Sedimentation is a somewhat unexpected phenomenon in which cancer cells encapsulated in a 3D hydrogel matrix migrate towards the bottom of the space. Despite the fundamental importance of 3D culture experiments in cancer research, the mechanisms underlying this phenomenon remain still elusive. Our study pursued a multidisciplinary approach combining 3D cell culture experiments, spatiotemporal modeling and genomic analysis to examine different plausible mechanisms that explain this phenomenon. The 3D cell culture experiments included mono-cultures of triple-negative breast cancer cells during the presence (different doses) and absence of the migrastatic drug Paclitaxel, co-cultures with cancer-associated fibroblasts, and mono-cultures with different adhesion properties of the surrounding environment. The collected data consisted of 3D imaging samples and bulk RNA-sequencing samples taken at various time points and treatment conditions. Our hypothesis on the reason behind migration was further tested using a hybrid discrete-continuum mathematical model which was validated using a recently developed computational framework [1]. Our results suggested that passive migration mechanisms such as gravity and compression of the hydrogel are insufficient to bring the cells at the bottom. Instead, migration inhibition experiments with Paclitaxel showed that cells actively migrate towards the bottom of the space in non-treatment conditions. Transcriptomic analysis suggested that the movement is regulated by TGF-β, MAPK, and Adherens Junctions pathways that are related to collective migration mechanisms [2]. Based on the experimental evidence, the multiscale model examined the hypothesis of chemotactic migration based on signal gradients originating from cells already attached at the bottom. The continuum part of the model was calibrated with the experimental data (control: average NRMSE < 40%, treatment: average NRMSE < 10%). The discrete part was used for the validation using spatial statistical analysis which revealed clustering patterns in both experiments and simulations that become more pronounced as the treatment dose increases. The chemotactic hypothesis was further tested with co-culture experiments with fibroblasts and adhesion experiments. The co-culture experiments showed that migration is inhibited in presence of fibroblasts owing to antagonistic signals that allow them to mix with cancer cells. The adhesion experiments revealed that migration is inhibited in presence of low adhesion environments. Overall, our results suggested that sedimentation is a result of active migration based chemotactic signals produced by cells attached to an adhesive surface [3].