Gene Expression Profiling of MCF7 Cells Grown in 3D Liquid Versus 3D Matrigel

: Insights into Stemness Regulation

Abstract

Background: Metastases are the primary cause of cancer-related mortality. Although metastatic tumour cells encounter various mechanical and environmental stresses such as a switch between tissue-embedded and fluid-suspended conditions, they have evolved strategies to survive. The cellular mechanisms enabling these cells to endure such hostile conditions remain poorly understood. This study hypothesises that suspension condition promotes stem-like features in cancer cells, enhancing their survival capacity. Methods: Breast cancer MCF7 cells were cultured in 3D liquid suspension or within Matrigel to simulate metastasising and solid tumour microenvironments, respectively. Cell polarity was assessed via immunofluorescence. Transcriptomic profiles of spheroids under different culture conditions were obtained using RNA sequencing. Stemness-related gene expression was evaluated by RT-qPCR. Genes related to the Hippo pathway and ER stress were differentially expressed among others between liquid and Matrigel cultures. siRNA-mediated knockdown, plasmid-based overexpression, and chemical modulation experiments were performed to investigate the roles of YAP/TAZ and ER stress in regulating stemness. Results: MCF7 cells in liquid culture formed apical-out spheroids, exhibiting elevated expression of stemness-associated genes, higher endogenous LATS1/2 kinase activity and lower ER stress, compared with Matrigel-cultured cells. siRNA mediated knockdown or ectopic overexpression of TAZ significantly reduced and enhanced 2D colony formation, respectively, whereas activation of YAP/TAZ via LATS inhibition was insufficient to induce stemness features. Pharmacological induction of ER stress markedly increased TAZ expression specifically in Matrigel, while reducing SOX2 expression in liquid culture. Notably, embedding liquid-cultured spheroids into Matrigel progressively reduced SOX2
expression and increased ER stress. Conclusion: These findings demonstrate that the tumour cell transcriptome and phenotype are strongly influenced by the surrounding microenvironment. Liquid culture promotes a stem cell-like, stress-buffering state, potentially enhancing tumour cell survival under hostile conditions encountered during metastasis.

Date of Award	20 Jan 2026
Original language	English
Awarding Institution	University of Bristol
Sponsors	China Scholarship Council
Supervisor	Nobue Itasaki (Supervisor) & Ann C Williams (Supervisor)