Gelatin-based 3D biomimetic scaffolds platform potentiates culture of cancer stem cells in esophageal squamous cell carcinoma

Cancer stem cells (CSCs) are crucial for tumorigenesis, metastasis, and therapy resistance in esophageal squamous cell carcinoma (ESCC). To further elucidate the mechanism underlying characteristics of CSCs and develop CSCs-targeted therapy, an efficient culture system that could expand and maintain CSCs is needed. CSCs reside in a complex tumor microenvironment, and three-dimensional (3D) culture systems of biomimetic scaffolds are expected to better support the growth of CSCs by recapitulating the biophysical properties of the extracellular matrix (ECM). Here, we established gelatin-based 3D biomimetic scaffolds mimicking the stiffness and collagen content of ESCC, which could enrich ESCC CSCs efficiently. Biological changes of ESCC cells laden in scaffolds with three different viscoelasticity emulating physiological stiffness of esophageal tissues were thoroughly investigated in varied aspects such as cell morphology, viability, cell phenotype markers, and transcriptomic profiling. The results demonstrated the priming effects of viscoelasticity on the stemness of ESCC. The highly viscous scaffolds (G': 6-403 Pa; G": 2-75 Pa) better supported the enrichment of ESCC CSCs, and the TGF-beta signaling pathway might be involved in regulating the stemness of ESCC cells. Compared to two-dimensional (2D) cultures, highly viscous scaffolds significantly promoted the clonal expansion of ESCC cells in vitro and tumor formation ability in vivo. Our findings highlight the crucial role of biomaterials' viscoelasticity for the 3D culture of ESCC CSCs in vitro, and this newly-established culture system represents a valuable platform to support their growth, which could facilitate the CSCs-targeted therapy in the future.