Mitriashkin Aleksandr, Yap Josephine Yu Yan, Fernando Elekuttige Anton Kanishka, Iyer N Gopalakrishna, Grenci Gianluca, Fong Eliza Li Shan
Translational Tumor Engineering Laboratory, Department of Biomedical Engineering, National University of Singapore, Singapore 119276, Singapore; Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore.
Translational Tumor Engineering Laboratory, Department of Biomedical Engineering, National University of Singapore, Singapore 119276, Singapore.
Acta Biomater. 2025 Jan 15;192:61-76. doi: 10.1016/j.actbio.2024.12.007. Epub 2024 Dec 4.
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.
单细胞研究的最新进展揭示了癌症相关成纤维细胞（CAF）巨大的转录组异质性，每个亚群可能在肿瘤微环境中具有独特作用。然而，目前仍不清楚如何在体外培养不同的CAF亚群以重现其体内表型。CAF固有的可塑性，即它们响应不同环境刺激动态改变表型的能力，使得在体外诱导和维持特定的CAF状态极具挑战性。在本研究中，我们调查了细胞形状以及在不同硬度的二维培养底物上的受限情况如何影响CAF的转录组图谱和表型。利用聚丙烯酰胺水凝胶的微图案化来诱导细胞形态的形状和受限依赖性变化，我们观察到微图案化的CAF表现出向更具促结缔组织增生性和炎性的CAF亚群的表型转变。此外，微图案化能够控制一系列CAF特异性标志物和信号通路。最后，我们报告了微图案化和非微图案化的CAF对抗癌药物的反应如何不同，强调了考虑CAF可塑性和调控网络的面向表型治疗的重要性。对CAF形态的控制为在体外建立高度稳定的CAF表型提供了独特机会，有助于更深入地了解CAF的可塑性、异质性以及开发新的治疗靶点。重要性声明：癌症相关成纤维细胞（CAF）是许多癌症中主要的基质细胞类型，最近的研究表明它们高度异质且包含几个亚群。目前仍不清楚如何在体外培养不同的CAF亚群以重现其体内表型。在本研究中，我们调查了细胞形状和受限情况如何影响CAF的转录组图谱和表型。我们报告称，微图案化的CAF类似于体内观察到的促结缔组织增生性和炎性CAF亚群，并且与非图案化CAF相比，对抗癌药物的反应不同。对CAF形态的控制能够在体外产生高度稳定的CAF表型，有助于更深入地了解CAF的可塑性和异质性。
