A Guide to Fibroblast Markers

Fibroblasts are a major cell type in the body, serving as the predominant cells in connective tissues. These cells play a central role in the maintenance of the extracellular matrix (ECM) and are involved in both normal physiological processes and pathological conditions, including wound healing, fibrosis, inflammation, autoimmune disorders, and cancer. Cancer-associated fibroblasts, an important component found in the tumor microenvironment, contribute to tumor progression and metastasis. In laboratory culture, they are particularly useful tools due to their accessibility and ability to be reprogrammed into pluripotent stem cells. These features highlight the necessity for distinct biomarkers that can accurately characterize fibroblasts, which can benefit cell sorting and functional studies aimed at elucidating their diverse roles and heterogeneous populations.

Functional roles of fibroblasts

The main functions of fibroblasts are centered on the ECM, including the synthesis and regulation of components such as collagens, elastins, laminins, nidogens, fibronectin, and tenascin. They contribute to ECM turnover and water retention, ensuring structural integrity. In the dermis, fibroblasts maintain tissue architecture and are activated during injury to support wound healing. They also exhibit immunoregulatory functions through an inflammatory secretome of chemokines, interleukins, prostaglandins, and TGFβ, influencing immune cell activity and angiogenesis. Additionally, fibroblasts express toll-like receptors and produce antimicrobial peptides like defensins.

Fibroblast heterogeneity

Fibroblast subpopulations play diverse roles across development, tissue maintenance, and disease. This phenotypic heterogeneity is apparent not only between different tissues but also within the same tissue. Known as intra-organ heterogeneity, multiple fibroblast populations coexist within a single organ. Meanwhile, inter-organ heterogeneity of fibroblasts in different anatomical locations and at various developmental stages can be observed by unique ECM compositions. This differential expression of ECM-related genes, or the matrisome, is a key aspect of fibroblast transcriptomic diversity, suggesting that the functional heterogeneity of fibroblasts is contextually specific to their tissue environment.

Primary fibroblasts originate during embryogenesis and give rise to resident quiescent fibroblasts (RQF), which maintain ECM homeostasis and support tissue-specific functions. Dermal fibroblasts are specialized fibroblasts of various types of skin tissue. Fibrosis-associated fibroblasts are linked to fibrogenesis and chronic scarring, often due to deregulated gene expression. Wound healing-associated fibroblasts mediate tissue repair by proliferating and coordinating wound responses. Cancer-associated fibroblasts are found in solid tumors, exhibiting both tumor-promoting and suppressing behaviors. Aging-associated fibroblasts emerge from aging tissues and environmental stress, with contributions from non-residential fibroblasts.

Fibroblast heterogeneity is increasingly understood through single-cell transcriptomic efforts, providing insights into the molecular profiles of fibroblasts beyond traditional anatomical and morphological characteristics. Based on scRNA-seq reports, it appears that no single marker can universally distinguish all fibroblasts from other cell types across different organs, but rather, a combination of markers is necessary for accurate identification.

Cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) play a crucial role in tumor progression by influencing the tumor microenvironment and are often correlated with poorer prognosis and lower survival rates in cancer patients. These fibroblast phenotypes are a key component of the tumor stroma, producing ECM with diverse molecular compositions and secreting factors that promote tumor growth and metastasis. They also induce drug resistance, angiogenesis, and facilitate pathways such as TGF-β1 and PDGF/PDGFR signaling, which can drive ECM remodeling, immune evasion, and cell invasion. For instance, in lung cancer, CAFs secrete inflammatory mediators like interleukins, TNF, and VEGF, further supporting tumor growth and immune evasion. The characterization of CAF phenotypes continues to be an active area of research.

Considerations for using fibroblast markers

When studying fibroblasts in culture, researchers must remember the heterogeneity of fibroblasts in an in vivo context. While diverse subpopulations can exist within a given tissue and exhibit unique characteristics, functions, and marker profiles, these identifiers can be nuanced. 

