Establishment and characterization of a cholangiocarcinoma cell line (RMCCA-1) from a Thai patient - PubMed (original) (raw)
Establishment and characterization of a cholangiocarcinoma cell line (RMCCA-1) from a Thai patient
Panthip Rattanasinganchan et al. World J Gastroenterol. 2006.
Abstract
Aim: To establish and characterize a new cell line derived from peripheral cholangiocarcinoma of a Thai patient.
Methods: The peripheral cholangiocarcinoma specimen surgically obtained from the patient was aseptically processed by washing and mincing before culturing in Ham's F12 medium containing 10% fetal bovine serum. After 3 mo, when the cell line has become homogeneous and stabilized, several features were investigated, including growth characteristics, immunofluorescence staining for cytokeratins, expression of tumor markers, chromosomal analysis by G-banding and multicolour fluorescence in situ hybridization (mFISH), in vitro migration and invasion characteristics.
Results: The RMCCA-1 cell line has been established. These cells proliferated as a monolayer with a population doubling time of 48 h. Immunofluorescence staining showed positive staining for human cytokeratin 7 and 19 verifying the biliary epithelial origin. RMCCA-1 secreted carbohydrate antigen 19-9 (CA19-9), but insignificant levels of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Chromosome analysis identified aneuploidy karyotypes with a modal chromosome number of 59. RMCCA-1 exhibited a low level of in vitro invasiveness, but a high degree of motility. The cell line exhibited a significant number of chromosomal aberrations as shown by mFISH and G-banding methods.
Conclusion: A new cell line derived from peripheral cholangiocarcinoma of a Thai patient has been established. This cell line shows a low level of in vitro invasiveness, but a high degree of motility. It will serve as a valuable tool for further studies on tumor biology, molecular pathogenesis, metastatic mechanism and response to therapeutic drugs of cholangiocarcinoma.
Figures
Figure 1
Hematoxylin and Eosin staining of the liver specimen (10 × magnification). The histopathological analysis of the specimen indicates a well-differentiated peripheral cholangio-carcinoma with no vascular invasion.
Figure 2
The RMCCA-1 culture under a phase contrast microscope at 20 × magnification. (A) at 16th passage; (B) at 30th passage. The RMCCA-1 cells exhibited circular to spindle shape with many processes and ornamental fringes. The nucleus and cytoplasm appeared granulated.
Figure 3
Growth kinetics of the RMCCA-1 cell line in vitro as analyzed by MTT assay. The tumor doubling time during the exponential phase of growth was 48 h. Each point represents mean ± SE from 3 independent experiments, each performed in triplicates.
Figure 4
Immunofluoresence staining of RMCCA-1 cells with antibodies against (A) Cytokeratin 7; (B) AE-1/AE-3 at 40 × magnification.
Figure 5
Representative karyotypes of RMCCA-1 cell line as assessed by (A) G-banding; (B) mFISH technique. The karyotype showed 54-61(3n)XX, -Y, der(1), t(1;2;7)(q31;q31;q22), t(2;17)(q33;q12), der(3)t(3;15)(q21;q?), t(4;17)(p14;q?), der(7)t(7;15)(p22;q?), der(8)t(8;7)(p12;?), der(10)t(10;13)(q21;q11), der(12)t(11;12)(q?,p11.2), der(13)t(13;9)(q11;q11), der(14)t(15;14)(q34;q32), der(16)t(13;16)(q11;p13.3), der(17)t(X;10;17)(p?,p?,p11.2), der(19)t(10;19)(q11.2;p13.3), der(19)t(17;19)(q?;q?), der(21)t(1;9;21)(?;?;q11), der(22)t(1;15;22)(?,?,?).
Figure 6
Invasion and migration rates of RMCCA-1 compared with KKU-213, KKU-100 and HuCCA-1 cell lines as determined by in vitro invasion and motility assay. Approximately 1 × 105 cells were seeded into the upper chamber of a transwell, and incubated for 6 h before the filter was fixed, stained, and the invading/migrating cells were counted under microscope.
Figure 7
MMPs activity as determined by gelatin zymography. An intense band at Mr 72 000 was shown in the FBS (fetal bovine serum) and the conditioned medium of KKU-100, but not of the RMCCA-1 cells. The Mr 72 000 band in KKU-100 has previously been shown to correspond to MMP-2 activity in many cell lines[11,12].
References
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