Figure 1
Neutrophils infiltrate into colitis-associated cancer (CAC) lesions and contribute to tumor growth. (a) The colon tissues of mice with or without CAC were removed at days 35 and 100 respectively and fixed with formalin and then processed conventionally. Infiltrating neutrophils were identified by staining with anti-Gr-1 antibody (brown). Six pairs of mice were analyzed. Scale bar=200 μm. The cell number per field was counted in five randomly selected visual fields at original magnification × 400. Representative results of six mice are shown on the right panel. HPF, high-power field. (b) Mice about to undergo CAC induction were injected intraperitoneally with anti-Ly6G antibody or isotype control on days −3 and −2 before initiation of dextran sulfate sodium (DSS) drinking (day 0), and then twice a week (days 2 and 5 each week) throughout the entire experimental period. Colon tissues were isolated on day 100 and stained with hematoxylin and eosin (H&E). Eight pairs of mice were analyzed. Scale bar=200 μm. (c) Mice were treated as in b, and then the number of tumors in the colon was counted (left). Representative images of colon tissues are shown on the right. (d) Colon tissues of mice treated as in b were isolated and TUNEL (TdT-mediated dNTP nick end labeling) stained (brown) for apoptosis of intestinal epithelial cells (IECs). Five pairs of mice were examined. An example is shown on the left and the summarized results on the right. The arrows denote the apoptotic cells. Scale bar=200 μm. (e) IECs were isolated from the colons of mice treated as in b and proteins were extracted and Bcl-XL (B-cell lymphoma-extra large) levels were measured by western blotting. The data shown are representative of those obtained in two independent experiments. (f) IECs were isolated from the colons of mice treated as in b and proteins were extracted, and then phosphorylation of the nuclear factor (NF)-κB p65 subunit was detected by western blotting. The data shown are representative of those obtained in three independent experiments. (g) Neutrophils in the lesions of CAC-bearing mice or in the spleen of naive littermates were isolated by fluorescence-activated cell sorting (FACS) and then implanted into transplanted CT-26 tumors in nude mice. Control nude mice implanted with tumors were injected with phosphate-buffered saline (PBS). Tumor size was monitored every day. Each group consisted of 6–8 mice. Left, summarized results; right, representative images of transplanted tumor tissues. **P<0.01; ***P<0.001.
