Tumor-promoting Neutrophils Could Efficiently Suppress Tumor Growth After Cytokine Priming And In Presence Of Normal NK Cells

Objective:In tumor-bearing status, the function of neutrophils was converted from tumor-suppressing to tumor-promoting by G-CSF/IL-6. But neutrophils play an important role in the anti-tumor immunity. This study will to explore the method to recover the tumor-supprssing function of neutrophils and the mechanism underlying this functional conversion.Methods:(1) Non-inflammatory and inflammatory peritoneal neutrophils were recruited and purified. Neutrophils were inoculated with tumor cells, and tumors were weighted to evaluate the anti-tumor effect of neutrophils. IFN-y and TNF-a in non-inflammatory and inflammatory peritoneal washing liquid were detected by ELISA. Neutrophils were stimulated with IFN-y and TNF-a, and the antitumor effect was evaluated by tumor weight after inoculated with tumor cells to mice.(2) Neutrophils were stimulated with IFN-y and TNF-a and the cytotoxicity was detected by Flow cytometry. MPO release rate of IFN-y and TNF-a stimulated neutrophils was detected. Activation of PI3K and p38MAPK and TRAIL was detected by western blotting. Real-time PCR to detect the expression of Trail and Rab27a of neutrophils after stimulated with IFN-y, TNF-a and T-sMs.(3) Plasmid pCXCL1was injected locally to recruit neutrophils and pIFN-y/pTNF-a was injected in situ for the treatment of tumor, tumors were weighted to evaluated tumor inhibitory effect. Neutrophils were depleted in vivo to confirm the effect of neutrophils in the in vivo experiments.(4) Immunohistochemistry assays to detect the infiltration of NK cells after inoculation of tumor cells/neutrophils. NK cells were depleted by injection of monoclonal antibodies in vivo to confirm the effect of NK cells on primed neutrophils.(5) Real-time PCR to detect the expression of Ccl3of neutrophils after stimulated with IFN-y and TNF-a. Western blotting to detect the CCL3in tissue. Flow cytometry assays to detect the NK ratio in peripheral blood and spleen of mice.(6) Real-time PCR, ELISA to detect the expression of1l-18, Rael, Mult1, H60of neutrophils after stimulated with IFN-y and TNF-a. Flow cytometry assays to detect the cytotoxicity acvitity of neutrophils and NK cells; NK activating ligand RAE-1, MULT-1, H60on neutrophil; intracellular IFN-yof NK cells and the apoptosis rate of neutrophils.(7) Co-inoculation of inflammatory neutrophils/NK cells with tumor cells to mice or mice inoculated with tumor cells were treated with pCXCL1/pIFN-y/pTNF-awith NK cells injection in tumor situ, tumor-suppressing effect was evaluated by tumor weight and survival rate of mice.Results:(1) Neutrophils isolated from inflammaroty intraperitoneal of naive mice, tumor-bearing and pG/pI6-mice can all inhibit tumor growth effectively. IFN-y and TNF-ain inflammaroty intraperitoneal was much higher than that in non-inflammaroty intraperitoneal. IFN-y and TNF-a can convert tumor-promoting neutrophils from tumor-bearing and pG/pI6-mice to tumor-suppressing neutrophils.(2) After stimulated with IFN-y and TNF-a, neutrophils’cytotoixcity was enhanced. When T-sMs was used as the second stimulation, Rab27a and TRAIL expression, activation of p38MAPK and PI3K and degranulation of neutrophils were increased.(3) In naive mice, recruiting neutrophils to tumor and increasing the expression of IFN-y and TNF-a in tumor situ can exert strong tumor inhibitory effect mediated by neutrophils. But in pG/pI6-mice, the effect of this strategy was weaker.(4) Primed neutrophils can recruit more NK cells in tumor situ in naive mice than that in tumor-bearing mice and pG/pI6-mice, and these NK cells can assist neutrophils to kill tumor cells. In tumor-bearing and pG/pI6-mice, NK cells lost the ability to assist neutrophils.(5) Primed neutrophils possess the normal ability to recruit NK cells, but chemotaxis capability of NK cells of tumor-bearing and pG/pI6-mice was impaired.(6) Primed neutrophils can express similar level of IL-18and NK-activating ligand RAE-1, MULT-1and H60. These neutrophils can activate naive NK cells, but not NK cells from tumor-bearing and pG/pI6-mice. Cytotoxicity and reactivity of NK cells from tumor-bearing and pG/pI6-mice to IL-2were decreased. And naive NK cells but not tumor-bearing and pG/pI6NK cells can prolong the survival period of primed neutrophils.(7) In pG/pI6-mice, primed neutrophils combined with normal functional NK cells can effectively inhibit tumor and prolong the survival period of tumor mice.Conclusions:Neutrophils can be converted from tumor-promotional to tumor-suppressional by IFN-y and TNF-a, the main mechanism underlying this convertion was IFN-y and TNF-a can priming tumor-promoting neutrophils, primed neutronphils not only exhibit enhanced cytotoxicity, anti-tumor gene expression, degranulation ability but also can recruit and activate normal NK cells effectively. Impaired NK function in tumor-bearing and high serum G-CSF/IL-6status weaken the tumor inhibition effect of primed neutrophils. Combining normal functional NK cells with primed neutrophils can inhibit tumor effectively.