Myofibroblasts are distinguished from activated skin 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 a
Cancer 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 role in regulating the normal colorectal stem cell niche and facilitating tumor progression. Myofibroblasts previously have been distinguished from normal fibroblasts mostly by the expression of α smooth muscle actin (αSMA). We now have identified AOC3 (amine oxidase, copper containing 3), a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast-reacting mAb PR2D3. The normal and tumor tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by nonenzymatic procedures. Whole-genome microarray mRNA-expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly differentially expressed in these two cell types: NKX2-3 and LRRC17 in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. TGFβ substantially down-regulated AOC3 expression in myofibroblasts but in skin fibroblasts it dramatically increased the expression of αSMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and increased expression of the fibroblast-associated gene SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3, and other markers, are a distinctly different cell type from TGFβ-activated fibroblasts.
|
myofibroblasts α smooth muscle actin AOC3 NKX2-3
|
including cardiac and skeletal muscle. Ultrastructural studies showed that the pericryptal cells detected by PR2D3 had many features of smooth muscle cells, providing further support that these cells were MFs. This result was confirmed by Sappino et al. (8) using an anti-αSMA mAb that also showed very clear staining of pericryptal cells as well as smooth muscle. Following the demonstration by Desmoulière et al. (9) that connective tissue fibroblasts were stimulated to express αSMA by TGFβ, leading to the acquisition of MF-like properties, it was assumed that MFs could be defined as TGFβ-activated fibroblasts. Subsequently, MFs defined in this way were shown to be widely distributed in many different tissues, often surrounding glandular structures. Such MFs are presumed to play important roles in mesenchymal–epithelial interactions, wound healing, fibrosis, and even in immune responses (10, 11). In this paper, we identify the protein target of PR2D3 to be AOC3 (amine oxidase, copper containing 3), a member of the semicarbazide-sensitive amine oxidase/copper-containing amine oxidase (SSAO) family. AOC3 is often called “VAP-1” (vascular adhesion protein-1) because of its role in lymphocyte–endothelial interactions. The identification of AOC3 as the target of PR2D3 has enabled us to distinguish clearly between connective tissuederived fibroblasts activated by TGFβ and MFs isolated both from Significance
| tumor microenvironment |
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 development of colorectal and other epithelial cancers. The definition of myofibroblasts previously depended almost entirely on the expression of smooth muscle actin. We identified the surface enzyme AOC3 (amine oxidase, copper containing 3) as a new marker of myofibroblasts and as a result have discovered additional highly distinctive markers for myofibroblasts, including the transcription factor NKX2-3. The discovery of these new markers should greatly enhance the proper definition of myofibroblasts and related cell types and thus should contribute to the improved treatment of the many diseases, including cancer, that involve these cell types.
T
here has been long-standing interest in the pericryptal cells that form a sheath around the epithelial cells in the large intestine and a realization of their likely importance in the functional control of the gut epithelium. These pericryptal cells were originally described as fibroblasts, although it was realized that they may resemble the fibroblasts actively involved in shrinkage at healing skin wounds rather than the usually observed fibroblasts in connective tissue (1–3). Gabbiani et al. (4) showed that these “modified fibroblasts” had many properties similar to smooth muscle and suggested that they be called “myofibroblasts” (MFs) (5). They then showed that an autoimmune human serum, which reacted with α smooth muscle actin (αSMA, which is the protein product of the ACTA2 gene), detected these modified fibroblasts, including those associated with “the periphery of