| BackgroundVasculogenic mimicry(VM),reported by Maniotis et al.in 1999,is a new blood supply pattern for tumors.Numerous evidences indicated that VM is a functional microcirculation pattern in some high malignant tumor.The wall of VM channels is lined exclusively by tumor cells mimicking the presence and function of endothelial cells.Tumor cells contact to blood flow directly,hence tumor with VM is characterized by hematogenous metastasis in an early stage,poor prognosis.The 5-year survival rate of melanoma patients with VM is about O.The molecular mechanism of VM formation is still not clear.The purpose of this research included the structure basis of VM formation,relationship between VM and microenvironment, experimental-therapy.Aims1)To identify VM and LPPCN existence in human melanoma and indicate the clinical and pathological significance of VM and LPPCN through a retrospective study with large samples.Based on these,to investigate the role of VM and the molecular mechanism,signaling transduction pathway involving in LPPCN formation.Finally, disclose the expression of apoptosis-associated proteins in LPPCN cells and the influence of hypoxia,IFP and apoptosis inhibitors on Endo G/Dnase 1 expression in LPPCN cells.2)To investigate the time-dependent changes of different blood supply patterns, including VM,mosaic vessels and endothelium-dependent vessels,in space distribution and destiny,as B16 transplant tumors in C57 mice grow.3)To investigate the influence of hypoxic microenvironment and increased IFP on tumor microcirculation patterns and invasiveness capability and the related molecular mechanism.To discover the relationship between the changes of tumor invasive capability as well as VM formation and melanoma relapse and metastasis after undergoing SFP,and then provide theory for the failures of anti-vascular therapy targeting at endothelium-dependent vessels and SFP.4)to investigate the efficiency of anti-VM therapy aiming at key proteins in VM formation on melanoma using animal experiment,and provide a base for the treatment of malignant tumors with VM.Methods1)One hundred and twenty four melanoma cases undergoing surgery in Tianjin Medical University form 5th/1,1984 to 20th/12,2006 were collected in this study.The information of samples including age,gender,prognosis,metastasis,tumor size and location etc.were collected,and VM and LPPCN,their distribution and their clinical and pathological significance were detected.Mouse model beating B16melanoma was established,and activated carbon tracking technique was used to detect whether VM channels connect with endothelium-dependent vessels.Immunohistochemical staining,PAS-CD 31 double staining and electron microscope were performed to observe the structure and distribution of VM and LPPCN and their correlation in transplant B16 melanoma.2)The difference of MMP-2 and MMP-9 expression in human melanoma with and without VM was detected using immunohistochemical staining.TUNEL and Immunohistochemical staining were performed to identify the apoptosis-associated proteins expressed in LPPCN cells and analyze the signaling transduction pathway controlling LPPCN formation.The influence of hypoxia,IFP and apoptosis inhibitors on Endo G/Dnase 1 expression in LPPCN cells was observed by real-time PCR.3)Sixty C57mice were divided into 12 groups(5one/group)and injected with B16 melanoma.Animals were killed,one group everyday,at 11thday after the inoculation of tumor.PAS-CD 31 double staining,MVD counting and electron microscope were used to observe the time-dependent changes of different blood supply patterns, including VM,mosaic vessels and endothelium-dependent vessels,in space distribution and destiny,as B16 transplant tumors grew.4)C57 mouse model bearing B16 melanoma was established.Five days later, ischemia was produced in the left hindlimb by ligating the femoral artery of C57 mice. After 15 days of melanoma cells injection,mice were sacrificed.Tumor growth in the hypoxic microenvironment,the number and distribution of VM and the expression of tumor invasion and metastasis-associated proteins were detected.Additionally,B16 melanoma cells were engrafted into abdominal cavity and skeletal muscle in the hindlimb of a mouse,and immunohistochemical staining,PAS-CD 31 double staining and Real-time PCR were performed to investigate the influence of different microenvironments on tumor microcirculation patterns and invasiveness capability.5)Mouse model bearing B16melanoma was established to identify the key proteins and their roles in VM formation.Immunohistochemical staining,gelatinase