Microcirculation Patterns In Melanoma And The Influence Of Microenvironment On Melanoma Angiogenesis

Objects: To investigate the transformation from vasculogenic mimicry, through mosaic vessels to endothelium-dependent vessels as melanoma grows. Furthermore, to probe the function of endothelial cells and mesenchymal stem cells derived from bone marrow in melanoma angiogenesis. Moreover, to indicate the influence of hypoxia and microenvironment pressure on melanoma angiogenesis and the molecular mechanism involving it. Methods:1 Sixty C57 mice were inoculated with B16 melanoma cells. Mice were sacrificed in turn (5 mice per day) when the size of tumor tissue was about 0.5mm. Formalin-fixed and paraffin-embedded tumor tissues were sectioned. The density of vasculogenic mimicry, mosaic vessels and endothelium-dependent vessels was counted on the sections. The time trend of three microcirculation patterns was determined by microvessels density (MVD). Immunohistochemical doubling-stain with HMB45 and tissue factor VIII and electronic microscopy were both used to confirm vasculogenic mimicry and mosaic vessel in this study.2 Endothelial cells marked with BrdU were injected into the tail vein of SCID mice and mice were sacrificed after 17 days. The tumor mass was removed and thenimmunohistochemically stained after section. Furthermore, mesenchymal stem cells isolated from fetal bone marrow with age of 15-22 weeks were incubated into SCID mice after screening, proliferation, validation and staining with PKH26. The mice were sacrificed after 6 days and the tumor tissue was made into frozen section to investigate with fluorescence microscope.3 The femoral on left hindlimb was ligated to establish an ischemic microenvironment. Mouse melanoma B16 cells were inoculated into the ischemic limbs and non-ischemic controls respectively and the engrafted melanomas were removed subsequently. Vasculogenic mimicry in melanoma cells of the two groups was counted and the expression of CK18, HIF-la, MMP-2, MMP-9, VEGF and Epo were assessed by immunohistochemical staining. Formalin-fixed, paraffin-embedded tissues were used in immunohistochemical staining. Results:1 The number of vasculogenic mimicry decreased and the number of endothelial-dependent vessels increased with time. The number of MV increased to peak at middle life time of the tumor and then decreaed gradually. There is a highly positive correlation between the size of the tumors and the number of endothelium-dependent vessels(r=0.718, P=0.009). There is a highly negative correlation between the size of the tumors and the number of VM(r=0.77, P=0.003). The wall of mosaic vessel was made from tumor cells and endothelial cells.2 Endothelial cells marked with BrdU were found in tumor vessels with anti- BrdU immunohistochemical staining. FISH results showed that there were mesenchymal stem cells from fetal bone marrow stained with PKH26 and they have differentiated to mature endothelial cells. The mesenchymal stem cells stained with PKH26 could be induced to differentiate and concern with the procession of angiogenesis afterproliferation.3 In the early stage of engrafted melanoma growth, the sizes of melanomas in ischemic limbs increased more slowly than in the controls. However, later there was no obvious difference in their size. The number of vasculogenic mimicry in melanoma of ischemic group was more than in controls (P=0.039). More tumor samples expressed CK18 in ischemic group than controls. Similarly, the expression of HIF-la, MMP-2, MMP-9, VEGF and Epo was higher in ischemic group than in non-ischemic controls (P=0.024, 0.047, 0.007, 0.025 and 0.027 respectively). There was positive correlation in melanoma cells of ischemic group between HIF-la and the tumor size and HIF-la expression and the number of vasculogenic mimicry (r=0.472 and 0.391, P=0.015 and 0.050 respectively). Conclusions:1 In the early stage of tumor growth, vasculogenic mimicry is the main blood supply pattern. As the size of tumor tissue enlarging and the number of endothelial cells proliferating, vasculogenic mimicry will be substituted by endothelium dependent vessels. Mosaic vessel might be the intergradation between VM and endothelium-dependent vessels.2 Not only the endothelial from the peripheral tissue of tumor, but also mesenchymal stem cells derived from bone marrow can take part in tumor angiogenesis.3 Vasculogenic mimicry is the main microcirculation pattern in the hypoxic condition. Melanoma cells express CK18, secrete more invasion-related proteins and become more invasive in the hypoxic microenvironment.4 Melanoma cells in the hypoxic microenvironment improved the expression of VEGF and Epo induced by HIF-la expression and prompted the formation of vasculogenic mimicry to acquire an adequate blood supply.5 The expression of MMP-2 and MMP-9 in tumor tissue increased to form vasculogenic mimicry and enhance the invasion ability of melanoma cells.6 Melanoma obtains blood supply form Vasculogenic mimicry mainly in a microenvironment with high pressure while endothelium-dependent vessel is the main microcirculation pattern in a microenvironment with low pressure.