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

Recent advances in single-cell studies have revealed the vast transcriptomic heterogeneity of cancer-associated fibroblasts (CAFs), with each subset likely having unique roles in the tumor microenvironment. However, it is still unclear how different CAF subsets should be cultured in vitro to recapitulate their in vivo phenotype. The inherent plasticity of CAFs, or their ability to dynamically change their phenotype in response to different environmental stimuli, makes it highly challenging to induce and maintain a specific CAF state in vitro. In this study, we investigated how cell shape and confinement on two-dimensional culture substrates with different stiffnesses influence CAF transcriptomic profile and phenotype. Using micropatterning of polyacrylamide hydrogels to induce shape- and confinement-dependent changes in cell morphology, we observed that micropatterned CAFs exhibited phenotypic shifts towards more desmoplastic and inflammatory CAF subsets. Additionally, micropatterning enabled control over a range of CAF-specific markers and pathways. Lastly, we report how micropatterned and non-micropatterned CAFs respond differently to anti-cancer drugs, highlighting the importance of phenotype-oriented therapy that considers for CAF plasticity and regulatory networks. Control over CAF morphology offers a unique opportunity to establish highly robust CAF phenotypes in vitro, facilitating deeper understanding of CAF plasticity, heterogeneity, and development of novel therapeutic targets. STATEMENT OF SIGNIFICANCE: Cancer-associated fibroblasts (CAFs) are the dominant stromal cell type in many cancers, and recent studies have revealed that they are highly heterogeneous and comprise several subpopulations. It is still unclear how different subsets of CAFs should be cultured in vitro to recapitulate their in vivo phenotype. In this study, we investigated how cell shape and confinement affect CAF transcriptomic profile and phenotype. We report that micropatterned CAFs resemble desmoplastic and inflammatory CAF subsets observed in vivo and respond differently to anti-cancer drugs as compared to non-patterned CAFs. Control over CAF morphology enables the generation of highly robust CAF phenotypes in vitro, facilitating deeper understanding of CAF plasticity and heterogeneity.