In vitro model of tumor-associated macrophages

The tumor microenvironment is highly complex and consists af many non-tumor cells and factors. To simplify the complexity, we developed a simple in vitro system where tumor (neuroblastoma) 'spheres' ~1-2 mm in diameter were generated on concave surfaces of noble agar. After sphere development, bone marrow-derived macrophages were added to the well. Many macrophages crawled into the spheres (as shown by live cell microscopy). After 24-28 h, the macrophages were isolated from inside or outside the tumor spheres using tissue digestion and anti-CD11b MACs beads. The macrophages from inside or outside the spheres were then subject to microarray analysis. We used microarrays to measure gene expression profiles in macrophages isolated from tumor spheres, or the macrophages from the same cultures that did not enter the spheres. Two timepoints were chosen, in three replicates. Each replicate is an independent experiment where macrophages were pooled from multiple wells to obtain sufficient material for array analysis.