer. A green fluorescent protein (GFP) reporter plasmid was delivered in as much as 80% of treated cells, and expression of the GFP protein was observed in as much as 75% of cells, using a power intensity of 2 W/cm(2) with a 25% duty cycle. In addition, the relative transfection abilities of a lipid noncationic and cationic microbubble platform were investigated. As a positive control, cells were transfected using Lipofectamine reagent. Cell survival and transfection efficiency were inversely proportional to acoustic power and microbubble concentration. Our results further demonstrated that high-efficiency transfection could be achieved, but at the expense of cell loss. Moreover, direct conjugation of plasmid to the microbubble did not appear to significantly enhance transfection efficiency under the examined conditions, although this strategy may be important for targeted transfection in vivo.


Subject(s)
Green Fluorescent Proteins/genetics , Microbubbles , Ultrasonics , Cations , Cell Line , Cell Survival , Cells, Cultured , Flow Cytometry , Gene Expression Regulation , Gene Transfer Techniques , In Vitro Techniques , Microscopy, Fluorescence , Plasmids , Transducers , Transfection
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