Establishment of clear cell renal cell carcinoma lines of Han nationality in China / 第二军医大学学报

ABSTRACT

Objective:To establish clear cell renal cell carcinoma(ccRCC)cell lines from clinical ccRCC specimens of Han nationality in China and to characterize the biological features.Methods:From 2005 to 2007,fresh surgical samples of ccRCC were obtained from 43 patients;the samples included primary tumor in situ,osseous metastasis,lymph node metastasis,and cancerous embolus.The samples were cultured in vitro using explant-culture method within 30-60 rain after surgery.Analysis on cell growth and colony-forming efficiency was recorded for the lines which were passaged for over 50 generations.Chromosome examination,pathological examination and tumorigenesis in NOD-SCID mice were used to determine their malignancy.Flow cytometry was used to determine expression of CA9 and CD133.Results:Most of the primary cells could only be passaged for less than 5 generations;5 lines could be serially passaged for over 5 passages,3 lines for over 10 passages,and only 2 lines could be stably passaged.One line,named RCC05-TXJ,was from osseous metastatic ccRCC and had been serially passaged for 110 generations in 21 months;the average doubling time was 19.2 h,average chromosome number was 75,and colony forming efficiency was 41%.Another line,named RCC05-ZYJ,was from primary ccRCC specimen and had been serially passaged for 160 generations in 18 months;the average doubling time was 16.5 h,average chromosome number was 55,and the colony forming efficiency was 37%.Immunohistological analysis demonstrated that both lines expressed CA9 and CD133.Flow cytometry analysis found that expression levels of CA9 and CD133 increased with the passages.Both RCC05-ZYJ and RCC05-TXJ lines were able to form tumor and to metastasize in NOD-SCID mice;however,their metastatic ability was obviously different. Conclusion:We have established 2 ccRCC cell lines with different metastatic potentials from the clinical ccRCC specimens of Han nationality in China.The ratio of tumor stem cells increases with the passages.