Matrigel implants embedded with IDVCs (IDO1-dependent vascularizing cells) to study inflammatory neovascularization.

The pathological expansion of immature blood vessels through neovascularization contributes to the development of a variety of diseases. In cancer, neovascularization supports tumor outgrowth and influences how tumors respond to therapy. Our studies have revealed that a defined cell population termed IDVCs (IDO1-dependent vascularizing cells) expressing the tryptophan catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) can foster a local inflammatory environment that promotes neovascularization. A powerful tool for investigating the biological role of isolated IDVCs in this inflammatory neovascularization process has been the Matrigel plug assay. In this assay, isolated cells are incorporated into a subcutaneously implanted Matrigel plug which is subsequently evaluated by confocal immunofluorescence microscopy for blood vessel density. We have employed this assay to demonstrate that isolated IDVCs are capable of promoting local neovascularization in an IDO1-dependent manner. Furthermore, the use of genetically engineered mouse strains and pharmacological interventions has enabled us to carry out in-depth investigations into IDO1's function as a nodal modifier of the local inflammatory environment responsible for eliciting a shift in the cytokine milieu from a neovasculature-restrictive to a neovasculature-sustaining status. Here we present a detailed methodology describing the reagents and procedures developed to isolate IDVCs and perform quantitative neovascularization studies. This assay should have great utility as a means for conducting investigative studies delving into the cellular and molecular processes involved in the complex interplay between inflammation and neovascularization.