Spheroids fixed in 4% paraformaldehyde (Sigma-Aldrich) and rinsed in Dulbecco’s PBS (DPBS) were stained with NucBlue ReadyProbes reagent (Life Technologies) to visualize the cell nuclei (Fig. 3, A3 and A4); actin cytoskeletal fibers were stained with ActinGreen 488 ReadyProbes reagent according to the manufacturer’s instructions. Imaging and 3D reconstruction of a heterogeneous pyramid structure (Fig. 4A) was performed using a Zeiss confocal microscope (LSM 880, Carl Zeiss AG, Oberkochen, Germany) using a 5× lens. The 3D reconstruction was obtained using Zen blue software (Carl Zeiss AG). A diamond structure (Fig. 4D) was first imaged through Zeiss Axiozoom (Carl Zeiss AG, Oberkochen, Germany) to capture the entire 3D structure and then further imaged on a confocal microscope (Olympus FV1000, Olympus America Inc., Center Valley, PA) to closely image the DAPI (4′,6-diamidino-2-phenylindole), F-actin, and CD31 staining.

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