epithelial cells of the intestine” (6). However, the autoimmune serum, probably because it was not monospecific, also bound to “cultivated” fibroblasts and so did not clearly distinguish the pericryptal cells as MFs. The first unambiguous identification of the pericryptal cells as MFs by Richman et al. (7) was based on the discovery of a mouse mAb, PR2D3, made against fresh samples of normal large intestine. This antibody clearly bound to the pericryptal cells as well as to smooth muscle, but it did not bind connective tissue fibroblasts. PR2D3 also bound to a wide range of presumptive MFs in other tissues but did not bind other types of muscle, E2162–E2171 | PNAS | Published online March 28, 2016
Author contributions: L.-t.H. and W.F.B. designed research; L.-t.H., N.A., and L.M.W. performed research; L.-t.H., D.O., J.W., and W.F.B. analyzed data; and L.-t.H., J.W., and W.F.B. wrote the paper. Reviewers: C.K., Stanford University; and N.A.W., Barts and the London School of Medicine. The authors declare no conflict of interest. Data deposition: The sequence reported in this paper has been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE77474). 1
To whom correspondence should be addressed. Email: walter.bodmer@hertford. ox.ac.uk.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1603534113/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1603534113
fluorescence-labeled purified PR2D3 mAb very clearly stains pericryptal MFs from normal colorectal tissue as well as the underlying smooth muscle layers, as originally observed by Richman et al. (7). MFs in a colorectal cancer (CRC) are also clearly stained (Fig. 1A), although in this case there is more abundant PR2D3 staining, and the tight association with the tumor epithelial cells, counterstained with an anti-epithelial cell adhesion molecule (EpCAM) epithelialspecific mAb in red, is much less prominent. Western blotting of a human smooth muscle lysate with PR2D3 showed the expected band at about 150 kDa only under native and not under reducing conditions, as is consistent with previous published results (Fig. 1B) (7). A pull-down immunoprecipitation was used to purify and identify the protein bound to PR2D3 from a lysate of human smooth muscle. Fig. S1A shows a Coomassie blue-stained SDS-PAGE of the PR2D3-bound enriched material. Under reducing conditions, there are two specific bands of ∼100 kDa and 250 kDa, which are the putative PR2D3 target proteins. These bands were excised from a series of parallel gels, digested with trypsin, and submitted to MALDI-TOF mass spectrum peptide analysis that clearly identified the more prominent 100-kDa band as AOC3 and the 250-kDa band as myosin heavy chain 11 (MYH11) (Fig. S1A). The commercially available anti-AOC3 mAb TK8-14 (12) recognized a band of ∼150 kDa in a Western blot of a human smooth muscle lysate under nonreducing conditions, as would be expected if it and PR2D3 were detecting the same product (Fig. S1B). Confirmation that AOC3 was the target of PR2D3 was obtained by showing that the anti-AOC3 mAb TK8-14reacted in a Western blot with a PR2D3 immunoprecipitate from human smooth muscle (Fig. S1C). A commercial anti-AOC3 that can be used in paraffin-embedded sections (Clone 393112 from R&D Systems raised against recombinant human VAP-1/AOC3) was used for immunohistochemistry analysis to show that the tissue distribution of AOC3 substantially matched that determined for PR2D3. Fig. 2A clearly shows the expected pericryptal staining of MFs in normal colon, and Fig. 2B shows the expected overlap of staining of AOC3 and αSMA in the normal colon. Further examples of the distribution
A
Normal
PNAS PLUS
AOC3 Expression Clearly Distinguishes Cultured Colorectal-Derived MFs from Cultured Skin Fibroblasts. The CCD 18CO cell line