zymogram and electron microscope were finished to investigate the effects of endostatin and doxycycline on melanoma growth,MMPs expression and their activity and different microcirculation patterns in B16 melanoma.Results1)Except of uveal melanoma,VM channels existed in human melanomas from other locations.PAS-positive membrane covering on the wall of VM channels was or not continuous,and some melanoma cells secreted PAS-positive substance to take part into ECM remodeling in VM formation.Animal experiments showed that there were VM channels in transplant B16 melanoma.Activated carbon tracking experiment confirmed that VM channels connected with endothelium-dependent vessels and was functional vessels for tumor blood supply,and PAS-positive membrane was not necessary for VM formation.VM,mosaic vessels and endothelium-dependent vessels co-existed in human and mouse melanoma,all of them composed of microcirculation system of melanoma.Statistical results showed that 44.55%(54/124)of melanoma had VM.Kaplan-Meier survival analysis indicated that the prognosis of patients with VM was worse than the prognosis of those without VM(P=0.000).2)LPPCN appears in B16 melanoma.These LPPCN cells have characteristics of classic necrosis in their morphology,but their distribution is distinguished from both necrosis and apoptosis.They connect with each other as lines and networks,which is similar to the distribution of VM channels and endothelium-dependent vessels. Immunohistochemical staining for HMB45 demonstrated that these cells were melanoma cells.Some networks lined by LPPCN cells showed a trend to connect with endothelium-dependent vessels.Animal experiments showed that LPPCN cells were likely to occur in the early stage of B16 melanoma growth and localize in the center of solid tumor.In human melanoma,there were 40%cases with LPPCN cells. Kaplan-Meier survival analysis indicated that the prognosis of patients with LPPCN cells was worse than those without LPPCN(P=0.041).3)Melanoma cells secreted PAS-positive substance that were granular or lamellar in cytoplasm of melanoma cells.In addition,melanoma cells synthesized and secreted collagenⅣto take part into ECM remodeling in VM formation.MMP-2 and MMP-9 expressed in melanoma cells and some mesenchymal cells.There was significant difference in MMP-2 and MMP-9 expression between melanoma with VM and without VM(P＜0.05).The expression indexes of MMP-2 and MMP-9 in VM group were 65.63±7.55and63.25±7.86,respectively.The expression indexes of MMP-2 and MMP-9 in non-VM group were 48.40±5.49and49.79±6.38.4)LPPCN were negative for caspase-8,caspase-6,caspase-7,TNFα,TRAIL,FAS and FASL,while they were positive-for caspase-9,caspase-3 and Bax.Melanoma cells positive for TUNEL staining distributed randomly,but not showing a network shape.The results of Real time-PCR for cultured cells in different conditions showed that the level of Dnase1 mRNA expression in both hypoxic group and control group increased as culture time expanded(P＜0.05).However,there was more Dnasel mRNA expression in the hypoxic group than that in the control group(P＜0.05).The expression of EndoG mRNA of melanoma cells in the hypoxic group upregulated and reached to the peak at 8h,and it was significantly higher than that in the control group at 6hand8h(P＜0.05).animal experiment results found that EndoG mRNA expression was upregulated remarkably in the ischemic group compared with the control group (P＜0.05),while Dnasel mRNA expression decreased in the ischemic group (P＜0.05).In addition,melanoma cells in both two groups expressed more EndoG than Dnasel,which was consistent with the experiment in vitro.There was no remarkable difference in EndoGandDnasel mRNA expressions between the abdominal cavity and the skeletal muscle(P＞0.05).Melanoma cells in Caspase inhibitor group did not express more EndoG than those in the control group.Dnasel mRNA expression of melanoma cells in two groups had a similar trend.5)All three types of microcirculation patterns were observed during tumor development.In the early stage of tumor growth,vasculogenic mimicry is the main pattern of blood supply.As tumor size increased,the number of mosaic vessels and endothelium-dependent raised.The number of endothelium-dependent vessels correlated with the size of the tumor(r=0.718,P=0.009),while the number of VM was inversely correlated(r=0.77,P=0.003).6)In the early stage of engrafted melanoma growth,the size of melanomas in ischemic limbs increased slower than in the controls(t=0.858,P＜0.05).However, there was no obvious difference in their size when animals were killed.Melanoma