| Objective:Vasculogenic Mimicry(VM)is closely related to tumor invasion and metastasis,and is an important indicator to judge the degree of tumor malignancy and drug resistance.The extracellular matrix in the tumor microenvironment is a key regulator affecting the occurrence and development of angiogenesis mimicry,and due to the complexity of the microenvironment and the limitations of the VM model itself,there is an urgent need to establish a new tumor model to study it in depth.In this study,Collagen and Fibronectin were used as extracellular matrices in the tumor microenvironment to regulate the proliferation,migration,matrix reconstruction,phenotypic transformation and VM of tumor cells derived from different organs(B16F10,A549),and on this basis,in vitro animal model is constructed to reveal the molecular mechanism of extracellular matrix formation in the tumor microenvironment.Methods:1、Different concentrations(1/5/10/15μg/ml)and components of extracellular matrix protein(Collagen/Fibronectin)were prepared to culture different types of tumor cells(melanoma-B16F10,lung cancer-A549),then selected the optimal experimental concentration;2、Different extracellular matrix protein components were grouped at the final experimental concentration: Control group(PBS),CO(Collagen),FN(Fibronectin),Collagen/Fibronectin(Collagen Fibronectin)to study the effects of different types of tumor cell behavior(cell morphology,proliferation,clonal formation,migration,invasion);3、Different types of tumor cells were cultured based on different extracellular matrix protein components to induce the formation of the VM system,regulate cell behavior and detect cell tubularization ability,stem cell properties of tumor cells(Matrigel evaluation,fluorescent labeling,cell immunofluorescence);4、The tumor cells induced by different optimized extracellular matrix protein components(Control group,CO group,FN group,Collagen/Fibronectin group)were injected into the mouse subcutaneous respectively,a murine tumor animal model was constructed for tumorigenesis and VM construction verification and evaluation,tumor size and weight were measured,tissue sections were used for CD31/PAS double staining to detect the number of VM,tumor vascular morphology was determined by HE staining,immunohistochemical staining of VM related proteins and EMT related proteins were determined.5、The expression of VM and EMT related proteins was detected by WB and immunofluorescence.Comprehensive analysis of the influence and mechanism of extracellular protein matrix culture system on VM formation in the tumor microenvironment.Results:1、When the concentration of collagen or fibronectin was 5 μg/ml,the proliferation of tumor cells was significantly increased,and there was no significant difference between the 10 μg/ml and 15 μg/ml groups(P>0.05),and 5 μg/ml was selected as Experimental concentration(P<0.05,P<0.01).2、The effects of different extracellular matrix protein components on tumor cell behavior,induction of VM formation,and stemness expression were detected.Under the light microscope,there was no significant difference in the morphology of B16F10 and A549 cells in the Collagen/Fibronectin group,but the number of cells increased significantly.CCK-8 showed that tumor cells proliferated significantly in the Collagen/Fibronectin group(P<0.05).Ed U detection and Ki-67 cell immunofluorescence showed that the proliferation ability of A549 and B16F10 cells in the Collagen/Fibronectin group was enhanced,and the results of clone formation experiments were consistent with the above results.Cell scratch healing experiments and Trans-well experiments showed that the combined system of Collagen and Fibronectin could promote the migration of B16F10 and A549 cells(P<0.05).Matrigel was used to evaluate the tube-forming ability of tumor cells.B16F10 and A549 cells cultured on different matrices could form tubes,and the tube-forming ability of cells cultured on Collagen/Fibronectin matrix was improved(P<0.05).In addition,cell immunofluorescence showed that the expression of stemness factors in B16F10 and A549 cells cultured on Collagen/Fibronectin matrix was enhanced,which promoted the formation of VM(P<0.05,P<0.01).3、The effect of tumor cells induced by different extracellular matrix protein components on the formation of subcutaneous tumors and VM in mice was detected.In vivo experiments,the COFN system significantly enhanced tumorigenicity in terms of average tumor size and weight compared with the control group(P<0.001).At the same time,immunohistochemical staining of melanoma tissue was performed using Ki-67,and the results showed that the Collagen/Fibronectin group could better promote tumor proliferation(P<0.01).CD31-PAS double staining results of tumor tissues showed that Collagen/Fibronectin matrix treatment significantly increased the number of VM(P<0.001).Immunohistochemical results showed that the expressions of VE-cadherin,Eph A2,and Vimentin were increased(P<0.001).MMP-2 tissue immunofluorescence showed that Collagen/Fibronectin matrix treatment can better promote matrix remodeling and microvessel formation.At the same time,the tumor tissue was stained with HE.Compared with the control group,the Collagen/Fibronectin group formed more microvessels.The fluorescence results were consistent.4、To study intracellular signal transduction regulated by the matrix,the activation of VM-related pathways was assessed by western blot and immunofluorescent staining.WB results showed that the expressions of Integrin-β3,VE-cadherin,Eph A2,PI3 K,MMP-2,and Vimentin in B16F10 cells in the Collagen/Fibronectin group were significantly higher than those in the Control group(P<0.01,P<0.001).In addition,the expressions of Integrin-β3 and Vimentin were also detected by immunofluorescence,and the results were consistent with WB.The results indicated that ECM proteins could potentially regulate VM formation in tumors via the Integrin-β3/VE-cadherin/Eph A2/PI3K/MMP-2 axis and EMT-related signaling pathways.Conclusion:Studies have shown that ECM proteins can induce VM signature formation in tumors through Integrin-β3/VE-cadherin/Eph A2/PI3K/MMP-2 and EMT-related signaling pathways.The collagen/fibronectin matrix promotes tumor proliferation,migration,and induction of VM formation,and the results highlight the plasticity of tumor cells in response to Integrin-mediated cell-extracellular matrix interactions after collagen/fibronectin matrix treatment.Targeted collagen/fibronectin expression is a potential method to inhibit VM formation in tumors and reduce anti-angiogenic therapeutic resistance,and the use of ECM protein has the potential to construct in vitro tumor models for application in the field of VM research. |
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