Generating Anti-Angiogenic Tumor-Associated Neutrophils

Take a murine melanoma tumor comprising tumor-associated neutrophils, or TANs.

TANs display elevated activity of the enzyme nicotinamide phosphoribosyltransferase, or NAMPT, stimulating angiogenesis, the proliferation of blood vessels in tumors, and facilitating tumor growth.

Mince the tissue and add enzymes to degrade the tissue matrix, loosening the cells.

Filter the suspension through a strainer and centrifuge to pellet the cells.

Add a lysing buffer to lyse the red blood cells, then centrifuge and discard the supernatant containing cell debris.

Introduce antibodies to block cellular Fc receptors, thereby avoiding non-specific immunostaining.

Introduce a fluorophore-labeled antibody cocktail specific for surface markers on TANs and a dye to discriminate dead cells.

Using FACS, isolate the fluorophore-labeled viable TANs and transfer them onto a microplate.

Add an inhibitor blocking NAMPT activity to selected wells and incubate.

Use the cells to assess their angiogenesis-stimulating capacity. The inhibitor-treated TANs show reduced angiogenesis compared to the untreated TANs.

For tumor-associated neutrophil isolation, place five tumors per well into individual wells of a sterile