Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

This method can help answer key questions about the role of neutrophil subpopulations in tumor immunology especially about the mechanical involvement of neutrophil extracellular traps, or NETs, in tumor metastasis. The main advantage of this technique is that it allows visualization of the adhesive interactions of tumor cells to intact NETs. The implications of this technique extend towards the therapy of cancer.

As NETs derived from specific neutrofil subsets may promote tumor metastasis. Demonstrating the procedure will be Junko Shoniohara from our laboratory. To acquire the neutrophils first infuse one liter of normal sterile saline immediately before wound closure directly into the abdominal cavity of a patient who has just undergone abdominal surgery due to gastrointestinal malignancy and lavage the abdominal cavity extensively for at least one minute.

Then stir the saline within the cavity and recover 200 milliliters of lavaged fluid with four 50-milliliter syringes. In the lab transfer the peritoneal lavage fluid through a 100 micrometer nylon filter into individual 50 milliliter tubes for centrifugation. Re-suspend the pellets in 5 milliliters of PBS supplemented with 0.02%EDTA and carefully overlay each cell suspension onto three milliliters of density gradient solution.

After density gradient separation harvest two to three milliliters of the intermediate layer from each tube and wash the peritoneal fluid samples in 10 milliliters of fresh PBS plus EDTA per tube. Pool the pellets in 10 milliliters of fresh PBS plus EDTA for an additional wash and dilute the cells to a one times 10 to the seventh cells per 60 microliters of magnetic activated cell sorting, or MACS, buffer concentration. Block any nonspecific binding with 20 microliters of Fc block for 10 minutes at four degrees Celsius followed by the addition of 20 microliters of anti-CD66b magnetic beads.

After 10 minutes at four degrees Celsius wash and re-suspend the cells in 500 microliters of MACS buffer and add the cells to an appropriately sized magnetic column within the magnetic field of a suitable magnetic separator. When all of the CD66b negative cells have run through the column add 15 millilitres of fresh MACS buffer to the column reservoir and transfer the column from the magnetic separator into a new conical tube. Then immediately plunge the column to flush the magnetically labeled CD66b positive into the tube.

After centrifugation re-suspend the isolated low density neutrophils at a five times 10 to the sixth cells per milliliter of RPMI1640 medium supplemented with FBS concentration and seed 1 milliliter of neutrophils per well on the six well poly-L-lysine coated plate. After two hours at 37 degrees Celsius and 5%carbon dioxide stain the cells in each well with an appropriate fluorescent membrane impermeable nuclear and chromosomal dye according to the manufacturer's instructions and immediately observe the morphology of the NETs by fluorescence microscopy. To assess tumor cell adhesion to the NETs re-suspend gastric tumor cells stained with the contrasting fluorescent dye at a one times 10 to the sixth per milliliter of RPMI1640 supplemented with 0.1%bovine serum albumin concentration and add one milliliter of tumor cells to each well of the low density neutrophil cultures.

After five minutes at 37 degrees Celsius remove the supernatant from each well and gently wash the cells two times with 2 milliliters of pre warmed RPMI1640 medium supplemented with BSA per wash. A five minute incubation is long enough for detecting NET specific binding. Add the warmed medium to the side of each well slowly gently swirl the plate and remove the supernatant with an aspirator.

The NETs and the attached tumor cells can then be observed by fluorescence microscopy. For a time lapse video analysis of the trapped tumor cells culture and stain the peritoneal low density neutrophils in a 35 millimeter dish, poly-L-lysine coated dish, as demonstrated followed by a brief wash in two milliliters of 0.1%BSA plus RPMI1640. Replace the medium with one times 10 to the sixth unstained gastric tumor cells suspended in two milliliters of RPMI supplemented with BSA for a five minute incubation at room temperature.

At the end of the incubation rinse the cells in two milliliters of fresh RPMI supplemented with BSA for five minutes at room temperature followed by gentle swirling to remove any unattached tumor cells. Then replace the supernatant with DMEM supplemented with FBS and antibiotics and mount the dish in a whole view cell observation system for time lapse analysis of tumor cell trapping by the NETs. After two hours of culture CD66b positive low density neutrophils derived from peritoneal lavage fluid exhibit string structures by green fluorescent nuclear and chromosomal staining while CD66b negative mononuclear cells do not.

When the low density neutrophil cultures are pre-treated with 100 units per milliliter of DNAse I, however, the characteristics structures are destroyed indicating that the structures are composed of extracellular DNA expelled from the neutrophils. When the low density neutrophils are cultured on uncoated plastic plates many NET clusters can be observed floating within the medium with few NETs attached to the bottom of the dish. After five minutes of incubation with a fluorescently labeled gastric tumor cell line many tumor cells attach to the neutrophils within the NETs a phenomenon that is not observed after pre-treatment with DNAse I.Similar adhesion patterns are also seen for other tumor cells.

OKUM-1 and NUGC-4. Interestingly while the NETs gradually disappear within several hours of extended tumor cell co-culture the tumor cells trapped by the NETs begin to proliferate vigorously after NET release. During this procedure it's important to remember that because the NETs stick to the plate weakly the washing and dish transfer processes should be performed in an especially cautious manner.

After its development this technique paves the way for researchers in various fields of biomedical science to explore the causal relationship of NETs with various diseases such as thrombosis, systemic inflammatory response syndrome, and autoimmune diseases.