Cytogenetic characterization and gene expression profiling in the rat reflux-induced esophageal tumor model.
OBJECTIVE
The reasons for the increasing incidence of esophageal adenocarcinoma are not clear. A causal relation between gastroesophageal reflux disease and esophageal adenocarcinoma has been suggested. Support for this comes from the development of esophageal adenocarcinoma in the rat reflux model. However, to date, no systematic characterization of the tumors derived from this model has been reported.


METHODS
We induced biliary reflux by creating esophagojejunal anastomoses in 12 Sprague-Dawley rats. The experiment was terminated at 9 months, and rat esophagi were harvested for histopathologic documentation of reflux-associated changes and evidence of tumor formation. Three cell lines were established from 2 of the reflux-associated tumors. We tested the ability of these cells to grow in vitro in tissue culture and in vivo as xenografts in an orthotopic location at the gastroesophageal junction. Furthermore, we performed a cytogenetic analysis and determined the array-based gene expression profiles of these 3 rodent carcinoma lines compared with normal esophageal mucosa.


RESULTS
At 9 months, 12 of 12 rodents had histologic features of metaplastic columnar epithelium in the esophagus, with 7 having invasive carcinomas with glandular differentiation (either adenocarcinomas or adenosquamous carcinomas). The 3 cell lines established from 2 reflux-associated tumors were capable of sustained in vitro propagation and grew successfully as xenografts in both subcutaneous and orthotopic locations, confirming the tumorigenic nature of these lines. Despite their establishment from primary tumors with glandular features, the histology of the xenografts was that of well-differentiated squamous carcinomas. Karyotype analyses demonstrated cytogenetic heterogeneity and aneuploidy; furthermore, translocation (7:11) was present in all 3 lines. Array-based gene expression profiling confirmed upregulation of several cancer-related genes important in human esophageal cancer. Quantitative reverse transcription-polymerase chain reaction was used to confirm the differential expression of selected transcripts (vascular endothelial growth factor [VEGF], polo-like kinases [PLK], cyclin dependent kinase 4 [CDK4], hypoxia-inducible factor 1alpha [HIF1alpha], and insulin-like growth factor 1 [IGF-1]) in comparison with nonneoplastic esophageal mucosal scrapings.


CONCLUSIONS
The rodent reflux model is capable of inducing metaplastic epithelial changes simulating Barrett esophagus, as well as subsequent neoplastic transformation, at a high frequency. Cell lines have been established from these tumors that are capable of in vitro and in vivo passaging. The rodent reflux model should be a valuable model for studying therapy and chemoprevention efforts for Barrett esophagus, whereas the established cell lines provide a useful resource for drug discovery and other high-throughput studies.
Bonde
Pramod
P
Department of Cardiothoracic Surgery, Johns Hopkins University School of Medicine, Baltimore, Md, USA.
Sui
Guoping
G
Dhara
Surajit
S
Wang
Jiaai
J
Broor
Apoorv
A
Kim
Irene F
IF
Wiley
John E
JE
Marti
Guy
G
Duncan
Mark
M
Jaffee
Elizabeth
E
Montgomery
Elizabeth
E
Maitra
Anirban
A
Harmon
John W
JW
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000818:Animals; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000473:pathology*; D020732:Cytogenetic Analysis; D018076:DNA, Complementary / Q000032:analysis; D004195:Disease Models, Animal; D004938:Esophageal Neoplasms / Q000235:genetics* / Q000473:pathology; D005764:Gastroesophageal Reflux / Q000235:genetics* / Q000473:pathology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009374:Neoplasms, Experimental; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014183:Transplantation, Heterologous
D018076:DNA, Complementary