Effects Of Low-dose Metronomic Chemotherapy On Vasculogenic Mimicry Of Ovarian Cancer Xenograft Tumors And The Possible Mechanisms

Objective:Vasculogenic Mimicry?VM?is one sort of special blood-providing pattern in highly malignant tumors.It is reformed by tumor cells deformatation and the extracellular matrix remodeling.In recent years,the study found that VM is closely related to the tumor growth,metastasis and invasion.Membrane type 1 matrix metalloproteinases?MT1-MMP?as a member of the matrix metal protein enzymes plays an important role in malignant tumor growth,invasion and metastasis.Focal adhesion kinase?FAK?belongs to the protein tyrosine kinase,can change the cells adhesion and extracellular matrix,facilitate the movement of the tumor cells,infiltration,invasion and metastasis.To explore the effects of low-dose metronomic chemotherapy?LDM?on VM of ovarian cancer xenograft tumors and the possible mechanisms,by injecting cisplatin with LDM versus maximum tolerated-dose chemotherapy?MTD?in the nude mice bearing HO8910 ovarian cancer xenografts.And to explore the influence of LDM on the expression level of membrane type-1matrix metalloproteinase?MT1-MMP?and focal adhesion kinase?FAK?.Methods:1 To establish the nude mice xenograft tumor models,divide into three groups and cisplatin treatment.For subcutaneous?s.c.?tumor formation,HO8910 cells?1×107?were suspended in 0.2 ml phosphate-buffered saline?PBS?and s.c.injected into the right thigh of each nude mouse.Approximately 10 days later,the mice were randomly divided into three groups as follows: LDM group?intraperitoneal injection of 1mg/kg cisplatin daily for eighteen days?,MTD group?intraperitoneal injection of 3mg/kg cisplatin once every three days for six cycles?and control group nude mice were treated with the equal saline solution.2 Obtain the tumor samples.The mice were closely monitored,xenograft tumor volumes were measured every 2 days.According to a computer-generated radom number table every two days in each experimental group extracted 3 mice anesthesia overdose killed,stripped out the xenograft tumor in aseptic conditions,and divided into two separates,the one put in 4% paraformaldehyde,the other one stored in the fridge?at-80??.Calculated the tumor growth suppression rate respectively after 18 days of the chemotherapy.3 Immunohistochemical staining?IHC?and CD31/PAS double staining.Stained tumor samples obtained from formalin-fixed,paraffin-embedded,sliced,using IHC,CD31/PAS double staining observed the expression of cytokines.Under the microscope continuous counting tumor tissue 10high-power field of view?×400?on the VM,whichever is the mean of VM density,divided into the central area and the peripheral area.4 IHC and Western Blotting was used to measure the expression of MT1-MMP and FAK in three groups.Results:1 Tumor growth of nude miceDuring the process of the chemotherapy,subcutaneous xenograft tumors of control group,MTD-DDP group and LDM-DDP group were increasing constantly?PMTD-DDPgroup=0.002,PLDM-DDP group<0.001,Pcontrol group=0.001?.After the end of chemotherapy,tumor volumes in MTD-DDP group and LDM-DDP group were smaller than control group?P=0.042,P=0.000?,and those in LDM-DDP group were significantly smaller than MTD-DDP group?P=0.009?.The tumor growth suppression rate for MTD-DDP group and LDM-DDP group was 10.48% and 31.14%,respectively.2 The morphology of VM with CD31/PAS double stainingVM and endothelium dependent vascular were observed in xenografttumor under the microscope using CD31/PAS double staining.Endothelial dependent vascular was existed in the peripheral area more than the central area and intersected with the host,while VM was largely existed in the central area.At high magnification,there were PAS-positive pattened vessels in the xenograft tumor tissue.Some tumor cells mimic endothelial cells and lined inside PAS-positive vessel and form endothelial-like vessel,and erythroeytes might be seen inside the vessel.PAS positive blood sample pattern was straight,parallel,overlapping,ring,mesh and so on.3 The formation of VM in different chemotherapy models1)The formation of VM in the central areaDuring the 2st day of the chemotherapy,VM could be observed in the central area of the xenograft tumors among control group,MTD-DDP group and LDM-DDP group.The density of VM was no statistical difference among control group,MTD-DDP group and LDM-DDP group?P=0.468,P=0.472,P=0.359?.During the 4st day to 18 th day of the chemotherapy,the density of VM in the central region changed gradually.There was a statistical difference among the three groups.The density of VM in control group increased from the 2st day to 8th day of the chemotherapy,and declined from the 9st day to18 th day of the chemotherapy,whereas the density of VM in MTD-DDP group and LDM-DDP group increased from the 2st day to 10 th day of the chemotherapy,and declined from the 11 st day to 18 th day of the chemotherapy.From the begining to 8th day of the chemotherapy,the density of VM in MTD-DDP group was not statistically different from control group?P₂=0.810,P₄=0.479,P₆=0.363,P₈=0.822,all P>0.05?.From the 10 st day to 18 th day of the chemotherapy,the density of VM in MTD-DDP group was larger than control group(P₁₀=0.037,P₁₂=0.018,P₁₄=0.014,P16=0.043,P18=0.013).From the 4st day to 18 th day of the chemotherapy,the density of VM in LDM-DDP group was smaller than control group and MTD-DDP group(P₄=0.010,P₆=0.005,P₈=0.013,P₁₀=0.027,P₁₂=0.046,P₁₄=0.006,P16=0.005,P18=0.006,all P<0.05).2)The formation of VM in the