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 six-well plate, as they are harvested, and use sterile scissors to mince the tumors into 2- to 3-millimeter pieces. Next, digest the tumor fragments with 1 milliliter of disk-based collagenase, DNase I solution for 45 minutes at 37 degrees Celsius and 5% carbon dioxide with humidity, mixing the samples with a 10-milliliter syringe every 15 minutes.

At the end of the incubation, filter the cells through 100-micrometer strainers into one 15-milliliter tube per well to remove the undigested fibers, and add PBS to a final volume of 15 milliliters. Collect the dissociated cells by centrifugation, and lyse the red blood cells with 1 milliliter of lysis buffer per tube.

After mixing, combine the contents from each tube into a single 15-milliliter tube, stopping the reaction after two minutes with 11 milliliters of complete 4 degrees Celsius medium. After centrifugation, resuspend the pellet in 1 milliliter of PBBS, and block any nonspecific binding with three microliters of FC-blocked antibody for 15 minutes on ice.

At the end of the incubation, label the cells with anti-CD11b and Ly6G antibodies and any other antibodies of interest plus DAPI for a 30-minute incubation on ice protected from light. At the end of the incubation, wash the cells in 14 milliliters of PBS, and resuspend the pellets at a 1 x 10⁷ cells per milliliter of fresh complete medium concentration on ice.

Then, sort the CD11b-positive Ly6G-high DAPI-negative neutrophils on a fluorescence-activated cell sorter according to standard sorting protocols, checking the purity of the isolated neutrophils on the flow cytometer at the end of the sort. For in vitro tumor-associated, neutrophil inhibition, seed 1.5 times 10 to the fifth sorted neutrophils into each of two wells of a 96-well U-bottom plate, and add FK866 to a final concentration of 100 nanomolar in complete medium into one well and an equal volume of complete medium alone to the other well. After two hours in the cell culture incubator, wash the cells two times with PBS, and resuspend the pellets in PBS at the appropriate concentration for the subsequent injection.