Inhibition of heterophil influx and angiogenesis in HT-1080 onplants by cortisone. Onplants containing HT-1080 cells (5 × 104 cells per onplant) were placed on the top of the CAM of 10-day-old chick embryos, nontreated or injected with cortisone (0.5 mg/embryo). (A) At 72 hours, nontreated (left panels) and cortisone-treated (right panels) HT-1080 onplants with the underlying CAM were harvested, embedded in OCT compound, and frozen. Cryosections were immunohistochemically stained with mAb 29-7 (top panels) or anti–chMMP-9 antibody (bottom panels). As judged by the 29-7 staining (original magnification × 4), expanding HT-1080 cells (brown) are localized at the top of the onplants and in between grids. Boxed areas are presented at higher magnification (original magnification × 40) of the adjacent sections stained with the chMMP-9–specific antibody. The chMMP-9–positive staining is associated with single heterophils and heterophil clusters (arrows). (B) Cortisone reduces the heterophil influx (top graph) and angiogenic response (bottom graph) in the collagen onplants containing HT-1080 cells. Cryosections of nontreated and cortisone-treated collagen HT-1080 onplants harvested at 72 hours were stained with the chMMP-9 antibody. The chMMP-9–positive heterophils were scored in × 20 images. Data are presented as the mean ± SEM of chMMP-9–positive heterophil numbers per square. Levels of angiogenesis were scored at 66 hours and presented as percentage of grids with newly formed blood vessels. *P < .001. (C) Cortisone diminishes chMMP-9 protein levels in collagen CAM onplants containing HT-1080 cells. Nontreated (HT-1080) and cortisone-treated (HT-1080+cortisone) onplants were harvested at 72 hours and snap-frozen on dry ice. Extracted proteins were separated by SDS-PAGE on gelatin-containing 8% polyacrylamide gels for zymography analysis (5 μg/lane; top panel) or separated by 8% SDS-PAGE, transferred to a membrane support, and immunoblotted with anti–chMMP-9 antibody (25 μg/lane; bottom panel).