[American Type Culture Collection (ATCC): CRL-1459], originally derived from neonatal colonic mucosa, was defined as being an MF line by Valentich et al. (14). Newly derived putative MF cultures were established in the W.F.B. laboratory from normal and cancer-involved fresh colorectal tissue from a variety of sources, as described in Materials and Methods. Foreskin fibroblasts were obtained from the ATCC. Skin fibroblasts from two different adult individuals were previously established in the W.F.B. laboratory using then-conventional approaches. Fig. 3A shows that the MF line CCD 18CO clearly stains with both fluorescence-labeled AOC3 and PR2D3, whereas the foreskin fibroblasts were completely unstained. The presence of the AOC3 protein in a subset of MF lines and its absence in fibroblasts are shown in Fig. 3B. Once it was established that the surface expression of AOC3 is highly sensitive to proteolytic digestion (Fig. S3A), we proceeded to use EDTA-isolated single-cell suspensions for FACS analysis of AOC3 expression using both anti-AOC3 and PR2D3 mAbs. This analysis provided further evidence of the presence of clearly detectable AOC3 on the surface of MFs and its absence from fibroblasts and two representative epithelial cell lines derived from CRCs (Fig. 3C). Fig. S3B shows the similarity between the FACS profiles for anti-AOC3 and PR2D3 as detected in four
B
CRC
SM
:
Na tiv
5x
ed
Identification of AOC3 as the Primary Target of mAb PR2D3 and AOC3 Expression as a Potential MF Marker. As shown in Fig. 1A,
e : R ed uc
Results
of AOC3 in normal and cancer tissue in the gastrointestinal tract are shown in Fig. 2C, and examples in other cancer tissues are shown in Fig. S2. Fig. 2D shows that AOC3 also labels the presumed cancer-associated MFs in lymph node metastases of CRC and the presumed MFs surrounding the lymph node capsule (13). In contrast to these results, there is a notable absence of AOC3 staining of the cancer-associated fibroblasts in breast cancer (Fig. 2E). Tables S1 and S2 summarize the data obtained so far on the tissue distribution of AOC3 in a range of normal and tumor tissues. There generally is a strong correspondence between the AOC3 and αSMA staining wherever MFs are presumed to be present, but neither is expressed in normal skin. The notable exceptions, with αSMA staining but not AOC3, are breast carcinomas, squamous cell skin carcinomas, and salivary glands. Uterine cervical carcinomas, in contrast, stained for AOC3 but not for αSMA. This variation in staining patterns suggests further heterogeneity of MFs and fibroblasts in different tissues, in addition to that identified only by the presence or absence of AOC3.
SM
normal and cancerous colorectal tissues. Other markers, shown to be clearly associated with this AOC3-based distinction, provide new candidates for the identification of the complex of fibroblastrelated cell types found in many tissues and disease states.
150 kDa 20x
102 kDa 76 kDa
Tubulin
PR2D3 / AUA1 Fig. 1. The MF marker mAb PR2D3 recognizes the AOC3 protein in human colonic lysates. (A) Immunofluorescence staining of cryostat sections of normal colon and CRC tissue using PR2D3 (green) for MFs and AUA-1 (red) to identify epithelial cells. (B) The immunoblot of human colonic smooth muscle (SM) lysate using mAb PR2D3 shows a band of about 150 kDa in a native (nonreduced) sample.
Hsia et al.
PNAS | Published online March 28, 2016 | E2163
MEDICAL SCIENCES
WB: PR2D3
A
AOC3
SMA
D
i
Normal
C
B
ii
Merge
Cancers
Colon
Duodenum adenocarcinoma
Rectum
Rectum adenocarcinoma
Stomach
Stomach adenocarcinoma
Prostate
Prostate adenocarcinoma
iii
E
Breast cancer, AOC3
Breast cancer, SMA
Fig. 2. AOC3 is expressed on MFs in human normal and cancer tissues. (A) Confocal immunofluorescence of pericryptal MFs in a normal colon cryostat section. AOC3 (TK8-14) is shown in green and DAPI in blue in all cases where not otherwise mentioned. (Magnification: 100×.) The red arrow identifies the MFs. (B) Paraffin-embedded sections of normal colon were double-stained using AOC3 antibody (green) (393112; R&D Systems) and anti-αSMA (red) (1A4; Sigma). DAPI staining is shown in blue. The yellow staining in the merged image shows that the expression of AOC3 colocalizes with αSMA on MFs. (C) Anti-AOC3 (red) (393112; R&D Systems) is expressed on MFs in various FFPE normal and cancerous human tissues (colon, rectum, stomach, and prostate). (Magnification: 20×.) (D) AOC3 expression on MFs in FFPE CRC lymph node metastases. (i) Micrometastasis. (ii) Macrometastasis. (Magnification: 5×.) (iii) Macrometastasis. (Magnification: 20×.) AOC3 (393112; R&D) is shown in green, and AUA-1 is shown in red. (E) FFPE MFs in breast cancer are AOC3− but αSMA+. Several different cases were tested; one representative example is shown. (Magnification: 20×.) AOC3 (393112; R&D) is shown in red, and anti-αSMA (1A4; Sigma) is shown in green.
