Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

Our protocol provides a novel tool for evaluating the angiogenic potential of tumor-associated primary neutrophils in vivo without the need to use artificial neutrophil cell line models. Direct inhibition of nicotinamide phosphoribosyltransferase, shortly NAMPT, in tumor-associated neutrophils with their subsequent adoptive transfer into tumor-bearing animals allows the manipulation of neutrophils without systemic toxic side effects. We will demonstrate the therapeutic potential of manipulated anti-angiogenic tumor-associated neutrophils after transfer into tumor-bearing hosts for the study of potential neutrophil-based immunotherapies in different cancer models.

To set up an allogenic tumor mouse model, shave the skin of 10 eight to 12-week old female interferon alpha and beta-receptor subunit 1 knockout mice with an electric shaver and disinfect the skin with 70%ethanol, then load three times 10 to the six B16F10 melanoma cells per milliliter of PBS in a one-milliliter syringe equipped with a 0.4 by 19-millimeter needle and inject 100 microliters of the suspension subcutaneously on a rear flank into each shaved animal. For neutrophil-adoptive transfer, dilute the B16F10 melanoma cells to a six times 10 to the sixth cells per milliliter concentration in PBS and dilute neutrophils treated with NAMPT inhibitor FK866 to a six times 10 to the fifth cells per milliliter of PBS concentration. Then, mix untreated or inhibitor-treated neutrophils with the melanoma cells at a neutrophil-to-tumor ratio of 1:10 and subcutaneously inject 100 microliters of cells into up to five mice per group.

After the injections, place up to five injected female mice in a single cage and use calipers to measure the tumor length, width, and depth every day for 14 days. At Day 14 after injection, disinfect the skin of each injected animal with 70%ethanol and use scissors and forceps to harvest the tumors into a 50-milliliter conical tube containing complete medium on ice. To section the tumors for histological analysis, submerge the tumors in optimum cutting temperature compound and freeze the samples in liquid nitrogen for storage at minus 80 degrees Celsius.

On the day of the sectioning, thaw the samples to minus 20 degrees Celsius before using a cryotome to obtain five-micrometer sections. After fixation in non-specific binding, stain the tumor tissue sections with the primary antibodies of interest for one hour at 20 degrees Celsius, followed by three washes in PBS. After the last wash, stain the cells with an appropriate fluorescence-conjugated secondary antibody in a nuclear-staining dye like DAPI for one hour at 20 degrees Celsius protected from light.

At the end of the incubation, dry the slides for 20 minutes at 20 degrees Celsius protected from light before mounting the samples with an anhydrous mounting medium, then cover each slide with a cover slip and allow the mounting medium to dry for one hour at 37 degrees Celsius before imaging the tissues by fluorescence microscopy. 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 two to three-millimeter pieces. Next, digest the tumor fragments with one milliliter of dispase collagenase d dnase 1 solution for 45 minutes at 37 degrees Celsius in 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 one 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 four-degrees Celsius medium.

After centrifugation, resuspend the pellet in one milliliter of PBS and block any non-specific binding with three microliters of Fc-block 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 one times 10 to the seventh 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. NAMPT-inhibitor treated neutrophils have a significantly decreased capacity to stimulate aortic branch formation compared to untreated cells.

In addition, the subcutaneous injection of inhibitor-treated anti-angiogenic neutrophils induces a significant impairment in tumor growth compared to mice injected with untreated interferon alpha and beta receptor subunit 1 knockout neutrophils. Histological examination of the extracted tumors further confirms the significant suppression of angiogenesis in tumors isolated from mice treated with inhibitor-treated tumor-associated neutrophils compared to those injected with untreated knockout neutrophils. To assess the properties of FK866-treated tumor-associated neutrophils, quantitative PCR and Western Blot can be performed to study the activation of intracellular pathways involved in neutrophil polarization.

Since neutrophils are short-lived, it is necessary to perform all of the steps as quickly as possible and to keep neutrophils cold during the entire procedure. This technique paves the way for the study of the intracellular mechanisms and factors involved in the regulation of angiogenic and tumorigenic properties of tumor-associated neutrophils in vivo.