The Pilot Study On The Linearly Patterned Programmed Cell Necrosis And Vasculogenic Mimicry Of Melanoma

Objective Melanoma is a skin cancer with poor outcome associated with the strong invasiveness and high metastatic potential. VM correlates significantly with the invasion and metastasis of melanoma. LPPCN--linearly patterned programmed cell necrosis is a special kind of programmed cell death with no inflammatory cell infiltration. The LPPCN cells was disappeared and the empty spaces was left to provide spatial structure for the formation of vasculogenic mimicry（VM） and endothelial-dependent vessel（EDV）. In this present study, we collected 135 primary melanoma tissue specimens to detect the phenomenon of VM and LPPCN in melanoma.Methods 1. 135 primary melanoma tissue specimens were obtained from the Tumor Tissue Bank of the Tianjin Cancer Hospital. We detected the phenomena of LPPCN and VM in the tissues by HE staining and CD34/PAS double staining respectively. Immunohistochemical staining method was used to test the expression of c-myc in human melanoma tissues. SPSS software was used to clarify the relationship of c-myc and VM, LPPCN and clinicopathological data. Kaplan–Meier survival analysis and Log-rank test was also performed. 2. MTT methods, Matrigel 3D culture, invasion and migration assay, wound healing assay were performed to detect the impact of c-myc on the biological behavior of melanoma cells. Western Blot, RT-PCR and immunofluorescence methods were used to determine the impacts of c-myc on the expression of VM-related proteins. Co IP and ruciferase method were used to explore the mechanism of c-myc in regulation Snail and VE-cadherin expression. 3. Annexin V-FITC apoptosis methods and flow cytometry were used to test the roles of c-myc in apoptosis of melanoma. Western blot method and si RNA ofBcl2 and Bax were used to explore the mechanism of c-myc promoting cell death. Hypoxia treatments models were established. RT-PCR method and Western blot were used to test the influence of c-myc in the susceptibility of melanoma cell apoptosis. 4. Xenograft model of nude mouse was used to test the effect of c-myc on melanoma in vivo. HE staining and Endomucin/PAS double staining were performed to observe the LPPCN and VM in xenograft tumor. Immunohistochemical and was used to detect the protein expression related to VM and cell apoptosis. 5. We established a paraffin embedding method for multicellular spheroids.Mouse melanoma cell line B16F10 multicellular spheroids were cultured and embedded by paraffin. HE staining and IHC staining were used to observe the LPPCN phenomena.B16F10 multicellular spheroids were injected in C57 mice, at the fifth day the xenograft tumors were harvested and embedded. HE staining method was performed to test the formation of LPPCN. 6. We enriched melanoma stem-like cells by tumorophseres method（using serum-free medium culture system）. B16F10 cells cultured in the multicellular tumor spheroid（MTS） model was used as control. Using HE staining method detect morphological changes of melanoma cells in these two culture system. 7. The extracted RNA of melanoma stem-like cells was collected. We uses an Agilent Mouse Gene Expression to detect the gene expression profile of melanoma stem-like cells. Then quantitative real-time PCR was performed to identify the changes in expression levels. 8. To detect the role of Notch4 expression in melanoma, Notch4 high B16F10 cell was isolated by fluorescence-activated cell sorting（FACS）. The efficiency of isolation and the subsequent determination of VM-related protein expression were performed by Western Blot.9. Matrigel 3D culture, invasion assay and wound healing assay were performed to detect the impact of Notch4 on the biological behavior of melanoma cells. The effects of transfection and its impacts on expression of VM-related proteins and apoptosis proteins were tested by the Western Blot, RT-PCR and immunofluorescence. 10. We used DAPT, a γ-secretase inhibitor（GSI） to block Notch signaling and used Western Blot to detect Twist1, E-cadherin and E-cadherin expression. A full-length Twist1 complementary c DNA was transfected into A375-sh Notch4-3 and MUM-2B-sh Notch4-3 cells and Western Blot was used to detect Twist1, E-cadherin and E-cadherin expression. 11. 120 primary melanoma tissue specimens were obtained from the Tumor Tissue Bank of the Tianjin Cancer Hospital. Immunohistochemical staining method was used to test the expression of Notch4 in human melanoma tissues. SPSS software was used to clarify the relationship of Notch4 and clinicopathological data.Results 1. In the 135 cases of melanoma tissues, we found both LPPCN（48.15%） and VM（45.93%）. Both LPPCN and VM are significantly positively related to metastasis, Breslow’ depth and poor prognosis（p<0.05）.Most of human melanoma tissues showed c-myc positive expression. And the enhanced expression of c-myc was significantly related to the phenomena of LPPCN and VM, Breslow’ depth, lymphatic and distant metastasis（p<0.05）. Kaplan-Meier and Log-rank test showed patients with c-myc high expression had poor prognosis. 