different MF cultures; this similarity supports the evidence that these antibodies are detecting the same determinant. Affymetrix microarray gene-expression data comparing the mRNA-expression levels of ACTA2 and AOC3 in panels of MF primary cultures and skin-derived fibroblast cultures strongly supports the evidence that AOC3 is a specific marker for MFs. This specificity of AOC3 is in contrast to αSMA, which is clearly expressed in both colon-derived MFs and skin-derived fibroblasts (Fig. 3D). Further mRNA analysis by quantitative RT-PCR (RTqPCR) confirmed that AOC3 is expressed at the mRNA level in most MF lines and is absent from fibroblasts (Fig. S3C). The AOC3 protein belongs to the SSAO family (15, 16). When AOC3 was knocked down in CCD 18CO cells by siRNA, the MF SSAO enzyme activity was also eliminated (Fig. 3E). The inhibition of the enzyme activity by semicarbazide and the lack of inhibition by the selective monoamine oxidase inhibitor Clorgyline confirmed that the surface expression of AOC3 in the MF CCD 18CO cell line is associated with the expected SSAO enzyme activity. Surface Detection of AOC3 Enables FACS Separation of MFs from Epithelial Cells. To date there have been no surface markers
that could distinguish MFs from fibroblasts or epithelial cells, but now, using either anti-AOC3 or PR2D3 mAbs, MFs can be separated from other cells using flow cytometry for analysis or sorting. The use of anti-AOC3 and anti-EpCAM mAbs to obtain E2164 | www.pnas.org/cgi/doi/10.1073/pnas.1603534113
an unambiguous separation of cultured MFs and epithelial cells from a 1:1 mixture of primary Myo6544 cells and cells from SW1222 (a CRC-derived cell line) is illustrated in Fig. 4A. Initial attempts to obtain such a separation from fresh colorectal tissue proved unsuccessful, because AOC3 apparently was not expressed on the fresh tissue-derived MFs, notwithstanding its later expression on the cultured explants. However, collagenase is commonly used in the isolation of single cells from fresh colorectal and other tissues, and it seemed possible that the AOC3 surface protein was as sensitive to collagenase as it is to trypsin. This sensitivity to collagenase is shown in Fig. S4. The sensitivity is explained by the existence of two collagenase-target amino acid sequences in the outer membrane proximal region of AOC3. AOC3 surface expression is clearly observed when EDTA is used to obtain singlecell suspensions and is, as expected, lost after treatment with trypsin. The use of anti-AOC3 for MF separation from an EDTA nonenzymatic digest of normal human colon tissue is shown in Fig. 4B. AOC3+ cells sorted by FACS using fluorescence-labeled AOC3 were uniformly positive not only for anti-AOC3 and PR2D3 but also for vimentin (the product of the VIM gene, a widely used fibroblast marker) (Fig. 4C). Analysis of the Differences in Expression in MF and Fibroblast Cell Lines. Whole-genome microarray mRNA-expression profiles were
obtained from four MF and four fibroblast cell lines. Partek Genomics Suite software was used to identify significant differences Hsia et al.
PNAS PLUS
A
D Myofibroblasts Fibroblasts
CCD 18CO/PR2D3
CCD 18CO/AOC3 8
-1
B
D CC
CO M
yo
20
20
yo
98
19
M
M
yo
F. Fibro cells /AOC3 65
44
ro
ib
F.F
ro
ib
nF
i Sk
AOC3 Tubulin
Myofibroblasts CCD 18CO
Myo 6544
ns
E
Myo 1998
Count
C
p
Cancer 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 role in regulating the normal colorectal stem cell niche and facilitating tumor progression. Myofibroblasts previously have been distinguished from normal fibroblasts mostly by the expression of α smooth muscle actin (αSMA). We now have identified AOC3 (amine oxidase, copper containing 3), a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast-reacting mAb PR2D3. The normal and tumor tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by nonenzymatic procedures. Whole-genome microarray mRNA-expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly differentially expressed in these two cell types: NKX2-3 and LRRC17 in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. TGFβ substantially down-regulated AOC3 expression in myofibroblasts but in skin fibroblasts it dramatically increased the expression of αSMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and increased expression of the fibroblast-associated gene SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3, and other markers, are a distinctly different cell type from TGFβ-activated fibroblasts.
