Figure 1.

DISC-3D Protocol. Spheroids are formed in the presence of fluorescently labeled polystyrene beads, which provide a visual cue for locating spheroids in later steps. Spheroids are placed in collagen I solutions that are allowed to gel. Following spheroid implantation and initial cell invasion, the samples are overlaid with warmed gelatin. The gelatin is allowed to diffuse through the sample for 24 h as the cells continue to invade, and is then gelled for 1 h at 4 °C prior to flash freezing. The sample then can be cryosectioned and imaged via a variety of techniques. Variations of the procedure optimized for distinct experiments are described in Methods and in Fig. S1. A representative spheroid subjected to the DISC-3D protocol and imaged via brightfield microscopy is shown in Fig. S2b,c at the two points in the protocol outlined in blue boxes.