peripheral areaVM was appeared in the peripheral area of the ovarian cancer xenograft tumors.Similar to the cental area,the density of VM in the peripheral area changed gradually.During the 2st day of the chemotherapy,the density of VM were no statistical difference among control group,MTD-DDP group and LDM-DDP group?P=0.893,P=0.667,P=0.598?.The density of VM in MTD-DDP group and control group increased from the 2st day to 10 th day of the chemotherapy,and declined from the 11 st day to 18 th day of the chemotherapy.The density of VM in LDM-DDP group increased from the 2st day to 12 th day of the chemotherapy,and declined from the 13 st day of the chemotherapy.During the 2st day to 8th day of the chemotherapy,the density of VM in MTD-DDP group was not statistically different from control group?P₂=0.998,P₄=0.424,P₆=0.236,P₈=0.231,all P>0.05?.From the 10 st day to18 th day of the chemotherapy,the density of VM in MTD-DDP group was larger than control group(P₁₀=0.037,P₁₂=0.010,P₁₄=0.014,P16=0.043,P18=0.013,all P< 0.05).From the 4st day to 18 th day of the chemotherapy,the density of VM in LDM-DDP group was smaller than control group and MTD-DDP group(P₄=0.009,P₆=0.001,P₈=0.013,P₁₀=0.027,P₁₂=0.003,P₁₄=0.006,P16=0.034,P18=0.008,all P<0.05).3)Comparison of the dentity of VM in the peripheral area and in the central area of the three groupsFrom beginning to end of chemotherapy,the density of VM tended to reduce after the first increase both in the peripheral and central area in the control group,MTD-DDP and LDM-DDP group of ovarian cancer xenograft tumors.About in the middle of chemotherapy,there was a maximum density of VM.The density of VM in peripheral area was larger than central area.There was a statistical difference among the three groups?P<0.05?.4 The expression of MT1-MMP and FAK in the ovarian cancer xenograft tumors.Under the microscope,the expression of MT1-MMP and FAK was observed in tumor cells around the VM through immunohistochemical staining.Whereas MT1-MMP and FAK were not expressed in the area faraway from VM,or expressed a little.1)Expression of MT1-MMPIn the central area: On the second day of chemotherapy,there was a little expression of MT1-MMP in the central area of the three groups.The expression level of MT1-MMP was no statistical difference among the LDM-DDP group,MTD-DDP group and control group?P=0.184,P=0.417,P=0.417?.On the 10 th day of chemotherapy,as compared to control group,the expression level of MT1-MMP was higher in MTD-DDP group?P=0.002?,but MT1-MMP expression in LDM-DDP group was lower than control group?P<0.001?.On the 18 th day of chemotherapy,as compared to control group,the expression level of MT1-MMP was higher in MTD-DDP group?P=0.004?,but MT1-MMP expression in LDM-DDP group was lower than control group?P=0.002?.In the peripheral area: On the second day of chemotherapy,there was a little expression of MT1-MMP in the peripheral area of the three groups.The expression level of MT1-MMP was no statistical difference among the LDM-DDP group,MTD-DDP group and control group?P=0.326,P=0.710,P=0.184?.On the 10 th day of chemotherapy,as compared to control group,the expression level of MT1-MMP was higher in MTD-DDP group?P=0.0.004?,but MT1-MMP expression in LDM-DDP group was lower than control group?P=0.009?.On the 18 th day of chemotherapy,as compared to control group,the expression level of MT1-MMP was higher in MTD-DDP group?P=0.016?,but MT1-MMP expression in LDM-DDP group was lower than control group?P=0.018?.2)Expression of FAKIn the central area: On the second day of chemotherapy,there was a little expression of FAK in the central area of the three groups.The expression level of FAK was no statistical difference among the LDM-DDP group,MTD-DDP group and control group?P=0.082,P=0.196,P=0.420?.On the 10 th day of chemotherapy,as compared to control group,the expression level of FAK was higher in MTD-DDP group?P=0.001?,but FAK expression in LDM-DDPgroup was lower than control group?P=0.002?.On the 18 th day of chemotherapy,as compared to control group,the expression level of FAK was higher in MTD-DDP group?P=0.008?,but FAK expression in LDM-DDP group was lower than control group?P=0.004?.In the peripheral area: On the second day of chemotherapy,there was a little expression of FAK in the central area of the three groups.The expression level of FAK was no statistical difference among the LDM-DDP group,MTD-DDP group and control group?P=1.000,P=0.482,P=0.573?.On the10 th day of chemotherapy,as compared to control group,the expression level of FAK was higher in MTD-DDP group?P=0.021?,but FAK expression in LDM-DDP group was lower than control group?P=0.004?.On the 18 th day of chemotherapy,as compared to control group,the expression level of FAK was higher in MTD-DDP group?P=0.009?,but FAK expression in LDM-DDP group was lower than control group?P=0.006?.Conclusion:1 LDM-DDP resulted in greater tumor growth inhibition in the nude mice bearing HO-8910 ovarian cancer xenografts compared with MTD-DDP,at the same total cumulative dose.2 Ovarian cancer xenografts can form VM,and the growth of VM has certain rules.LDM can reduce the number of VM,and MTD can increase the number of VM.LDM can inhibit the formation of VM and MTD can promote the formation of VM.3 In LDM model,the expression level of MT1-MMP and FAK in the ovarian cancer xenografts is higher than that in MTD model,suggesting that LDM may through inhibit the expression of MT1-MMP and FAK to reduce the formation of VM,and to inhibit tumor growth,invasion,metastasis and recurrence.