3D spheroid culture to examine adaptive therapy response in invading tumor cells

3D in vitro culture models of cancer cells in extracellular matrix (ECM) have been developed to investigate drug targeting and resistance or, alternatively, mechanisms of invasion, however models allowing analysis of shared pathways mediating invasion and therapy resistance are lacking. To evaluate therapy response associated with cancer cell invasion, we here used 3D invasion culture of tumor spheroids in 3D fibrillar collagen and applied Ethanol-Ethyl cinnamate (EtOH-ECi) based optical clearing to detect both spheroid core and invasion zone by subcellular-resolved 3D microscopy. When subjected to a single dose of irradiation (4 Gy), we detected preferential cell survival in the invasion zone, compared to the spheroid core. By physical separation of core and invasion zone we identified differentially regulated genes between preferentially engaged in invading cells controlling cell division, repair and survival. This imaging-based 3D invasion culture may be useful for analysis of complex therapy-response patterns in cancer cells in drug discovery and invasion-associated resistance development. 50,000 MV3 H2B-GFP spheroids were embedded in collagen I for 3D invasion. Two days after embedding cells from core and invasion zone were enriched by punch biopsy and RNA was isolated for RNAseq.