Modulation of angiogenesis in HT-1080 onplants: inhibition of inflammatory cell influx by ibuprofen and its rescue by exogenous heterophils. (A) Collagen onplants containing 5 × 10⁴ HT-1080 cells were incubated on the CAM of nontreated embryos (left panels) or embryos treated systemically with ibuprofen (middle and right panels). In addition, a subset of ibuprofen-treated embryos was engrafted with HT-1080 onplants containing 5 × 10⁴ purified heterophils isolated from peripheral blood (right panels). At 72 hours, onplants were harvested and frozen in OCT compound or fixed in formalin. Cryosections were immunohistochemically stained with mAb 29-7 to visualize human cells (top panels) or chMMP-9–specific antibody to visualize heterophils (arrows, middle panels). Paraffin-embedded sections were immunohistochemically stained with chMMP-13–specific antibody to visualize monocytes/macrophages as brown rounded cells (bottom panels). (B) Angiogenic response and the heterophil and monocyte influxes were scored at 72 hours in control onplants (ctrl) and onplants containing 5 × 10⁴ HT-1080 cells (HT), which were placed on nontreated embryos or embryos systemically treated with ibuprofen (IB). Isolated heterophils (Het) or erythrocytes (Ery) were added at a concentration of 5 × 10⁴ cells per onplant to the 2 subsets of HT-1080 onplants grafted on the CAM of ibuprofen-treated embryos. Angiogenic response was determined as a fraction of onplant grids containing the newly formed blood vessels (top graph; bar indicates mean). Influx of heterophils was determined as a tissue density of chMMP-9–positive cells scored in × 20 tissue images (middle graph). Influx of monocytes was determined as a tissue density of chMMP-13–positive cells scored in × 20 tissue images (bottom graph). Data are presented as the mean ± SEM. Shown is a representative of 3 independent experiments. Statistical significance was confirmed (P < .05) for each variable in comparison with the previous experimental group as depicted in the scatter and bar graphs.