Mathieson Layla, Jones Phoebe, Koppensteiner Lilian, Neilson Liam, Dorward David A, O'Connor Richard, Akram Ahsan R
Centre for Inflammation Research, Institute of Regeneration and Repair, University of Edinburgh, Edinburgh, UK.
Department of Pathology, Royal Infirmary of Edinburgh, Edinburgh, UK.
BJC Rep. 2025 Jul 10;3(1):50. doi: 10.1038/s44276-025-00159-w.
Cancer-associated fibroblasts (CAFs) are the predominant cell type in the stroma of many solid organ malignancies, including non-small cell lung cancer (NSCLC). They exhibit considerable phenotypic and functional heterogeneity and are widely used in functional assays and co-culture models. CAF research frequently involves the in vitro expansion and maintenance of CAFs to facilitate functional assays and co-culture studies. However, less is known about how in vitro culture temporally affects CAF phenotype.
We characterised the phenotype of CAFs from NSCLC patients compared to non-cancerous lung fibroblasts using conventional in vitro conditions by tracking changes in CAF subset marker expression levels by flow cytometry. Additional transcriptomic and functional analyses were performed to determine differences between CAFs and non-cancerous fibroblasts.
We demonstrate that CAFs from NSCLC undergo phenotypic drift in culture, and that there is a convergence to a subset phenotype predominantly upregulated in non-cancerous lung. Additionally, we demonstrate the phenotype, transcriptome and function of fibroblasts converge between CAFs and fibroblasts from non-cancerous lung by the third culture passage, suggesting that in vitro conditions promote this phenotype.
We highlight the need to understand and monitor the culture phenotype during functional studies with CAFs, as the heterogeneity found in the tumour microenvironment is rapidly lost in cultured cells.
癌症相关成纤维细胞（CAFs）是包括非小细胞肺癌（NSCLC）在内的许多实体器官恶性肿瘤间质中的主要细胞类型。它们表现出相当大的表型和功能异质性，并广泛应用于功能测定和共培养模型。CAF研究经常涉及CAFs的体外扩增和维持，以促进功能测定和共培养研究。然而，关于体外培养如何随时间影响CAF表型的了解较少。
我们通过流式细胞术追踪CAF亚群标志物表达水平的变化，使用传统体外条件，将NSCLC患者的CAFs与非癌性肺成纤维细胞的表型进行了表征。进行了额外的转录组和功能分析，以确定CAFs与非癌性成纤维细胞之间的差异。
我们证明，NSCLC来源的CAFs在培养过程中发生表型漂移，并且会趋向于一种在非癌性肺中主要上调的亚群表型。此外，我们证明，到第三次传代时，CAFs与非癌性肺成纤维细胞的成纤维细胞表型、转录组和功能会趋同，这表明体外条件促进了这种表型。
我们强调在对CAFs进行功能研究期间，需要了解并监测培养表型，因为肿瘤微环境中发现的异质性在培养细胞中会迅速丧失。