“Fibroblasts share several characteristics with other cell types, such as myofibroblasts, pericytes, and smooth muscle cells. This similarity can complicate their identification, as they often lack unique, specific surface markers,” says Changsuk Moon, Ph.D., a Senior Scientist of Microphysiological Systems at ATCC. "Tissue-specific fibroblasts exhibit distinct characteristics and functions depending on their origin. The interactions between fibroblasts and other cell types can introduce variability that needs to be carefully controlled in experiments." 

Fibroblast behavior can also differ significantly when cultured outside of their native physiological environment. Culturing fibroblasts often homogenizes the population, with proliferative subtypes outcompeting and overshadowing more quiescent ones. This phenomenon can unintentionally enrich for fibroblasts with an activated phenotype. 

Culture-associated changes inevitably can be reflected in protein expression. “Dynamic phenotypic changes in fibroblasts can significantly influence marker expression, especially when cultured under varying conditions,” reminds Moon. 

Moon also shares useful insights on several fibroblast markers: “Vimentin, COL1A1, and α-SMA are commonly used to identify and study fibroblasts.”

Certain markers also tend to be used with particular applications. “CD90, Fibroblast Surface Proteins TE-7 and 1B10, α-SMA, and Vimentin are specific fibroblast surface proteins that provide additional specificity in indicating populations used in immunohistochemistry and immunofluorescence studies,” says Moon.

Other applications include Western blotting for α-SMA, FAPα, and HSP47; flow cytometry for ITGB1, DLK1, FAPα, and PDGFRα; ELISA for collagen, elastin, fibronectin, and laminin; and functional mRNA expression analysis for COL1A1, FAPα, α-SMA, and CTGF. 

Table of fibroblast markers

The table below lists protein markers of fibroblasts mentioned by recent literature for characterizing distinct subpopulations. Marker information is accompanied by links to relevant antibody and ELISA kit products, as these are commonly used in marker immunodetection. The associated products are offered by a variety of manufacturers and, when used in combination with other fibroblast markers, can serve as a useful reference for identifying or isolating fibroblast populations.

References

Lynch MD, Watt FM. Fibroblast heterogeneity: implications for human disease. J Clin Invest. 2018;128(1):26-35. doi:10.1172/JCI93555

LeBleu VS, Neilson EG. Origin and functional heterogeneity of fibroblasts. FASEB J. 2020;34(3):3519-3536. doi:10.1096/fj.201903188R

Lendahl U, Muhl L, Betsholtz C. Identification, discrimination and heterogeneity of fibroblasts. Nat Commun. 2022;13(1):3409. Published 2022 Jun 14. doi:10.1038/s41467-022-30633-9

Łuszczyński K, Soszyńska M, Komorowski M, et al. Markers of Dermal Fibroblast Subpopulations for Viable Cell Isolation via Cell Sorting: A Comprehensive Review. Cells. 2024;13(14):1206. Published 2024 Jul 17. doi:10.3390/cells13141206

Han C, Liu T, Yin R. Biomarkers for cancer-associated fibroblasts. Biomark Res. 2020;8(1):64. Published 2020 Nov 11. doi:10.1186/s40364-020-00245-w

Yamamoto Y, Kasashima H, Fukui Y, Tsujio G, Yashiro M, Maeda K. The heterogeneity of cancer-associated fibroblast subpopulations: Their origins, biomarkers, and roles in the tumor microenvironment. Cancer Sci. 2023;114(1):16-24. doi:10.1111/cas.15609

Tokhanbigli S, Haghi M, Dua K, Oliver BGG. Cancer-associated fibroblast cell surface markers as potential biomarkers or therapeutic targets in lung cancer. Cancer Drug Resist. 2024;7:32. Published 2024 Sep 10. doi:10.20517/cdr.2024.55

Oscar Franzén, Li-Ming Gan, Johan L M Björkegren.  PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data. Database, Volume 2019, 2019, baz046, https://doi.org/10.1093/database/baz046