Establishment And Oncobiological Characteristics Of Stable Fluorescent Protein-expressing Human HCC Xenograft Model In Nude Mice With High Metastatic Potential

Background:Hepatocellular carcinoma(HCC) is one of major health problems in China,and more than 50%of new diagnosed cases worldwide are found every year in China.Although therapeutic interventions such as surgical resection,chemotherapy, and radiotherapy are clinically available,about 60%of HCC patients will develop a recurrence or/and a metastasis in the first 5 years and their overall survival rate is still less than 5%.The dismal outcomes of HCC may be attributed to no subclinical sign and easy spread in the early stage,restrictively repeated use of existing therapeutical interventions due to severe side-effects,dysfunctions of normal liver tissues,and no efficient inhibitors to tumor metastasis.In order to elucidate molecular mechanisms of HCC recurrence and metastasis, the researchers of Liver Cancer Institute of Fudan University have succeeded in establishing human HCC xenografts in nude mice,namely LCI-D20 and LCI-D35,via orthotopic transplantation of HCC patient intact tumor tissues as well as in cloning several metastatic human HCC cell lines such as MHCC97,HCCLM3 and HCCLM6 from LCI-D20 xenograft.Although these in vitro and in vivo HCC models provide us a useful platform for the study of tumor growth and metastasis as well as for the pre-clinical screen of antitumor medicine,HCC growth after orthotopic transplantation in nude mice can not be observed in real time,and spontaneous metastasis to the liver and lung and peritoneal seeding can not be quantitatively analyzed,either.Therefore,it was imperative that a real-time HCC xenograft model be developed in nude mice.Fluorescent protein-expressing HCC cell lines and xenograft models in nude mice may provide the best solution to real-time observation of tumor growth and metastasis. Such fluorescent protein-expressing tumor models as melanoma and prostate cancer cells have been successfully established.However,there was a dearth of literature on metastatic fluorescent protein-expressing HCC cell lines and xenograft models.The present study pioneered the successful establishment of two stable green or red fluorescent protein-expressing HCC cell lines,HCCLM3-G and HCCLM3-R,via lentivirus and their relevant xenograft models in nude mice,for examining their onco-biological characteristics.Objectives:Establish and study onco-biological characteristics of stable RFP- or GFP-expressing HCCLM3 cell lines and those of their relevant xenograft models with high metastatic potential in nude mice.These models might be used for further molecular studies on tumor metastasis,angiogenesis and also be applied to anti-tumor drug screening in pre-clinical stage.1.Establishment and cell-biological characteristics of stable fluorescent protein-expressing hepatocellular carcinoma cell linesWe tried to infect HCCLM3 cells with enhanced GFP or RPF gene via pseudo-lentivirus,an efficient gene transfer vehicle for human tumor cells,and obtained two more stable fluorescent protein-expressing tumor cell lines,named HCCLM3-G/HCCLM3-R.Three hundred days after 60 consecutive in-vitro cell passages,HCCLM3-G and HCCLM3-R expressed green or red fluorescence,stable and intense.Our PCR results indicated that GFP and RFP encoding cDNA integrated into the genome of HCCLM3 cells introduced by pseudo-lentivirus.The study indicated that the main cell-biological characteristics of two new cells such as cell morphological,proliferation,karyotypes and expression pattern of biomarkers are similar to those of their parental cells.To develop a more objective and convenient method for cell proliferation analysis in fluorescent protein-expressing cells,we examined the cell proliferation of HCCLM3-G and HCCLM3-R by quantitating the fluorescent pixels and numbering the cells,with which the growth curves of HCCLM3-G and HCCLM3-R were found to be similar to each other.The results indicated that cell proliferation analysis based on fluorescent image was practicable.This approach allowed us to analyze cell proliferation in rapidity,sensitivity,repetitiveness and succession without any interference on cell growth and could be applied to anti-tumor drug screening in pre-clinical stage.2.Establishment and onco-biological characteristics of stable-fluorescent protein-expressing subcutaneous and orthotopic xenografts in nude mice with high metastatic potentialFollowing the subcutaneous injection or orthotopic tumor implantation,all the mice were anesthetized via intraperitoneal injections of sodium pentobarbital(50 mg/kg) for whole-animal fluorescent imaging.All the hepatic tumors could be observed on day 14 under a fluorescent stereomicroscope.Two hundred and seventy days after six consecutive tumor passages,HCCLM3-R and HCCLM3-G orthotopic xenograft models still expressed stable and intense red or green fluorescence.The rates and quantity of spontaneous metastasis of HCCLM3-G and HCCLM3-R reached 100%and 100%in the liver,100%and 100%in the lung,and 90%and 90%in the peritoneum,respectively,on day 42.The onco-biological characteristics of the new modles are similar to those of their parental models.Previously we could not detect abdomen metastasis in nude mice without pathology examination and the abdomen metastasis could not be quantitated.Now we developed a more objective method for in vivo orthotopic tumor volume analysis and for ex vivo lung and abdomen metastasis quantiatate in fluorescent protein-expressing tumor models.We quantitated the tumor volume by formula and IOD,and Pearson correlation analysis indicated that we could analyze the tumor volume and metastasis by quantitating the IOD.Lung metastasis is an important onco-biological character, but we could only semi-quantity them by serial sections.The new models are very useful for detecting and quantitating small lung metastasis in a objective way,and the abdomen metastasis could be quantitated now.