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Myofibroblasts Are Distinguished from Activated Skin Fibroblasts by The Myofibroblasts are distinguished from activated skin PNAS PLUS fibroblasts by the expression of AOC3 and other associated markers Lin-ting Hsiaa, Neil Ashleya, Djamila Ouareta, Lai Mun Wangb,c, Jennifer Wildinga, and Walter F. Bodmera,1 aCancer and Immunogenetics Laboratory, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; bDepartment of Cellular Pathology, University of Oxford, Oxford, United Kingdom; and cOxford Biomedical Research Centre, University of Oxford, Oxford, United Kingdom Contributed by Walter F. Bodmer, March 4, 2016 (sent for review January 20, 2016; reviewed by Calvin Kuo and Nicholas A. Wright) Pericryptal myofibroblasts in the colon and rectum play an important including cardiac and skeletal muscle. Ultrastructural studies role in regulating the normal colorectal stem cell niche and facilitating showed that the pericryptal cells detected by PR2D3 had many tumor progression. Myofibroblasts previously have been distinguished features of smooth muscle cells, providing further support that from normal fibroblasts mostly by the expression of α smooth muscle these cells were MFs. This result was confirmed by Sappino et al. actin (αSMA). We now have identified AOC3 (amine oxidase, copper (8) using an anti-αSMA mAb that also showed very clear staining containing 3), a surface monoamine oxidase, as a new marker of myo- of pericryptal cells as well as smooth muscle. Following the fibroblasts by showing that it is the target protein of the myofibro- demonstration by Desmoulière et al. (9) that connective tissue blast-reacting mAb PR2D3. The normal and tumor tissue distribution fibroblasts were stimulated to express αSMA by TGFβ, leading and the cell line reactivity of AOC3 match that expected for myofibro- to the acquisition of MF-like properties, it was assumed that MFs blasts. We have shown that the surface expression of AOC3 is sensitive could be defined as TGFβ-activated fibroblasts. Subsequently, to digestion by trypsin and collagenase and that anti-AOC3 antibodies MFs defined in this way were shown to be widely distributed in can be used for FACS sorting of myofibroblasts obtained by nonenzy- many different tissues, often surrounding glandular structures. matic procedures. Whole-genome microarray mRNA-expression pro- Such MFs are presumed to play important roles in mesenchy- files of myofibroblasts and skin fibroblasts revealed four additional mal–epithelial interactions, wound healing, fibrosis, and even in MEDICAL SCIENCES genes that are significantly differentially expressed in these two cell immune responses (10, 11). types: NKX2-3 and LRRC17 in myofibroblasts and SHOX2 and TBX5 in In this paper, we identify the protein target of PR2D3 to be skin fibroblasts. TGFβ substantially down-regulated AOC3 expression AOC3 (amine oxidase, copper containing 3), a member of the in myofibroblasts but in skin fibroblasts it dramatically increased the semicarbazide-sensitive amine oxidase/copper-containing amine expression of αSMA. A knockdown of NKX2-3 in myofibroblasts oxidase (SSAO) family. AOC3 is often called “VAP-1” (vascular caused a decrease of myofibroblast-related gene expression and in- adhesion protein-1) because of its role in lymphocyte–endothelial SHOX2 creased expression of the fibroblast-associated gene ,suggest- interactions. The identification of AOC3 as the target of PR2D3 ing that NKX2-3 is a key mediator for maintaining myofibroblast has enabled us to distinguish clearly between connective tissue- characteristics. Our results show that colorectal myofibroblasts, as de- derived fibroblasts activated by TGFβ and MFs isolated both from fined by the expression of AOC3, NKX2-3, and other markers, are a distinctly different cell type from TGFβ-activated fibroblasts. Significance myofibroblasts | α smooth muscle actin | tumor microenvironment | AOC3 | NKX2-3 Myofibroblasts surround the epithelial cells of the crypts that form the surface of the gut. They play an important role in controlling the normal epithelium and influence the develop- here has been long-standing interest in the pericryptal cells that ment of colorectal and other epithelial cancers. The definition Tform a sheath around the epithelial cells in the large intestine of myofibroblasts previously depended almost entirely on the and a realization of their likely importance in the functional control expression of smooth muscle actin. We identified the surface of the gut epithelium. These pericryptal cells were originally de- enzyme AOC3 (amine oxidase, copper containing 3) as a new scribed as fibroblasts, although it was realized that they may re- marker of myofibroblasts and as a result have discovered ad- semble the fibroblasts