2. Overexpression c-myc enhanced the ability of VM formation, invasion and migration and the sensitivity of apoptosis of A375, MUM-2B and MUM-2C cell lines（p<0.05）. Western Blot, RT-PCR and immunofluorescence results showed c-myc overexpression increased the expression of Vimentin, VE-cadherin, Snailand decreased the expression of E-cadherin（p<0.05）. Reporter gene assays suggested that c-myc can combine with the promoter of VE-cadherin and Snail to enhance their activity of transcription. 3. Results of hypoxia treatments showed enhanced expression of c-myc increased the protein Bax expression and decreased the ratio of Bcl2/Bax in severe hypoxic conditions（p<0.05）. Consequently overexpression c-myc increases in melanoma cells sensitive to hypoxia. 4. Animal experiments showed that overexpression c-myc increased the tumor growth, volume and the phenomena of LPPCN and VM in xenograft tumors. The expressions of VE-cadherin, Snail, Bcl2 were up-regulated by the overexpression of c-myc, in contrast, the protein expression of HIF-1α and E-cadherin was down-regulated by the c-myc overexpression. 5. The HE staining of multicellular spheroids showed the three layers: proliferating cells, resting cells and dying/necrotic cells from outer to center. In the dying/necrotic cell layer, melanoma cells was displayed as SPPCN in which cells were darker staining, concentrated cytoplasm and loss of cell-cell adhesion. At the fifth day, the xenograft tumors in C57 mice were harvested. The xenografted melanoma without blood vessel or VM contain LPPCN like cells not for linearly patterned. 6. The gene expression analysis showed 3760 differentially expressed genes, including 1699 up-regulated gene and 2061 down-regulated gene in MCSLCs. The expression levels of Notch3（NM₀08716）, Notch4（NM₀10929）, Dtx4（NM₁72442）, JAG2（NM₀10588） Pofut（NM₀80463）, Sox4（NM₀09238）, Wnt10a（NM₀09518）, NGFR（NM₀33217）, ABCA8（NM₀13851） and ABCA1（NM₀13454） were assessed. The quantitative real-time PCR identified the changes in expression levels and showed that MCSLCs expressed higherlevels Notch4 and the cancer stem cell-related gene NGFR 7. Western Blot results showed Notch4highB16F10 expressed high level of Notch4, Jagg2, Twist1, VE-cadherin, Sox2 and low level of E-cadherin。 8. A375 and MUM-2B cells expressed high levels of Notch4 and demonstrated the capacity of vasculogenic mimicry（VM） formation. Notch4 silencing reduced cell invasion, migration and VM formation. Western Blot results and quantitative real-time PCR revealed Notch4 suppression decreased Twist1 and VE-cadherin（CDH5） expression and increased E-cadherin expression. 9. DAPT inhibited Twist1 and VE-cadherin（CDH5） expression and increased E-cadherin（CDH1） expression. Notch4 supression in cells increased VE-cadherin（CDH5） expression and decreased E-cadherin（CDH1） expression after the re-overexpression of Twist1 by transfection of cells with Twist1 expression clone. 10. Immuhistochemistry method showed 72 cases of melanoma patients expressed high expression of Notch4 was found to be related to metastasis of melanoma and indicated a poor prognosis.Conclusions 1. Human melanoma tissues have LPPCN and VM. There are significant relationships between both of LPPCN and VM with metastasis, Breslow’ depth and poor prognosis. c-myc is highly expressed in malignant melanoma and related to Breslow’ depth, lymphatic and distant metastasis, poor prognosis and the phenomena of LPPCN and VM. 2. c-myc promotes the formation of VM by enhancing the mesenchymal phenotype of melanoma via up-regulated Vimentin and VE-cadherin expression and weakening epithelia phenotype via TGF-b/Snail/E-cadherin. 3. c-myc promotes LPPCN by sensitizing melanoma cells to severe hypoxia via upregulating Bax expression.4. In the multicellular spheroids without blood supply, there are LPPCN like cell death, but not linearly patterned distribution. Melanoma cells cannot form LPPCN without blood vesssel. 5. The gene expression analysis showed 3760 differentially expressed genes, including 1699 up-regulated gene and 2061 down-regulated gene in MCSLCs. Melanoma stem-like cells expressed high level of Notch4. Notch4 promotes invasion, migration and the formation of VM by regulating the Twist1 expression. Notch4 is up-regulated in metastatic melanoma and related to poor prognosis.