Angiogenesis and heterophil influx induced by human tumor cells in collagen CAM onplants. (A) Schematic presentation of tissue and cellular components of a collagen onplant containing HT-1080 cells 3 days after grafting on the CAM. Tumor cells could be visualized in the upper portions of the onplant, around and between 2 layers of grids from the nylon meshes. The grids are often displaced during tissue processing, leaving empty circles in the tissue sections. Large, preexisting blood vessels containing nucleated erythrocytes and leukocytes are located in the underlying CAM, which is bordered by the endoderm layer. Newly formed blood vessels, which are the ones scored in the angiogenic assay, are identified mostly between or directly below the grids and usually are close to tumor cells. Angiogenic vessels are often filled with blood cells, indicating the existence of an established, complete circulation. Stromal fibroblast-like cells of the CAM mesoderm are numerous and infiltrate the entire collagen onplant, including the very top portions of the onplant. Lower and middle portions of the onplant are infiltrated with inflammatory cells such as monocytes/macrophages and heterophils. These cells could be identified morphologically by immunohistochemical staining with chMMP-13– and chMMP-9–specific antibodies, respectively. The matrix components could be identified as a fine network of fibrils present throughout the entire onplant and a thick ribbonlike structure at the collagen/air interface on the top of onplant. (B) Collagen onplants supplemented with buffer alone (control) or 5 × 10⁴ HT-1080 cells (HT-1080) were placed on the CAM and scored 72 hours later for levels of angiogenesis. Recombinant chicken TIMP-2 was incorporated into onplants at 2.85 μM. Hydroxamate MMP inhibitor GM6001 was added either topically (top; 5 μLof 25 μM solution) or systemically (syst; 10 μL of 1.25 mM solution) at the time of onplant grafting and 48 hours later. At 72 hours, the angiogenic response was determined as a fraction of grids containing newly formed blood vessels. Statistical significance between the groups of onplants was determined by comparison with nontreated HT-1080 cell–containing onplants. ^**P < .001; ^*P = .004. (C) At 72 hours (day 3), HT-1080 cell–containing onplants were harvested, embedded in OCT compound, and frozen. HT-1080 cells were identified in cryosections (left panel) after immunohistochemical staining with mAb 29-7 recognizing a human cell surface antigen (original magnification × 20). Human tumor cells (brown) are located at the top and in between the grids of onplants, whereas underlying CAM appears mainly devoid of tumor cells. Displaced nylon mesh grids appear as empty circular structures. Some newly formed blood vessels are indicated by arrows. By day 6 of incubation, highly vascularized HT-1080 tumors are generated on the top of the onplant grids (right panel). (D) Kinetics of heterophil influx into collagen CAM onplants supplemented with HT-1080 cells. The numbers of chMMP-9–positive heterophils were determined over a 72-hour time course in the × 20 images of tissue cryosections stained with the chMMP-9 antibody as described in Figure 3. For comparison, samples of 72-hour control onplants (control) and normal CAM (NCAM) were included in quantitation. Data are presented as the mean ± SEM of heterophil numbers per square scored in 7 to 14 images per time point. ^**P < .001.