| Cell fusion,also named as cell hybrid,is a process which two or more cells become one by fusion of cytoplasm.In recent years,there is a growing body of evidence demonstrating the association of cell fusion with cancer progression.Meanwhile,fusogenic proteins play important roles in cell fusion.As one of the important members of fusogenic protein,syncytin-1 has been highly detected in a great variety of tumor tissues,and has been proved to participate in the fusion event with different tumor cells such as breast cancer,choriocarcinoma,lung cancer et al.Nevertheless,the underlying specific regulating effect and mechanism are still unclear.Recently,inflammation has also been suggestted as a possible trigger for cell fusion.Chronic inflammation can lead to recruitment of human pluripotent stem cells(HPSCs),mesenchymal stromal cells(MSCs),and cells of the myelomonocytic lineage and subsequently tissue restoration by cell fusion in the late phase of physiological wound healing.The frequency of cell fusion events was also found about 10 to 100-folds increase in several tissues in chronic inflammatory conditions.The inflammatory cells such as macrophage,leukocyte,dendritic cell et al can promote the progression of tumor through fuse with tumor cells.Since chronic inflammation is one of the most critical characteristics in tumor microenvironment,it is not difficult to imagine a correlation among inflammation,cell fusion and cancer.However,the signal pathways and effector molecules involved in TNF-a-mediated cell fusion remained poorly understood.In the present study,we show that the pro-inflammatory factor TNF-a can activate Wnt/?-catenin signal pathway and thereby enhance the fusion between oral squamous cell carcinoma cells and endothelial cells via up-regulation of fusogenic protein syncytin-1.These results provide new insight into the interaction between oral cancer cells and endothelial cells,demonstrate a signal transduction pathway that links inflammation,Wnt/?-catenin signal pathway and cell fusion in tumor microenvironment,and predict a novel potential role of chronic inflammation in tumor progression.Part I:The role of TNF-a in spontaneous fusion between SCC-9 and HUVEC cellsObjective:To investigate the morphology and characteristics of fused cells between SCC-9 and HUVEC,and the role of TNF-a played in the fusion event.Material and methods:The cultured SCC-9 were added with lentiviral supernatants of lentiviral particles whcih contained RFP vector.Alike,the cultured HUVECs were added with lentiviral supernatants which contained GFP vector.After transduction for 72h,the stable transfected cells of RFP-SCC-9 and GFP-HUVECs were sorted by FACS.Then we co-cultured equal numbers of RFP-labeled SCC-9 and GFP-tagged HUVEC cells for 24 h without any fusogenic reagents.Double fluorescent staining showed that hybrid cells were observed in co-culture system by FM.After then the nuclei were stained by DAPI,and fused cells were observed by LSCM.Under the stimulation of 10ng/ml TNF-a for 24h,10?g/ml puromycin was used to screen for fluorescence positive cells,and then fused cells were assessed by number counting and FCAS on 1,3,7 d,respectively in 3 random fields in a 200×magnification.After 2 weeks,the fusion rates were assessed by FACS.Results:The results of FM showed that the orange-like double fluorescent staining cells were observed in co-culture system,demonstrating an existence of spontaneous fusion between human SCC-9 and HUVEC cells.The hybrid cells increased in a culture time-dependent pattern while the rate of spontaneous cell fusion kept constantly.Meanwhile,most of hybrid cells contained stable fluorescence intensity,with irregular shapes,appeared healthy and showed no signs of apoptosis or nuclear condensation in co-culture system.Some of the hybrids contained only one nucleus,while a few contained two,three and/or multiple nuclei.The distance between nuclei varied in different hybrid cells.In addition,the fused cells could be cultured for at least 7-8 weeks.The observation results of LSCM showed that the the cytomembrane of fused cells showed continuous and intact,no cellular shrinkage,foaming or bubbling phenomenon happened.Simultaneously,the nucleus was apparent,stable stained with DAPI,and the nucleus showed no signs of condensation.By counting double fluorescence-positive cells in 3 random fields in a 200×magnification,we found that cell fusion was enhanced by up to 2-3(2.18±0.32)folds under the lOng/ml TNF-a stimulation compared with untreated cells in vitro.FACS analysis also showed that the stimulation of TNF-a increased the percentage of bi-fluorescent cells to 1.78±0.053%,compared with untreated cells(0.748±0.024%).The difference was statistically significant(P<0.01);Conclusion:Results from FM,LSCM,artificial cell counting and FACS analysis suggested spontaneous cell fusion between SCC-9 and HUVEC could happen.The fused cells with orange-like color could live stable in the co-culture system with different form and pattern,distinct number of nuclei,various distance between nuclei and showed no indication of apoptosis.The inflammatory factor TNF-a stimulation actively enhance the fusion between SCC-9 and HUVEC cells and evidently promote the fusion event.Part II:The role of syncytin-1 in the TNF-?-enhanced fusion between SCC-9 and HUVECObjective:To clarify the potential role of fusogenic protein syncytin-1 in the TNF-a-enhanced fusion between SCC-9 and HUVEC.Critical focus on the crosstalk between TNF-? and syncytin-1,syncytin-1 and cell fusion.Material and methods:Expression of syncytin-1 was detected in surgical specimens from 28 patients with Oral Squamous Cell Carcinoma(OSCC).Meanwhile,the expression of syncytin-1 and the receptor ASCT-2 were detected by Western Blot.To investigate whether TNF-? could regulate the expression of fusogenic protein syncytin-1 and its receptor ASCT-2,we examined the syncytin-1 and ASCT-2 expression in TNF-? stimulation group and control group for 6,12 and 24h,respectively.The endogenous expression of syncytin-1 in SSC-9 cells by short:hairpin RNA(sh-RNA)to investigate the biological function of syncytin-1 during cell fusion.After knockdown of syncytin-1,RFP-tagged HUVECs were co-cultured with GFP-labeled sh-RNA of syncytin-1 in SCC-9 and GFP-labeled SCC-9,respectively.The number of fused cells were assessed by artificial cell counting on day 1,3,7,and the fusion rates were measured by FACS.Results:Immunohistochemistry showed that syncytin-1 was detected in 26 out of 28 human OSCC specimens(93%)and mainly localized in cellular membrane and/or cytoplasm.But the level of syncytin-1 expression showed different;According to our statistics,In these 28 human OSCC specimens,22 cases of them were detected with a relatively higher expression of syncytin-1(Scored 4-7),while other 6 cases showed a lower expression(2 of them showed no expression)(Scored 0-3).Western blot using an antiserum against the extracellular domain of syncytin-1 further revealed a 60-kDa band in extracts of SCC-9 as well as HUVEC cells,corresponding in size to a syncytin-1 component present in the placenta.ASCT-2 was documented in HUVECs and SCC-9 extracts by using western blot analysis.Obviously,the expression of syncytin-1 in HUVEC and ASCT-2 in SCC-9 showed evidently lower than syncytin-1 in SCC-9 and ASCT-2 in HUVEC.Western blot analysis showed that the constant stimulation of lOng/ml TNF-a actually induced a continuous and dramatic increase of syncytin-1 in SCC-9 and ASCT-2 in HUVECs during an observation period of 24h.Quantitative analysis showed that the ratio of syncytin-1/GAPDH and ASCT-2/GAPDH are significantly higher than the control groups at each time-point(P<0.01).After knockdown of syncytin-1,Cell counting analysis showed the transfection of syncytin-1 sh-RNA obviously inhibited the number of fused cells between SCC-9 and HUVEC in co-culture systems on day 1,3,7.FACS assay also confirmed an evidently lower fusion rate in the syncytin-1 knockdown co-culture group(0.196±0.008%)than that in control group(0.768±0.012%),showing a significantly statistical difference(P<0.01).Conclusion:Syncytin-1 was highly expressed in the OSCC tissue and SCC-9,while the receptor,ASCT-2 was highly expressed in HUVEC.Besides,Syncytin-1 and ASCT-2 were positively related to TNF-a.The fusion rate sharply decreased when syncytin-1 was silenced,which means that syncytin-1 played important role in the fusion event between SCC-9 and HUVEC.In summary,there are very closely relationship and crosstalk between syncytin-1 and TNF-a,syncytin-1 and cell fusion.Part ?:The role of Wnt/?-catenin in the TNF-a-enhanced fusion between SCC-9 and HUVECObjective:To verify the underlying molecular mechanism of cell fusion between SCC-9 and HUVEC,Especially focus on the mechanism how could syncytin-1 modulated by TNF-a through Wnt/?-catenin signal pathway and investigate the role Wnt/?-catenin pathway played in the fusion process between SCC-9 and HUVEC.Material and methods:To investigate whether syncytin-1 was responsive to Wnt/?-catenin pathway activation,the protein level of syncytin-1 was detected in 6,12 and 24 h when SCC-9 cells were stimulated with different reagents including TNF-a(lOng/ml);DKK-1(100ng/ml),the suppressor of Wnt/?-catenin signal pathway;and the pathway agonist,Wnt3a(100ng/ml).P-?-catenin,P-catenin and syncytin-1 were detected by Western Blot.We then silenced and/or enhanced the expression of ?-catenin gene by sh-RNA and lentivirus until the stable transfected cells of sh-?-catenin-SCC-9 and en-?-catenin-SCC-9 obtained.Then 10 ng/ml TNF-a were added,respectively.After then Western Blot was used to check the expression of syncytin-1 at the time point of 6,12 and 24 h.Simultaneously,equal number of RFP-SCC-9 and GFP-HUVEC were mixed in the co-culture system.After activation or inhibition of Wnt/?-catenin signal pathway by TNF-?(10ng/ml),DKK-1(100ng/ml),Wnt3a(100ng/ml),fusion between SCC-9 and HUVECs were evaluated by using artificial cell counting on co-culture day 1,3,7,and FACS was used to acquire the fusion rate between SCC-9 and HUVEC in this study;Similarly,RFP-HUVEC were co-cultured with equal number of sh-?-catenin-SCC-9 and en-?-catenin-SCC-9,respectively,the number of fusion cells and the fusion rate were calculated by artificial cell counting on day 1,3,7 and FACS on day 14.Results:The results of Western Blot analysis showed that compared to the control group without any stimulation,the constant stimulation of TNF-a actually induced a continuous and dramatic increase of syncytin-1 in SCC-9,the expression of phosphorylation of ?-catenin(P-?-catenin)decreased sharply,while the amount of?-catenin increased evidently(P<0.01).Moreover,sustained treatment with DKK-1 led to an obviously decrease of total ?-catenin and increase of P-?-catenin which followed by a significant down-regulation of syncytin-1 expression in SCC-9 cells.After incubation of SCC-9 cells with sh-?-catenin,we also found that syncytin-1 expression presented a time-dependent pattern of decrease and significant difference compared with control group.In contrast,an obvious enhancement of syncytin-1 expression was documented in SCC-9 cells compared to the non-treatment group when the recombinant Wnt3a was used to stabilize and refrain p-catenin from de-phosphorylation and disintegration.Interestingly,salvage experiment with sustained 10ng/ml TNF-a stimulation could restore syncytin-1 expression in either sh-?-catenin or en-?-catenin-treated SCC-9 cells,implying that the expression of syncytin-1 was also controled by some other signal pathway.The number of fused cells increased obviously compared to the control group when the recombinant human Wnt3? was added to the co-culture system.On the contrary,DKK-1 could significantly reduce the fusion events.FACS analysis showed that the rate of fusion between SCC-9 and HUVEC reduced to as low as 0.367±0.016%when DKK-1 was added to the co-culture system.However,the fusion rate increased up to 1.76±0.07%when the co-culture system was incubated with Wnt3a.Both of the DKK-1 group and Wnt3a group showed statistical difference in comparison to the control group(P<0.01).When SCC-9 cells were pre-treated with ?-catenin-specific sh-RNA(sh-?-catenin-SCC-9),the number of double-labeled fluorescent staining cells fell sharply to nearly 1/3-1/2 of the control co-culture group.In accordance with this,the bio-fluorescently-stained cells increased 2?3 folds when ?-catenin was overexpressed/enhanced by transfection of en-RNA in SCC-9(en-?-catenin-SCC-9).The FACS results also revealed that the fusion rate reduced to 0.30±0.029%in GFP-SCC-9/RFP-HUVEC co-cultured with presence of sh-?-catenin,but the rate of fused cells would be 1.80±0.033%when en-?-catenin was introduced to co-culture with RFP-HUVEC.Both of sh-and en-groups showed significant statistical difference in comparison to the control group(P<0.01).Conclusion:Our results showed that TNF-? treatment dramatically led to the activation of Wnt/?-catenin signal pathway in SCC-9;Activation of Wnt/?-catenin signal pathway could control the expression of syncytin-1,and contribute to the enhancement in cancer-endothelial cell fusion induced by the pro-inflammatory factor TNF-?.In summary:In this study,we confirmed that inflammatory factor TNF-a could enhance fusion between squamous cell carcinoma cells 9(SCC-9)and human umbilical vein endothelial cells(HUVEC).Further study revealed that TNF-a could promote the expression of syncytin-1 in SCC-9 and its receptor neutral amino acid transporter type 2(ASCT-2)in HUVEC.Syncytin-1 acted as an important downstream effector in TNF-a-enhanced cancer-endothelial cell fusion.TNF-a treatment also led to the activation of Wnt/?-catenin signal pathway in SCC-9.Our results suggest that Wnt/?-catenin signal pathway activation-dependent up-regulation of syncytin-1 contributes to the pro-inflammatory factor TNF-a-enhanced fusion between oral squamous cell carcinoma cells and endothelial cells.These findings not only provide new insights into the links between inflammation and cancer from view of cell fusion,but also uncover partially the mechanisms underlying the cell fusion in inflammatory tumor microenvironment.The present study may also predict a new therapeutic target for cancer prevention and treatment. |
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