The Impact Of Local Microenvironment On Linerly Patterned Programmed Cell Necrosis In Mouse Melanoma And Molecular Mechanism

ObjectiveTo observe how hypoxia and microenvironment pressure changes influence the number and distribution of LPPCN (Linearly Patterned Programmed Cell Necrosis) in mouse melanoma. To analyze the growth rate of transplanted tumors, patterns of angiogenesis and the ability of tumor invasion in different local microenvironments.To illustrate the related signal transduction pathways in LPPCN and the significance of LPPCN as the spatial structure of Vasculogenic Mimicry (VM).To enrich the theory of tumor angiogenesis.Methods1. Building of animal modelThe left femoral arteries of C57 mice were ligated and then B16F10 melanoma cell suspension was injected into the left ischemic skeletal muscles and the non-ischemic right controls respectively to build the ischemic animal model; Biomechanical animal model was built by injecting the B16F10 melanoma cell suspension into the the abdominal cavity and left skeletal muscles of C57 mice.2. Analyzing of the related molecular mechanism of the formation of LPPCNHE staining and immunohistochemistry staining were performed to compare the growth pattern, LPPCN distribution,VM number and the invasion ability among groups of different local hypoxic condition and pressure situation.Results1. LPPCN exists in mouse melanomaLinerly or network-like LPPCN cells exist in C57 B16 melanoma.LPPCN cells were deeply stained and karyorhexis can be found. The banded structure is often surrounded by endothelium-dependent blood vessels and sometimes the cable channels encircled by LPPCN cells can be seen and red blood cells were in the channels.2. The difference of LPPCN number among groups In the ischemia group,LPPCN count (11.25±6.04) and LPPCN cell number (51.73±25.06) were much higher than the control group (5.50±3.34,16.50±4.01) and the difference was statistically significant (P<0.05); In the biomechanical model, LPPCN count (13.80±6.05) and LPPCN cell number (56.46±28.36) were much greater than the abdominal group (6.67±3.12,16.98±8.69) and the difference was significant (P<0.05).3. LPPCN immunohistochemical stainingBcl-2 staining index of SPPCN(Scattered Patterned Programmed Cell Necrosis, the early stages of the formation of LPPCN) cells(3.68±1.3.7) were higher than LPPCN cell staining index (2.15±1.16); Bax staining index of SPPCN cells (2.10±0.79)was lower than LPPCN cells(4.35±1.20).Differences were statistically significant (P<0.05). Caspase-3 staining index of LPPCN cells was higher than the Caspase-3 staining index of the tumor cells around LPPCN. So were the staining index of Caspase-9 and Bax.The differences were all statistically significant (P <0.05).4. The expression of invasion-related proteinHIF-1αstaining index (3.38±1.93) of ischemia group was higher than the control group (1.75±0.71); HIF-1αstaining index of the hindlimb group (3.10±1.60) was greater than the abdominal group (1.89±0.60) (P<0.05); MMP-2 staining index of the ischemic group (3.00±0.76) were higher than the control group (1.38±0.52); MMP-2 staining index of hindlimb group (4.10±0.88) was higher than abdominal group (3.00±1.12), and the difference was significant (P<0.05); MMP-9 staining index of ischemic group (4.00±0.93) were higher than the control group (2.50±0.76); MMP-9 staining index of hindlimb group (4.40±0.70) was higher than abdominal group (2.22±0.44), and the difference was significant (P<0.05); MMP-2, MMP-9 staining index was positively correlated with LPPCN count (r= 0.402, P= 0.017;r= 0.533, P= 0.001); Twistl staining index in ischemia group (3.25±0.89) and hindlimb group (4.00±0.94) were significantly higher than the control group (1.88±0.84) and the abdominal group (2.44±0.88).5. The difference of VM count among groupsVM count of ischemia group (6.13±0.99) was significantly higher than the control group (2.13±1.13); VM count in hindlimb group (9.40±4.48) was significantly higher than abdominal group (3.44±1.01).6. LPPCN count, LPPCN cell number and the number of VM were positively correlated respectively (r= 0.551, P= 0.001;r= 0.613, P= 0.000).Conclusion1. Hypoxia and pressure changes in tumor can induce the formation of LPPCN; LPPCN exists as a new pattern of cell death which is different from the traditional apoptosis and necrosis. The molecular events of this death may be similar to apoptosis regulated by mitochondria pathway.2. Hypoxic microenvironment and local IFP (Interstitial Fluid Pressure) may have great impact on tumor invasion and the formation of VM. In the hypoxic and high IFP environment, HIF-la was highly expressed which stimulates the MMP-2, MMP-9 and Twistl expression and increases the invasion ability of tumor cells which were involved in the formation of VM.3. By the regulation of apoptosis-related proteins, some tumor cells can form LPPCN and LPPCN may serve as the basis of the spatial structure of VM and endothelium-dependent vessels.As a special pattern of cell death, the mechanism and significance of LPPCN are not fully clear. The theory of cell death model can be enriched through the research on the molecular mechanism of LPPCN. Besides, the possible mechanisms of how endothelial cells around tumor penetrate into the tumor region which has higher pressure can be illustrated,thus provides a theoretical basis for vasculogenic mimicry theory. At the same time,if the key elements of LPPCN formation can be inhibited, it is possible to find new anti-vascular therapy approaches and new targets to tackle the problem of poor clinical effects of traditional anti-angiogenesis treatment.