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. 2013 Nov;62(11):1745-56.
doi: 10.1007/s00262-013-1476-9. Epub 2013 Oct 4.

Tumor-associated neutrophils (TAN) develop pro-tumorigenic properties during tumor progression

Inbal Mishalian  1 , Rachel Bayuh, Liran Levy, Lida Zolotarov, Janna Michaeli, Zvi Gregorio Fridlender
Affiliations

"VSports在线直播" Tumor-associated neutrophils (TAN) develop pro-tumorigenic properties during tumor progression

Inbal Mishalian et al. Cancer Immunol Immunother. 2013 Nov.

Abstract

The role and characteristics of tumor-associated neutrophils (TAN) in cancer are poorly defined. We have recently shown that TAN can have anti-tumorigenic (N1) or pro-tumorigenic (N2) functions. An interesting unanswered question is how the phenotype of TAN is influenced by the ongoing evolvement of tumor microenvironment. We therefore studied the phenotype and effects of TAN at different time points during tumor progression. We used two models of murine tumor cancer cell lines-Lewis lung carcinoma (LLC) and AB12 (mesothelioma). Neutrophils were studied at early and late stages and compared to each other and to neutrophils from bone marrow/periphery of naïve mice. Although there was no difference in the number of neutrophils entering the tumor, we found that at early stages of tumor development, neutrophils were almost exclusively at the periphery of the tumor VSports手机版. Only at later stages, neutrophils were also found scattered among the tumor cells. We further found that TAN from early tumors are more cytotoxic toward tumor cells and produce higher levels of TNF-α, NO and H2O2. In established tumors, these functions are down-regulated and TAN acquire a more pro-tumorigenic phenotype. In line with this phenotype, only depletion of neutrophils at later stages of tumor development inhibited tumor growth, possibly due to their central location in the tumor. Our work adds another important layer to the understanding of neutrophils in cancer by further characterizing the changes in TAN during time. Additional research on the functional role of TAN and differences between subsets of TAN is currently underway. .

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Conflict of interest statement (VSports手机版)

There are no financial or other interests that might be construed as conflict of interest.

Figures

Fig. 1
Fig. 1
TAN influx during tumor development. The percentage of CD11b+Ly6G+ (TAN) cells out of all tumor cells after AB12 (a) or LLC (b) cell inoculation. Each bar represents the mean ± SD of 9 mice, combined from 2 separated experiments. Percentage of ICAM-1 in CD11b+LY6G+ cells as assessed using flow cytometry at early and late stages of AB12 (c) and LLC (d), compared to the expression in neutrophils from blood of syngeneic naïve mice (BN). Each bar represents the mean ± SD of 9 mice, combined from 2 separate experiments. e Shows representative FACS tracing of ICAM-1 in CD11b+LY6G+ cells. The number in the gate is the percentage out of CD11b+LY6G+ cells, *p < 0.05
Fig. 2
Fig. 2
TAN distribution within tumor. Representative photomicrographs of tumor sections labeled with anti-Ly6G mAb (immunoperoxidase method) at early (7 day—top) and late (14 day—bottom) stages after AB12 cells inoculation. The periphery of the tumor is shown in the left panel and the central part of the tumor in the central and right panel. This staining was done in 3 separate mice of each group with similar results
Fig. 3
Fig. 3
TAN are capable of killing tumor cells ex vivo. TAN were purified from pooled tumors (n = 5–6) at early (7 day—white) and late (14 day—black) stages after AB12 cells inoculation. Isolated TAN were cocultured with AB12-Luciferase cells at different ratios for 24 h, and the percentage of tumor cells killing was calculated. Each bar represents the mean ± SD at each ratio of 3 (early) and 6 (late) samples, combined from 2 separate experiments, * p < 0.05
Fig. 4
Fig. 4
Potential cytotoxic secretion of ROS and TNF-α expression in TAN increases with tumor progression. TAN were purified from pooled tumors (n = 5–6) at early (7 day—white) and late (14 day—black) stages after AB12 or LLC cells inoculation. Blood neutrophils (BN) were purified from blood of naïve BALB/c mice (n = 5). Detection of NO released from AB12 (a) and LLC (b) tumors, after culturing 2.5 × 105 cells in medium for 24 h. Each dot represents one sample. Samples were combined from 2 separate experiments. Mean ± SD is shown, *p < 0.01. Experiment was repeated two times for each time point. Detection of H2O2 released from TAN purified from AB12 (c) and LLC (d) tumors or from naïve Balb/C mice (BN). 2.5 × 105 cells were activated in medium with PMA, prior to the measurement. Each dot represents one sample. Samples were combined from 2 separate experiments (AB12, c) or taken from a single experiment (LLC, d). Mean ± SD is shown, *p < 0.01. Flow cytometry analysis of naïve blood and digested tumor at early (7 day) and late (14 day) stages after AB12 cell inoculation. The percentage of intracellular TNF-α staining in isolated CD11b+Ly6G+ cells is shown. Each bar represents the mean ± SD of 8 mice, combined from 2 separated experiments, *p < 0.01 (e). Representative FACS tracing of TNF-α in CD11b+LY6G+ cells is shown (f). The number in the bar is the percentage out of CD11b+LY6G+ cells. The black area represents proper isotype
Fig. 5
Fig. 5
Systemic depletion of neutrophils inhibits tumor growth only if done at late stages. Tumors were generated by s.c. inoculation of 2 × 106 AB12 cells into mice right flank. a Tumors were inoculated on day 0, and 300 μg of anti-Ly6G monoclonal antibody or control IgG was injected on days −1, 1, 4 and 7. Tumor sizes were measured twice a week using calipers. Each dot represents the mean ± SD of 5 mice, *p < 0.05. b Mice bearing relatively large tumors (~250 mm3) were injected intraperitoneally with 300 μg of anti-Ly6G monoclonal antibody or control IgG on days 12, 14 and 16. Tumor sizes were measured twice a week using calipers. Each dot represents the mean ± SD of 5 mice, *p < 0.05
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