Fig. 6

Recombinant cleaved JAM-A treatment promotes invasion of drug-sensitive SK-BR-3 cells in vitro and a semi-in vivo model. SK-BR-3–sensitive (SK-BR-3-Sens) cells (100,000) were plated per well in six-well plates for invasion assays; 24 h later, cells were treated in serum-free media with vehicle control (phosphate-buffered saline, or PBS) or specified concentrations of recombinant cleaved JAM-A (rcJAM-A; ab151859, Abcam); 72 h later, 200,000 cells from each condition were inserted into the top chamber of a Matrigel invasion assay in serum-free media with PBS or rcJAM-A treatment; 5% fetal bovine serum in the lower chamber was used as a chemoattractant; 16 h later, non-invaded cells were scrubbed off, and invaded cells were fixed and stained with 0.5% crystal violet. An image was taken in each quadrant of the membrane at 20× magnification, and invaded cells were counted by an independent party. (a) Quantification of drug-sensitive SK-BR-3 cellular invasion in response to recombinant cleaved JAM-A treatment. *P <0.05 by one-way analysis of variance with Tukey’s multiple comparison test, n = 4 independent experiments. (b–d) SK-BR-3-Sens cells (2 × 10⁶) were seeded on the chorioallantoic membrane (CAM) of chick embryos in a silica ring. The following day, tumor xenografts were treated with 40 ng rcJAM-A or PBS daily for 4 days. The tumor and surrounding CAM were then excised, fixed in 4% paraformaldehyde overnight, and paraffin-embedded. (B) In the majority (71%) of rcJAM-A treated xenografts, invasion of neoplastic cells deep into the intermediate mesodermal layer of the CAM was observed, whereas this pattern was seen in only 43% of PBS-treated tumors. Photomicrographs show representative examples of superficial (neoplastic cells only infiltrating the chorionic epithelium) and deep (neoplastic cells present within the intermediate mesodermal layer) tumor invasion. Hemotoxylin and eosin (H&E) and pan-cytokeratin, 200× magnification. (C, D) Tumor xenografts were stained for the proliferation marker Ki67. The proliferation index (percentage Ki67-positive cells per 500 tumor cells) was significantly higher in rcJAM-A–treated tumor xenografts than in controls. Representative microscopic images are shown of low and high Ki67 expresssion in controls and treated tumors. Ki67 (Mib 1) 200× magnification. *P <0.05 by two-tailed equal variance unpaired t test