Description of the ADC library screen over four concentrations in 3D vs. 2D co-cultures.

<p><b>A</b>, immunofluorescent images of fixed and stained 2D (left panels) and fixed, sectioned, and stained 3D (right panels) co-cultures stained for the endothelial cell marker CD31 (green), the fibroblast-rich protein vimentin (red), and all the cells’ nuclei (blue), <i>bars,</i> 200 microns. The top most panels are the BT-474+HF+HE co-cultures, seeded with 1500 BT-474+750 fibroblasts+750 endothelial cells per well. The bottom panels are the HF+HE co-cultures, seeded with 750 fibroblasts+750 endothelial cells. <b>B</b>, schematic of the four-concentration screening protocol in BT-474+HF+HE and HF+HE cells cultured in 3D or 2D. <b>C</b>, Z’ values generated from one or two 96-well plates set, incubated, and handled as described for the screen for the 2D BT-474+HF+HE cells (top left panel), 2D HF+HE cells (top right panel), 3D BT-474+HF+HE cells (bottom left panel), 3D HF+HE cells (bottom right panel).</p>