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 a) in vitro migration, invasion and proliferation, b) tumor proliferation, invasion and metastasis in a murine model, and c) stromal cell composition in the PDA microenvironment. Migration assays were performed 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 are activated and expressed in patient-derived PDA tumors, stromal components and human PDA cell lines. PF-562,271 blocked phosphorylation of FAK 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 did decrease tumor cell proliferation and resulted in fewer tumor-associated macrophages and fibroblasts compared to 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.

SUMMARY Mitochondria undergo fission and fusion to maintain homeostasis, and tumors exhibit the dysregulation of mitochondrial dynamics. We recently demonstrated that ectopic HRas G12V promotes mitochondrial fragmentation and tumor growth through Erk phosphorylation of the mitochondrial fission GTPase Dynamin-related protein 1 (Drp1). However, the role of Drp1 in the setting of endogenous oncogenic KRas remains unknown. Here, we show that Drp1 is required for KRas-driven anchorage-independent growth in fibroblasts and patient-derived pancreatic cancer cell lines, and it promotes glycolytic flux, in part through the regulation of hexokinase 2 (HK2). Furthermore, Drp1 deletion imparts a significant survival advantage in a model of KRas-driven pancreatic cancer, and tumors exhibit a strong selective pressure against complete Drp1 deletion. Rare tumors that arise in the absence of Drp1 have restored glycolysis but exhibit defective mitochondrial metabolism. This work demonstrates that Drp1 plays dual roles in KRas-driven tumor growth: supporting both glycolysis and mitochondrial function through independent mechanisms.

Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and adjuvant chemotherapy have an expected survival of only 2 years due to disease recurrence, frequently in the liver. We investigated the role of liver macrophages in progression of PDAC micrometastases to identify adjuvant treatment strategies that could prolong survival. A murine splenic injection model of hepatic micrometastatic PDAC was used with five patient-derived PDAC tumors. The impact of liver macrophages on tumor growth was assessed by (i) depleting mouse macrophages in nude mice with liposomal clodronate injection, and (ii) injecting tumor cells into nude versus NOD--gamma mice. Immunohistochemistry and flow cytometry were used to measure CD47 ("don't eat me signal") expression on tumor cells and characterize macrophages in the tumor microenvironment. engulfment assays and mouse experiments were performed with CD47-blocking antibodies to assess macrophage engulfment of tumor cells, progression of micrometastases in the liver and mouse survival. clodronate depletion experiments and NOD--gamma mouse experiments demonstrated that liver macrophages suppress the progression of PDAC micrometastases. Five patient-derived PDAC cell lines expressed variable levels of CD47. In engulfment assays, CD47-blocking antibodies increased the efficiency of PDAC cell clearance by macrophages in a manner which correlated with CD47 receptor surface density. Treatment of mice with CD47-blocking antibodies resulted in increased time-to-progression of metastatic tumors and prolonged survival. These findings suggest that following surgical resection of PDAC, adjuvant immunotherapy with anti-CD47 antibody could lead to substantially improved outcomes for patients. .

BackgroundRelevant 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.MethodsHuman 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.ResultsFifteen 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.ConclusionsThe 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.