crizotinib and Carcinoma--Pancreatic-Ductal

The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations.

Topics: Adenocarcinoma; Animals; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Proliferation; Chickens; Chorioallantoic Membrane; Crizotinib; Deoxycytidine; DNA Mutational Analysis; Drug Evaluation, Preclinical; Gemcitabine; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 6; Luminescent Measurements; MicroRNAs; Models, Biological; Pancreatic Neoplasms; Sequence Analysis, DNA; SOX9 Transcription Factor; Tumor Cells, Cultured

Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclinical tests fail in these patients, emphasizing the need of improved preclinical models to test novel anticancer strategies. Here, we developed four orthotopic mouse models using primary human PDAC cells genetically engineered to express firefly- and Gaussia luciferase, simplifying the ability to monitor tumor growth and metastasis longitudinally in individual animals with MRI and high-frequency ultrasound. In these models, we conducted detailed histopathologic and immunohistochemical analyses on paraffin-embedded pancreatic tissues and metastatic lesions in liver, lungs, and lymph nodes. Genetic characteristics were compared with the originator tumor and primary tumor cells using array-based comparative genomic hybridization, using frozen specimens obtained by laser microdissection. Notably, the orthotopic human xenografts in these models recapitulated the phenotype of human PDACs, including hypovascular and hypoxic areas. Pursuing genomic and immunohistochemical evidence revealed an increased copy number and overexpression of c-Met in one of the models; we examined the preclinical efficacy of c-Met inhibitors in vitro and in vivo. In particular, we found that crizotinib decreased tumor dimension, prolonged survival, and increased blood and tissue concentrations of gemcitabine, synergizing with a cytidine deaminase-mediated mechanism of action. Together, these more readily imaged orthotopic PDAC models displayed genetic, histopathologic, and metastatic features similar to their human tumors of origin. Moreover, their use pointed to c-Met as a candidate therapeutic target in PDAC and highlighted crizotinib and gemcitabine as a synergistic combination of drugs warranting clinical evaluation for PDAC treatment.

Topics: Animals; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Crizotinib; Deoxycytidine; Female; Gemcitabine; Humans; Inactivation, Metabolic; Mice; Mice, Nude; Pancreatic Neoplasms; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Pancreatic-ductal-adenocarcinoma (PDAC) is amongst the most lethal malignancies, mainly because of its metastatic spread and multifactorial chemoresistance. Since c-Met is a marker of pancreatic-cancer-stem-cells (CSC), playing a key role in metastasis and chemoresistance, this study evaluated the therapeutic potential of the novel c-Met/ALK inhibitor crizotinib against PDAC cells, including the Capan-1-gemcitabine-resistant cells (Capan-1-R). Crizotinib inhibited PDAC cell-growth with IC50 of 1.5 μM in Capan-1-R, and synergistically enhanced the antiproliferative and proapoptotic activity of gemcitabine, as detected by sulforhodamine-B-assay, flow cytometry and combination-index method. Capan-1-R had higher expression of the CSC markers CD44+/CD133+/CD326+, but their combined expression was significantly reduced by crizotinib, as detected by quantitative-RT-PCR and FACS-analysis. Similarly, Capan-1-R cells had significantly higher protein-expression of c-Met (≈2-fold), and increased migratory activity, which was reduced by crizotinib (e.g., > 50% reduction of cell-migration in Capan-1-R after 8-hour exposure, compared to untreated-cells), in association with reduced vimentin expression. Capan-1-R had also significantly higher mRNA expression of the gemcitabine catabolism-enzyme CDA, potentially explaining the higher CDA activity and statistically significant lower levels of gemcitabine-nucleotides in Capan-1-R compared to Capan-1, as detected by Liquid-chromatography-massspectrometry. Conversely, crizotinib significantly reduced CDA expression in both Capan-1 and Capan-1-R cells. In aggregate, these data show the ability of crizotinib to specifically target CSC-like-subpopulations, interfere with cell-proliferation, induce apoptosis, reduce migration and synergistically interact with gemcitabine, supporting further studies on this novel therapeutic approach for PDAC.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromatography, Liquid; Crizotinib; Deoxycytidine; Drug Resistance, Neoplasm; Drug Synergism; Gemcitabine; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Mass Spectrometry; Neoplastic Stem Cells; Pancreatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines