The Effect Of P5D2 On The Model That Cervical Squamous Carcinoma Cell Line HCE1 Multicellular Spheroids Invade Umbilical Vein Endothelium Cell Monolayers

Objectives:To establish a model of human umbilical vein endothelial cell line HUVEC monolayer which is invaded by the multicellular spheroids of human cervical squamous carcinoma cell line HCE1.The expression of the integrinβ1 in multicellular spheroids,the relationship between integrinβ1 and the invaded endothelial cell and the effects of monoclonal mAb against theβ1 integrin P5D2 on HCE1 multicellular spheroids invading umbilical vein endothelium cells have been preliminary studied and discussed.Methods:(Ⅰ):A model of human umbilical vein endothelial cell line HUVEC monolayer invaded by human cervical squamous carcinoma cell line HCE1 multicellular spheroids has been established(called a cervical squamous carcinoma cell HCE1 infiltrating model in brief):the HCE1/MCS and the HUVEC monolayer cell have been co-cultured and infiltration has been digitally photographed on the same day and the 1st, 4th,7th days from the initial plating.The invasive areas of the HCE1/MCS were determined by using Motic med.(Ⅱ):The expressions ofβ1 integrin on HCE1 monolayer cell, HCE1/MCS and HVUEC monolayer cell were detected by immunohistochemistry. (Ⅲ):The effects of P5D2 on HCE1 multicellular spheroids invading umbilical vein endothelium cells1.Groups:2 groups have been established.P5D2 group:treated with 1.0ug/ml of P5D2.Control group:treated with the same concentration of mIgG.2.Observation:The infiltration of two models has been digitally photographed on the same day and the 1st,4th,7th days from the initial plating respectively.The invasive areas of the HCE1/MCS were determined by using Motic med.3.The expressions ofβ1 integrin on the 4th day on P5D2 and on the control group were detected by immunohistochemistry.Results:(Ⅰ):Establishment of a cervical squamous carcinoma cell HCE1 infiltrating model.1.Culture of HCE1 monolayer cells:We have observed that the growth of HCE1 monolayer cells was in a good condition,with bright cytoplasm.Cells became irregular polygonal after cell attachment.2.Culture of HCE1 multicellular spheroids:After a 24h-rotating-culture,cells began to aggregate into multicellular spheroids,and become compacter and larger in size in the process of time.Central necrosis core has been observed on the 4th day of culture.3.Culture of HUVEC cells:Cells were polygonal,mutually conjoined,with a paving-stone-form.4.Cervical squamous carcinoma cell HCE1 infiltrating model:Due to the size of the spheroids,HCE1 multicellular spheroids were distinguishable readily from the HUVEC monolayer cells.After 1 hour of co-culture,it has been observed that HCE1 multicellular spheroids adhered to HUVEC monolayer cells,non separable by shaking,but they were not on the same focal plane.24h after the model establishment,the spheroids appeared to disaggregate and spread across the HUVEC monolayer cells.However,on the 4th day,the spheroid cells have invaded the HUVEC monolayer cell and rapidly established invasion foci.Central necrosis core and outgrowth of HCE1 monolayer cells have been observed.As the HUVEC cells retracted,it was clear that invading HCE1 cells were in the same plane as the HUVEC monolayer cell.On the 7th day,the area occupied by the original spheroids has become larger,the HUVE cells receded and the cervical cancer cells proliferated on the exposed tissue culture surface.(Ⅱ):Expression ofβ1 integrin was detected by immunohistochemistry:β1 integrin expression is negative in HUVE cell monolayer but positive expression in HCE1/monolayer and HCE1/MCS. The expression ofβ1 integrin is significantly stronger in HCE1/MCS than in HCE1/monolayer(P＜0.005).(Ⅲ):The effects of P5D2 on HCE1 multicellular spheroids invading umbilical vein endothelium cells1.Cervical squamous carcinoma cell HCE1 infiltrating model treated by P5D2:During a 7-day observation,filtration level of the model treated by medicaments is obviously lower than the control group.After 24h in the presence of mAb against theβ1 integrin subunit,the HCE1 spheroids disaggregated,with a smaller invasion area compared to mIgG group.There was a reduction of 32.16%in the area of invasion compared to the mIgG control(P＜0.001).On the 4th day,a marked inhibition has been noticed with the presence of theβ1 integrin mAb,there was a reduction of 60.77%in the area of invasion compared to the mIgG control(P＜0.001).On the 7th day,invasion of spheroids into the HUVEC monolayer cell had been notably blocked by theβ1 integrin mAb with a reduction of 85.5%compared to the mIgG control(P＜0.001).2.Expression ofβ1 integrin(on the 4th day) was detected by immunohistochemistry:expression ofβ1 integrin is significantly stronger in control group than in P5D2 group(P＜0.005). Conclusions:1.The establishment of a model of human umbilical vein endothelial cell line HUVE monolayer invaded by human cervical squamous carcinoma cell line HCE1 multicellular spheroids was successful.2.The expressions ofβ1 integrin in multicellular spheroids and in filtration model may relate to the strength of adhesion and invasion.3.P5D2,a monoclonal antibody against theβ1 integrin subunit,can partly block the invasion of cervical squamous carcinoma cell line HCE1 multicellular spheroids into human umbilical vein endothelium cell line HUVE monolayer cell.