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Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung.

Kalinina T, Güngör C, Thieltges S, Möller-Krull M, Penas EM, Wicklein D, Streichert T, Schumacher U, Kalinin V, Simon R, Otto B, Dierlamm J, Schwarzenbach H, Effenberger KE, Bockhorn M, Izbicki JR, Yekebas EF - BMC Cancer (2010)

Bottom Line: Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3).Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6.Subcutaneous transplantation of PaCa 5061 into pfp-/-/rag2-/- mice resulted in formation of primary tumors and spontaneous lung metastasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of General, Visceral and Thoracic Surgery, University Hospital Hamburg, Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. tkalinin@uke.de

ABSTRACT

Background: Pancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line.

Methods: The newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-ras, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp-/-/rag2-/- mice. Sensitivity towards chemotherapy was analysed by MTT assay.

Results: PaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with multiple numerical and unbalanced structural aberrations. Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3). Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6. Subcutaneous transplantation of PaCa 5061 into pfp-/-/rag2-/- mice resulted in formation of primary tumors and spontaneous lung metastasis.

Conclusion: The established PaCa 5061 cell line and its injection into pfp-/-/rag2-/- mice can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.

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Cell morphology and growth characteristics of PaCa 5061 cells. 1A. Morphological examination of non-confluent and confluent PaCa 5061 cells by phase contrast microscopy. The cells grew in a monolayer with polygonal shape, epithelial morphology and large nuclei. 1B. Growth curve of PaCa 5061 in culture. Proliferation of cells (1000/well) was determined every 24 hours and relative growth rates were analyzed over time (0-144 h) by MTT assay. The results shown are for an experiment representative of three independent assays.
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Figure 1: Cell morphology and growth characteristics of PaCa 5061 cells. 1A. Morphological examination of non-confluent and confluent PaCa 5061 cells by phase contrast microscopy. The cells grew in a monolayer with polygonal shape, epithelial morphology and large nuclei. 1B. Growth curve of PaCa 5061 in culture. Proliferation of cells (1000/well) was determined every 24 hours and relative growth rates were analyzed over time (0-144 h) by MTT assay. The results shown are for an experiment representative of three independent assays.

Mentions: A new pancreatic carcinoma cell line designated PaCa 5061 was generated from a resected PDAC. Within the first 2 weeks, the tumor cell clusters adhered to the surface of the cell culture flasks and gradually formed cell colonies. Initially, contaminating fibroblastic cells proliferated and surrounded the tumor cell colonies. During serial passages, the number of fibroblastic cells gradually decreased and was replaced entirely by tumor cells (Figure 1A). All cells adhered tightly to the bottom of the cell culture flasks in a monolayer, and were characterized as polygonal epithelial-like cells with large nuclei containing several nucleoli. The appearance of PaCa 5061 cells was polygonal. The population doubling time ranged from 30 to 48 hours (Figure 1B). The epithelial phenotype was confirmed by the positive immunoreactivity for cytokeratin and the expression of the pancreatic tumor markers CEA and CA 19-9 (Figure 2A). The same pattern of immunoreactivity was observed in the primary tumor of patient. FACS analysis of PaCa 5061 cell population revealed homogeneous expression levels for EpCAM (98.7%), CD44 (100%), EGFR (83.5%) as well as CEACAM 1 (93.3%) whereas heterogeneous expression levels was observed for CEACAM 5 (24.7%) and CEACAM 6 (69.7%) (Figure 2B).


Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung.

Kalinina T, Güngör C, Thieltges S, Möller-Krull M, Penas EM, Wicklein D, Streichert T, Schumacher U, Kalinin V, Simon R, Otto B, Dierlamm J, Schwarzenbach H, Effenberger KE, Bockhorn M, Izbicki JR, Yekebas EF - BMC Cancer (2010)

Cell morphology and growth characteristics of PaCa 5061 cells. 1A. Morphological examination of non-confluent and confluent PaCa 5061 cells by phase contrast microscopy. The cells grew in a monolayer with polygonal shape, epithelial morphology and large nuclei. 1B. Growth curve of PaCa 5061 in culture. Proliferation of cells (1000/well) was determined every 24 hours and relative growth rates were analyzed over time (0-144 h) by MTT assay. The results shown are for an experiment representative of three independent assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927995&req=5

Figure 1: Cell morphology and growth characteristics of PaCa 5061 cells. 1A. Morphological examination of non-confluent and confluent PaCa 5061 cells by phase contrast microscopy. The cells grew in a monolayer with polygonal shape, epithelial morphology and large nuclei. 1B. Growth curve of PaCa 5061 in culture. Proliferation of cells (1000/well) was determined every 24 hours and relative growth rates were analyzed over time (0-144 h) by MTT assay. The results shown are for an experiment representative of three independent assays.
Mentions: A new pancreatic carcinoma cell line designated PaCa 5061 was generated from a resected PDAC. Within the first 2 weeks, the tumor cell clusters adhered to the surface of the cell culture flasks and gradually formed cell colonies. Initially, contaminating fibroblastic cells proliferated and surrounded the tumor cell colonies. During serial passages, the number of fibroblastic cells gradually decreased and was replaced entirely by tumor cells (Figure 1A). All cells adhered tightly to the bottom of the cell culture flasks in a monolayer, and were characterized as polygonal epithelial-like cells with large nuclei containing several nucleoli. The appearance of PaCa 5061 cells was polygonal. The population doubling time ranged from 30 to 48 hours (Figure 1B). The epithelial phenotype was confirmed by the positive immunoreactivity for cytokeratin and the expression of the pancreatic tumor markers CEA and CA 19-9 (Figure 2A). The same pattern of immunoreactivity was observed in the primary tumor of patient. FACS analysis of PaCa 5061 cell population revealed homogeneous expression levels for EpCAM (98.7%), CD44 (100%), EGFR (83.5%) as well as CEACAM 1 (93.3%) whereas heterogeneous expression levels was observed for CEACAM 5 (24.7%) and CEACAM 6 (69.7%) (Figure 2B).

Bottom Line: Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3).Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6.Subcutaneous transplantation of PaCa 5061 into pfp-/-/rag2-/- mice resulted in formation of primary tumors and spontaneous lung metastasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of General, Visceral and Thoracic Surgery, University Hospital Hamburg, Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. tkalinin@uke.de

ABSTRACT

Background: Pancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line.

Methods: The newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-ras, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp-/-/rag2-/- mice. Sensitivity towards chemotherapy was analysed by MTT assay.

Results: PaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with multiple numerical and unbalanced structural aberrations. Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3). Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6. Subcutaneous transplantation of PaCa 5061 into pfp-/-/rag2-/- mice resulted in formation of primary tumors and spontaneous lung metastasis.

Conclusion: The established PaCa 5061 cell line and its injection into pfp-/-/rag2-/- mice can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.

Show MeSH
Related in: MedlinePlus