Overexpressing DLL1 Induces Tumor Vascular Normalization And Inhibits Tumor Growth

Objective: Notch signal pathway is an ancient and conservative signal pathway,and plays crucial roles in a range of life processes.In mammalian,Notch signaling pathway consists of four Notch receptors(Notch 1-4)and five ligands(DLL1,DLL3,DLL4,Jagged1,and Jagged2).Previous studies showed that notch ligand DLL4 involved in tumor angiogenesis.Although blockade of DLL4 suppressed tumor growth,it induced vascular neoplasm.Thus,we investigated the effects and mechanisms of DLL1 on tumor angiogenesis in this study.Methods: We developed breast carcinoma cell line(EO771)and lung carcinoma cell line(LAP0297)with DLL1 overexpression or empty construct vector by using a retrovirus system.EO771 tumor cells were orthotopically inoculated into the mammary fat pads of C57BL/6 mice.LAP0297 tumor cells were inoculated into the right back flank of FVB mice.We used the caliper to measure the tumor size every three days when tumors became palpable.When tumor size reached 6-8 mm in diameter,tumor tissues were harvested and cut into four pieces.One piece was fixed with 4% paraformaldehyde for histological analysis.Another piece was dissociated to obtain single cell suspensions for flow cytometric analysis.The remaining two pieces were frozen in liquid nitrogen for protein or m RNA analysis,respectively.In vivo T cell depletion was used to investigate the mechanism of the impact of DLL1 overexpression on tumor growth and tumor angiogenesis.Result: We successfully constructed the breast carcinoma cell line(EO771)and lung carcinoma cell line(LAP0297)with DLL1 overexpression or empty vector.Overexpressing DLL1 in tumor cells inhibited tumor growth and even induced regression in some of tumors in both tumor models,compared with tumor cells with the control vector.Overexpressing DLL1 increased the number of tumor-infiltrating CD8~+ T cells and promoted their IFNγ production.Moreover,overexpressing DLL1 reduced tumor vessel density,increased pericyte coverage,and improved vascular perfusion,suggesting that overexpressing DLL1 induces tumor vascular normalization.In addition,overexpressing DLL1 promoted the polarization of tumor associated macrophages to an M1-like phenotype.Furthermore,in vivo depletion of CD8~+ T cells reversed tumor growth inhibition and vascular function enhancement by DLL1 overexpression.Simultaneously depletion of CD8~+ and CD4~+ T cells reversed tumor vascular normalization induced by DLL1 overexpression.Conclusion and significance: Overexpressing DLL1 in the tumor microenvironment induces tumor vascular normalization and inhibits tumor growth via T cell activation.Our findings suggest that DLL1 could be served as a new target for tumor immunotherapy.