Retrosflt-1 Decelerates The Growth Of A Murine Experimental Osteosarcoma And The Expression Of Correlated Proto-Oncogene

Partâ… RetrosFlt-1 Decelerates the Growth of a Murine Experimental OsteosarcomaBackgroundOsteosarcoma is the most common malignant tumor seen in orthopaedic surgery. Despite intensive treatment,including adjuvant chemotherapy,wide excision of tumors and amputation of the diseased limbs,approximately half of such patients die within 5 years.On this basis,it is clear that novel therapeutic approaches are needed for improved osteosarcoma treatment.One recent novel interventional strategy for cancer is based upon interruption of tumor angiogenesis.Indeed,Angiogenesis plays a critical role in neoplastic processes and is essential for growth,invasion and metastasis of solid tumors. Among many factors regulating angiogenesis,vascular endothelial growth factor (VEGF)may be one of the most potent.Enhanced VEGF gene expression has been identified in a number of malignant tumors from osteosarcoma,breast,lung,ovarian and liver in comparison with normal tissue.This recognition leads to potential strategy to target the VEGF pathway as a prospective anti-osteosarcoma therapy.VEGF exerts its biological effects on endothelial cells by binding to its cell surface receptors.Receptors identified to bind VEGF include VEGFR-I(Flt-1)and VEGFR-2(Flk-1).Soluble Flt-1 is an alternatively spliced form of the Flt-1 VEGF receptor.This form of Flt-1 binds to VEGF with the same affinity and equivalent specificity as that of the full-length receptor,but this binding does not initiate signal transduction because sFlt-1 lacks its intracellular tyrosine kinase domains and the cell association.Indeed,there are numbers of studies trying to apply the sFlt-1 gene transfer technique to halt growth and metastasis of different solid tumors.Generally, these studies have established the concept that gene therapy may offer a technical means to realize the potential benefits of antiangiogenesis approaches.ObjectiveTo investigate the in vitro transduction efficiency of retroviral vectors encoding sFlt-1 to osteosarcoma G-292 cells and to evaluate the effects of sFlt-1 modification on the tumor growth of a mutine orthotopic osteosarcoma.Materials and methodsAnimals Severe combined immunodeficient(SCID)mice at four weeks of age were used as hosts for the experimental sarcoma.The animals were housed in a pathogen-free environment and given free access to autoclaved chow and water.All mice were quarantined for one week prior to experimentation.Osteosarcoma cell lines Human osteosarcoma cell lines,G-292(CRL-1423)were cultured and maintained by subculture at a ratio of 1:6-1:8.Construct retroviral vectors and gene transduction The original retroviral vector coding for sFlt-1 and retrovirus coding for bacteria LacZ were used for in vitro gene transduction.500μl of 10⁶cfu retrosFlt-1(or MFG-LacZ)was added onto G-292 cells with 500μl of fresh medium including polybrene at final concentration of 8μg/ml. Cells were incubated at 37℃for 8 hours before the 2nd addition of the same amount of the retroviral vectors.The cells were then maintained at 37℃incubator with 5%CO₂. The incorporation of sFlt-1 in genetic DNA of the transduced cells was evaluated by ELISA.LacZ transgene expression was detected using X-gal staining.Establishment of the orthotopic osteosarcoma SCID Mice were randomly divided into 3 groups with 10 animals per group,to receive wide-type G-292 cells,sFlt-1 transduced cells,and LacZ-transduced cells,respectively.Animals were sacrificed at 8 weeks after tumor cells innoculation by CO₂ asphyxiation.Legs containing orthotopic tumors were harvested for histological and molecular evaluations.MicroCT Evaluation An eXplore Locus MicroCT system was used to monitor the tumor growth and characters of bone lesions.Mice were scanned immediately following tumor cell implantations,and every 2 weeks thereafter.The length and width of tumor were measured and the tumor volumes were calculated.RNA Extraction and Real Time Quantitative PCR for Gene expression Total RNA extraction was performed using a commercial kit.The precipitated RNA was then treated with DNase and passed through a spin column for further purification.Reverse transcription and real time PCR for the expression of VEGF and sFlt-1 was performed using the ABI Prism 7700 Sequence Detector.The comparative gene expressions of the experimental tumor groups over the sham controls were calculated.Histology process and immunohistochemical(IHC)examination Proximal tibiae bearing osteosarcoma tissue and receiving sham operation were collected.Tissues were fixed in buffered formalin,decalcified in 12%EDTA and embedded in paraffin at consistent orientation.All tissues were stained with hematoxylin and eosin,and examined under a Zeiss light microscope.Digital photomicrographs were captured and analyzed using Image-Pro Plus analysis software.IHC was performed to detect the expression levels of VEGF,sFlt-1 and CD34.Digital images were captured and analyzed using the Image-Pro software package.The level of positive staining and localization was evaluated in six different fields,and expressed as integrated optical density(IOD).The comparison of the IODs among groups was carried out using the Image-Pro analysis tool.The numbers of the Microvessel density were calculated using CD34 staining sections.Statistical Analyses Statistical analysis between different groups was performed by Student T-test,or the ANOVA test;with the Schafer formula for post hoc multiple comparisons,using the SPSS software package.A p value of less than 0.05 was considered as significant difference.Data are expressed as mean±standard deviation.ResultsCell culture Cells were fusiform shape and polygon,Inequality of size.No acute cytotoxicity in cell culture was noticed following 2 times viral infection. Transgene Expression The expression of sFlt-1 protein levels was measured using ELISA.Extensive elevation of sFlt-1 levels was detected 24 hours after viral infection and reached peak levels at 14 days.The elevated transgene product was maintained for at least 6 weeks.Tumors retrieved from sFlt-1 transduced groups revealed strong positive Immunohistochemical