The analysis of Radiation-induced, tumor-infiltrating neutrophils, which has the antitumor characteristics

It is now becoming clear that interactions between tumor cells and host tissue stoma play a key role in tumor progression. Understanding the composition of the stromal cells in the tumor microenvironment immediately after tumor irradiation might be an important first step in immunomodulation by radiation therapy. To explore this, we harvested tumor mass, draining lymph nodes (DLNs), spleen and peripheral blood mononuclear cells (PBMCs) at different times after a single 15 Gy dose of focused irradiation of RM-9 mouse prostate tumor grafts growing in the hind leg of syngeneic C57BL/6 mice. Subpopulations of lymphocytes and granulocytes (CD4+, CD8+, CD4+CD25+, CD11c+, CD11b+Gr-1+mid and CD11b+Gr-1+ highLy-6G+ cells) were analyzed in the harvested tissue by flow cytometry. An infiltration of CD11b+Gr-1+ highLy-6G+ neutrophils reached a peak within the tumor microenvironment at 24 h after tumor irradiation while no significant changes in the other subpopulations were noted. Increased neutrophil infiltration after irradiation was also observed when the mammary gland cancer cell line 4T1 was implanted in BALB/c mice. To investigate the effect of neutrophils on tumor growth, we compared the tumor size in mice treated with the neutrophil-depleting anti-Ly-6G monoclonal antibody (Mab) to that of mice treated with an isotype –matched control antibody. Interestingly, in RM-9, and 4T1 tumor models, the therapeutic effect of irradiation was significantly attenuated in the mice with depleted neutrophils. To evaluate whether the early infiltration of neutrophils had significant tumor-specific immune response, we used EG7 (OVA gene-transfected)-tumor-bearing mice for detecting OVA-specific CTL. Then, these tumors (with and without neutrophil depletion by anti-Ly-6G MAb) were irradiated focally with 15 Gy. 7 days after irradiation, we observed the frequency of OVA-tetramer+CD8+ cells (OVA-specific CD8+) in tumor-bearing mice. A decrease in OVA-tetramer+CD8+ cells was observed in the DLN of the neutrophil-depleted mice. This result is consistent with the tumor growth curve. Hence neutrophil infiltration might be a very early but important step in initiating a robust adaptive tumor specific response in the DLN. Now we are investing how this neutrophil works for inducing tumor-specific CTL and for showing antitumor effect.The 7th GI-CoRE Medical Science and Engineering Symposiu