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Biological characteristics of fluorescent protein-expressing human hepatocellular carcinoma xenograft model in nude mice

Objectives To study biological characteristics of stable red fluorescent protein (RFP)-expressing or green fluorescent protein (GFP)-expressing HCCLM3 cell lines and those of their relevant xenograft models in nude mice. Methods HCCLM3, a human hepatocellular carcinoma cell line with high metastatic potential was infected with RFP or GFP full-length cDNA via lentivirus. Stable RFP-expressing or GFP-expressing HCCLM3 cells, namely HCCLM3-R and HCCLM3-G, were subcutaneously injected and two patient-like metastatic models of HCCLM3-R and HCCLM3-G in nude mice were established using surgical orthotopic implantation from subcutaneous tumor tissues. Cell proliferation, karyotype, biomarker expression, tumor growth, and metastasis of HCCLM3-R and HCCLM3-G were analyzed in vitro and in vivo. Results RFP and GFP genes were integrated in genomic DNA of HCCLM3. HCCLM3-R and HCCLM3-G expressed red and green fluorescence, stable and intense, 300 days after 60 consecutive passages, and also positively expressed CK8+, P16+, AFP+ and negatively expressed HBsAg−. Their biomarker expression and karyotype were found to be similar to those of the parental HCCLM3, and their tumorigenesis occurred in 10 nude mice without exception after a subcutaneous injection and did the same in 20 nude mice after an orthotopic implantation. The results showed that the rate of spontaneous metastasis to the liver and lung and peritoneal seeding was 100, 100, and 90%, respectively. Conclusion Stable fluorescent protein-expressing HCCLM3-R and HCCLM3-G xenografts in nude mice could be of two useful models for studying mechanisms of hepatocellular carcinoma growth and metastasis in real time.