GelMA ‐based 3D spheroids recapitulate transcriptomic and functional hallmarks of myeloid sarcoma

Myeloid sarcoma (MS) is a rare extramedullary manifestation of acute myeloid leukemia (AML). Conventional two‐dimensional cultures fail to replicate the constraints of the MS microenvironment. We demonstrated that 5% gelatin methacrylate (GelMA) hydrogels provide a tunable three‐dimensional matrix for in vitro modeling of MS. GelMA hydrogels exhibited tissue‐like stiffness and oxygen diffusion properties, supporting the formation of viable spheroids from myeloid leukemia cell lines. The 3D context induced G1 cell cycle arrest and apoptotic progression, reflecting dormancy at extramedullary sites. RNA sequencing revealed transcriptional reprogramming in GelMA‐embedded spheroids, with an enrichment in ECM remodeling and metabolic pathways. Furthermore, transcriptomic profiles from 3D‐cultured cells aligned with patient‐derived MS samples, validating the model's relevance to MS. Thus, we present a GelMA‐based system which provides a clinically relevant platform for investigating MS biology and screening potential therapeutic strategies.