Clinical, molecular and genetic validation of a murine orthotopic xenograft model of pancreatic adenocarcinoma using fresh human specimens.

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

Inhibition of the Growth of Patient-Derived Pancreatic Cancer Xenografts with the MEK Inhibitor Trametinib Is Augmented by Combined Treatment with the Epidermal Growth Factor Receptor/HER2 Inhibitor Lapatinib

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

Comparison of gene expression profiles for human tumors and mouse xenografts.

<p>Unsupervised clustering of gene expression profiles for three normal pancreatic specimens (N580, N190, N67), four pancreatic cancer cell lines (BxPC-3, L3.6pl, PANC-1, MPanc96) and each orthotopically xenografted human PDAC. A hierarchical clustering analysis with the Euclidian distance metric was used to generate agglomerative clusters with average linkage. Hierarchical clustering results are visualized as a dendrogram where closer branches represent samples with similar gene expression. Cell lines are denoted with “C” prefix and tumor lysates denoted with “T” prefix.</p

Overall model schema.

<p>After resection, human pancreatic ductal adenocarcinomas are orthotopically implanted into the pancreases of immunocompromised mice and propagated in subsequent generations. Tumors undergo genetic and proteomic assessment. Clinical and pathologic data are collected for each individual human tumor.</p

Pathological comparisons of human and mouse tumors.

<p>Hematoxylin and eosin (H&E) staining (100×) of human (F₀), early (F₁), and late (F₇, F₉) passage mouse tumors are shown (A), demonstrating preserved histologic architecture and stroma. H&E staining (200×) of a surgically implanted human tumor in the mouse pancreas (B); large arrowheads demonstrate normal pancreatic lobules, stars show interlobular fibrosis/chronic pancreatitis near the invasive tumor front, and smaller arrows demonstrate invading cancer cells.</p

Growth rate and metastatic rate of xenografts predicts patient survival.

<p>Kaplan-Meyer curves of patient survival based on rate of tumor growth (A), and metastasis (B) in F₁ mice.</p

Clinical and pathological comparisons of human and mouse tumors.

1<p>Well-Mod: mild to moderate cytologic atypia, gland forming growth; Poor: marked cellular atypia, solid and single cell growth.</p