sFlt-1 staining.Quantification analysis of sFlt-1 expression indicated significantly stronger positive stains in sFlt-1 transduced groups in comparison with non-viral controls(wide type G-292)or LacZ transduced groups. X-gal staining on LacZ-transduced cells exhibited strong blue colors,indicating the successful transfer and transduction efficiency was 78.6±6.8%.Inhibitory effects of sFlt-1 on growth of experimental osteosarcoma.Analyses of microCT data using Micro View program indicated all mice developed orthotopic bone tumors at 2 weeks after tumor cell inoculation and the individual tumor volume among groups was not significantly different.At 4,6,and 8 weeks,however,the average tumor volumes of the non-modified G-292 and LacZ-transduced groups appeared significantly larger than those of sFlt-1 transduced group(p＜0.05). Histological and Immunohistochemical Assessment Histology showed typical osteosarcoma characteristics including severe cellular pleomorphism,bone erosions, and neo-vascularization.Immunohistochemical staining illustrated strong positive VEGF staining on G-292 and LacZ-transduced groups.However,the experimental tumors derived from the cells with sFlt-1 gene modification exhibited dramatically more transgene expression against sFlt-1 antibody(p＜0.05).Microvessel density Significantly less blood vessel presence in sFlt-1 transduced group compared with G-292 and LacZ-transduced groups(p＜0.05).Molecular Assessment RNA samples were examined for the gene expression of VEGF and sFlt-1.Data showed strong expression of VEGF in the primary tumor tissue. Significant higher sFlt-1 expression in sFlt-1 transduced groups was obvious in comparison with G-292 or LacZ-treated groups(p＜0.05).Conclusions 1,Human osteosarcoma cell lines G-292 cells were transduced with retroviral vectors encoding sFlt-1 before transplanted into the host proximal tibia.It appears that the gene transfer was successful.2,Data showed strong expression of VEGF in the primary tumor tissue and further support to the idea that VEGF plays a critical role in tumor angiogenesis.3,The development of osteosarcoma was markedly suppressed in the sFlt-1 group,the average tumor volumes significantly smaller than G-292 and LacZ transduced groups at 4,6,8 weeks.It appears that the soluble Flt-1 may be an excellent candidate of therapeutic agents for the longer and effective inhibition of angiogenesis during tumor development/progression.4,Singificantly less blood vessel presence in sFlt-1 transduced group compared with G-292 and LacZ-transduced groups.It further supports to the idea that retrosFlt- 1 Decelerates the Growth of a Murine Experimental Osteosarcoma by VEGF blockade.Partâ…¡The expression of c-myc and c-fos proto-oncogene on murine osteosarcomaBackgroundWith the recent expansion of molecular biology techniques,the genetic alterations in the development and metastasis of malignant tumors have been observed.Cellular oncogenes were found activated by DNA rearrangements(proviral insertions,chromosome translocations and DNA amplifications).These alterations may result in an increased or deregulated gene regulation.Although numerous oncogenes and tumor suppressor genes have been identified in osteogenic sarcomas, the following seem to be expressed with relative high incidence:C-myc proto-oncogene on chromosome 8 encodes a transcription factor,which involves in the regulation of cell growth,DNA replication,and transcriptional regulation of specific target genes.C-los proto-oncogene,the cellular homologue of v-fos,is associated with many biological processes,ranging from transformation to cell-cycle progression and cell differentiation.In particular,it seems that the c-fos oncogene is involved in osteoblast and chondrocytes differentiation.ObjectiveTo investigate the expression of c-myc and c-fos proto-oncogene on murine orthotopic osteosarcoma and the correlations of these oncogenes with VEGF,sFlt-1 and osteosarcoma development.Materials and methodsReal Time Quantitative PCR for Gene expression Reverse transcription and real time PCR for the expression of c-myc,and c-fos was performed using the ABI Prism 7700 Sequence Detector.The comparative gene expressions of the experimental tumor groups over the sham controls were calculated.immunohistochemical(IHC)examination Proximal tibiae beating osteosarcoma tissue and receiving sham operation were collected.Tissues were fixed in buffered formalin, decalcified in 12%EDTA and embedded in paraffin at consistent orientation.IHC was performed to detect the expression levels of c-myc and c-fos.Digital images were captured and analyzed using the Image-Pro software package.The level of positive staining and localization was evaluated in six different fields,and expressed as integrated optical density(IOD).The comparison of the IODs among groups was carried out using the Image-Pro analysis tool. Statistical Analyses Statistical analysis between different groups was performed by Student T-test,or the ANOVA test;with the Schafer formula for post hoc multiple comparisons,using the SPSS software package.A p value of less than 0.05 was considered as significant difference.Data are expressed as mean±standard deviation.ResultsImmunohistochemical Assessment Immunohistochemical staining illustrated strong positive c-myc and c-fos staining on G-292 and LacZ-transduced groups.In sFlt-1 transduced groups,c-myc was found lower expression compared with G-292 or LacZ-treated groups,the difference are significant(p＜0.05);c-fos was found lower expression compared with G-292 or LacZ-treated groups,the difference are not significant.Molecular Assessment Real-time polymerase chain reaction indicated strong expression of c-myc and c-fos on G-292 and LacZ-transduced groups.Significant lower c-myc expression in sFlt-1 transduced groups was obvious in comparison with G-292 or LacZ-treated groups(p＜0.05).Conclusions1,In G-292 and G-292-LacZ experimental osteosarcoma model,c-myc and c-fos oncogenes markedly expressed in the tumor tissues supplementing with the high VEGF expression and osteosarcoma development.2,In sFlt-1 transduced groups,c-myc was found lower expression compared with G-292 or LacZ-treated groups,the difference are significant.