actively involved in shrinkage at healing skin ditional highly distinctive markers for myofibroblasts, including wounds rather than the usually observed fibroblasts in connective – “ the transcription factor NKX2-3. The discovery of these new tissue (1 3). Gabbiani et al. (4) showed that these modified fi- markers should greatly enhance the proper definition of broblasts” had many properties similar to smooth muscle and “ ” myofibroblasts and related cell types and thus should con- suggested that they be called myofibroblasts (MFs) (5). They tribute to the improved treatment of the many diseases, in- then showed that an autoimmune human serum, which reacted cluding cancer, that involve these cell types. with α smooth muscle actin (αSMA, which is the protein product of ACTA2 the gene), detected these modified fibroblasts, including Author contributions: L.-t.H. and W.F.B. designed research; L.-t.H., N.A., and L.M.W. per- those associated with “the periphery of epithelial cells of the in- formed research; L.-t.H., D.O., J.W., and W.F.B. analyzed data; and L.-t.H., J.W., and W.F.B. testine” (6). However, the autoimmune serum, probably because it wrote the paper. was not monospecific, also bound to “cultivated” fibroblasts and so Reviewers: C.K., Stanford University; and N.A.W., Barts and the London School of did not clearly distinguish the pericryptal cells as MFs. Medicine. The first unambiguous identification of the pericryptal cells as The authors declare no conflict of interest. MFs by Richman et al. (7) was based on the discovery of a mouse Data deposition: The sequence reported in this paper has been deposited in the Gene mAb, PR2D3, made against fresh samples of normal large in- Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE77474). testine. This antibody clearly bound to the pericryptal cells as 1To whom correspondence should be addressed. Email: walter.bodmer@hertford. well as to smooth muscle, but it did not bind connective tissue ox.ac.uk. fibroblasts. PR2D3 also bound to a wide range of presumptive This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MFs in other tissues but did not bind other types of muscle, 1073/pnas.1603534113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1603534113 PNAS Early Edition | 1of10 Downloaded by guest on September 27, 2021 normal and cancerous colorectal tissues. Other markers, shown to of AOC3 in normal and cancer tissue in the gastrointestinal tract be clearly associated with this AOC3-based distinction, provide are shown in Fig. 2C, and examples in other cancer tissues are new candidates for the identification of the complex of fibroblast- shown in Fig. S2.Fig.2D shows that AOC3 also labels the pre- related cell types found in many tissues and disease states. sumed cancer-associated MFs in lymph node metastases of CRC and the presumed MFs surrounding the lymph node capsule (13). Results In contrast to these results, there is a notable absence of AOC3 Identification of AOC3 as the Primary Target of mAb PR2D3 and staining of the cancer-associated fibroblasts in breast cancer (Fig. AOC3 Expression as a Potential MF Marker. AsshowninFig.1A, 2E). Tables S1 and S2 summarize the data obtained so far on the fluorescence-labeled purified PR2D3 mAb very clearly stains peri- tissue distribution of AOC3 in a range of normal and tumor tis- cryptal MFs from normal colorectal tissue as well as the underlying sues. There generally is a strong correspondence between the smooth muscle layers, as originally observed by Richman et al. (7). AOC3 and αSMA staining wherever MFs are presumed to be MFs in a colorectal cancer (CRC) are also clearly stained (Fig. 1A), present, but neither is expressed in normal skin. The notable ex- although in this case there is more abundant PR2D3 staining, and ceptions, with αSMA staining but not AOC3, are breast carcino- the tight association with the tumor epithelial cells, counterstained mas, squamous cell skin carcinomas, and salivary glands. Uterine with an anti-epithelial cell adhesion molecule (EpCAM) epithelial- cervical carcinomas, in contrast, stained for AOC3 but not for specific mAb in red, is much less prominent. Western blotting of a αSMA. This variation in staining patterns suggests further het- human smooth muscle lysate with PR2D3 showed the expected band erogeneity of MFs and fibroblasts in different tissues, in addition at about 150 kDa only under native and not under reducing condi- to that identified only by the presence or absence of AOC3. tions, as is consistent with previous published results (Fig. 1B)(7).A pull-down immunoprecipitation was used to purify and identify the AOC3 Expression Clearly Distinguishes Cultured Colorectal-Derived protein bound to PR2D3 from a lysate of human smooth muscle. MFs
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