Abstract LT03: Cell confinement by micropatterning induces phenotypic changes in cancer-associated fibroblasts

Recent advances in single-cell studies have unveiled the extensive transcriptomic heterogeneity of cancer-associated fibroblasts (CAFs), with each subset potentially playing unique roles in the tumor microenvironment. Despite these insights, it remains unclear how to culture different subsets of CAFs in vitro while preserving their in vivo phenotype. The inherent plasticity of CAFs, characterized by their ability to dynamically alter their phenotype in response to various environmental stimuli, presents significant challenges in inducing and maintaining specific CAF states in vitro. In this study, we investigated the effects of cell shape and confinement on two-dimensional culture substrates on the transcriptomic profile and phenotype of cultured CAFs. By inducing cytoskeletal rearrangement and activating mechanotransductory pathways through micropatterning of polyacrylamide hydrogels, we observed significant shape- and confinement-dependent changes in cell morphology. Our results showed that micropatterned CAFs exhibited phenotypic shifts towards more myofibroblastic, desmoplastic and inflammatory subsets. Additionally, micropatterning allowed for control over a range of CAF-specific markers and pathways, significantly altering their transcriptome landscape and behavior. Lastly, we demonstrated that micropatterned and non-micropatterned CAFs responded differently to anti-cancer drugs, underscoring the importance of phenotype-oriented therapy that accounts for CAF plasticity and regulatory networks. Control over CAF morphology through micropatterning presents a unique opportunity to establish highly robust CAF phenotypes in vitro, thereby facilitating a deeper understanding of CAF plasticity and heterogeneity. This approach also holds promise for the development of novel therapeutic targets, as it allows for the precise manipulation of CAF states to better model and study their interactions within the tumor microenvironment. Citation Format: Aleksandr Mitriashkin, Josephine Yu Yan Yap , N. Gopalakrishna Iyer , Gianluca Grenci, Eliza Li Shan Fong. Cell confinement by micropatterning induces phenotypic changes in cancer-associated fibroblasts [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr LT03.