tumors in the ischemic group had more vasculogenic mimicry channels than those in the controls(P＞0.05).The positive rate of HIF-1αwas 68.78%in the ischemic group that was significantly higher than that in the control(45.62%,P＜0.05). Similarly,the expression of MMP-2,MMP-9,and VEGF was higher in the ischemic group than in the controls(P=0.015,0.008,and 0.019,respectively).In the ischemic group,there was statistical significance for the correlation between HIF-1a and tumor size and VM number(r=0.472,0.391,P=0.015,0.050)7)There were VM channels,mosaic vessels and endothelium-dependent vessels in melanoma from both the abdominal cavity and the skeletal muscle,t-test showed that the skeletal muscle tumors had more VM channels than the abdominal cavity tumors (t=6.33,P=0.001),whereas endothelium-dependent vessels tended to be the main blood supply pattern in melanoma in the abdominal cavity.There was no difference in the number of MV and endothelium-dependent vessels between the abdominal tumors and the skeletal muscle tumors(t=1.10,3.90,P=0.282,0.069).CK-18 expression in the skeletal muscle tumors was significantly higher than in tumors in the abdominal cavity(P＜0.05).Compared with melanoma in the abdominal cavity,both MMP-2 and MMP-9 in mRNA and protein level were overexpressed in melanoma in skeletal muscle(P＜0.05).Melanoma growing in the skeletal muscle expressed more HIF-1αthan that in the abdominal cavity(25.22±8.50 and 45.80±14.87,P＜0.05)8)After treatment with doxycycline and endostatin,melanoma sizes in the doxycycline,endostatin,combination,and control groups were distinguished(P＜0.05).MV and VM counts in the different treatments were less than those in the control groups(P＜0.05).The expression of MMP-2 and MMP-9 in melanoma cells decreased from doxycycline,endostatin,combination to control group in turn,and the difference was statistically significant(F=12.791,F=5.560,P＜0.05).TIMP-2 expression increased from doxycycline,endostatin,combination to control group in turn,and the difference was statistically significant(F=4.638,P＜0.05).Gelatinase zymogram showed that the activity of MMP-9andMMP-2 in the doxycycline treatment group was statistically significant was higher than that in the combination group.The result of electron microscope demonstrated that endothelial cells in melanoma treated with endostatin were immature.Conclusions1)VM,a novel microcirculation pattern,exist in both human and mouse melanoma. Tumors with VM,due to its special structure,are likely to spread via blood vessels in the early stage,and the patients faced an inefficient curative effect and a poor prognosis.There are special molecular mechanisms involving in VM formation. Both of MMPs secreted-by tumor cells and ECM contribute to ECM-remodeling in VM formation.2)under the control of apoptosis-associated proteins,some melanoma cells undergo LPPCN that provide space for the formation of VM and endothelium-dependent vessels.LPPCN occurs in the high malignant melanoma.Patients with LPPCN have a worse outcome than those without VM.Two endonucleases,EndoGandDnase 1,are responsible for chromatin fragment,karyopyknosis,and chromatin margination in the process of LPPCN.The upregulation of their expressions in melanoma cells is closely related to hypoxia.3)According to the evidences from human and mouse melanoma,there is a three-stage phenomenon among VM channels,mosaic blood vessels,and endothelium-dependent blood vessels sustaining the blood supply.4)Tumor microenvironment is the most essential factor contributing to VM formation. Melanoma cells in the hypoxic condition express HIF-1αwith a high level,which upregulates MMP-2andMMP-9 expressions and leads to the digestion of ECM.This facilitates the tumor cells invasion and provides the space for VM formation.In addition to hypoxia,IFP in tumor tissue is another essential environmental factor influencing tumor growth and invasiveness.Tumor cells in a high-pressure microenvironment must secrete more MMPs to degrade the adjacent skeletal muscle to enable continual growth.Under selection from high interstitial fluid pressure in the tumor and mechanical pressure between the tumor and muscle tissue,tumor cells become more invasive.5)Doxycycline and endostatin inhibit the expression of MMP-2 and MMP-9 and the maturation of endothelial cells,which results in the regression of VM and endothelium-dependent vessels formation and the inhibition of melanoma growth. Hence the combination of doxycycline and endostatin has potential effect on melanoma with VM channels. |
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