|
myofibroblasts α smooth muscle actin AOC3 NKX2-3
|
including cardiac and skeletal muscle. Ultrastructural studies showed that the pericryptal cells detected by PR2D3 had many features of smooth muscle cells, providing further support that these cells were MFs. This result was confirmed by Sappino et al. (8) using an anti-αSMA mAb that also showed very clear staining of pericryptal cells as well as smooth muscle. Following the demonstration by Desmoulière et al. (9) that connective tissue fibroblasts were stimulated to express αSMA by TGFβ, leading to the acquisition of MF-like properties, it was assumed that MFs could be defined as TGFβ-activated fibroblasts. Subsequently, MFs defined in this way were shown to be widely distributed in many different tissues, often surrounding glandular structures. Such MFs are presumed to play important roles in mesenchymal–epithelial interactions, wound healing, fibrosis, and even in immune responses (10, 11). In this paper, we identify the protein target of PR2D3 to be AOC3 (amine oxidase, copper containing 3), a member of the semicarbazide-sensitive amine oxidase/copper-containing amine oxidase (SSAO) family. AOC3 is often called “VAP-1” (vascular adhesion protein-1) because of its role in lymphocyte–endothelial interactions. The identification of AOC3 as the target of PR2D3 has enabled us to distinguish clearly between connective tissuederived fibroblasts activated by TGFβ and MFs isolated both from Significance
| tumor microenvironment |
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 development of colorectal and other epithelial cancers. The definition of myofibroblasts previously depended almost entirely on the expression of smooth muscle actin. We identified the surface enzyme AOC3 (amine oxidase, copper containing 3) as a new marker of myofibroblasts and as a result have discovered additional highly distinctive markers for myofibroblasts, including the transcription factor NKX2-3. The discovery of these new markers should greatly enhance the proper definition of myofibroblasts and related cell types and thus should contribute to the improved treatment of the many diseases, including cancer, that involve these cell types.
T
here has been long-standing interest in the pericryptal cells that form a sheath around the epithelial cells in the large intestine and a realization of their likely importance in the functional control of the gut epithelium. These pericryptal cells were originally described as fibroblasts, although it was realized that they may resemble the fibroblasts actively involved in shrinkage at healing skin wounds rather than the usually observed fibroblasts in connective tissue (1–3). Gabbiani et al. (4) showed that these “modified fibroblasts” had many properties similar to smooth muscle and suggested that they be called “myofibroblasts” (MFs) (5). They then showed that an autoimmune human serum, which reacted with α smooth muscle actin (αSMA, which is the protein product of the ACTA2 gene), detected these modified fibroblasts, including those associated with “the periphery of epithelial cells of the intestine” (6). However, the autoimmune serum, probably because it was not monospecific, also bound to “cultivated” fibroblasts and so did not clearly distinguish the pericryptal cells as MFs. The first unambiguous identification of the pericryptal cells as MFs by Richman et al. (7) was based on the discovery of a mouse mAb, PR2D3, made against fresh samples of normal large intestine. This antibody clearly bound to the pericryptal cells as well as to smooth muscle, but it did not bind connective tissue fibroblasts. PR2D3 also bound to a wide range of presumptive MFs in other tissues but did not bind other types of muscle, E2162–E2171 | PNAS | Published online March 28, 2016
Author contributions: L.-t.H. and W.F.B. designed research; L.-t.H., N.A., and L.M.W. performed research; L.-t.H., D.O., J.W., and W.F.B. analyzed data; and L.-t.H., J.W., and W.F.B. wrote the paper. Reviewers: C.K., Stanford University; and N.A.W., Barts and the London School of Medicine. The authors declare no conflict of interest. Data deposition: The sequence reported in this paper has been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE77474). 1
To whom correspondence should be addressed. Email: walter.bodmer@hertford. ox.ac.uk.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1603534113/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1603534113
fluorescence-labeled purified PR2D3 mAb very clearly stains pericryptal MFs from normal colorectal tissue as well as the underlying smooth muscle layers, as originally observed by Richman et al. (7). MFs in a colorectal cancer (CRC) are also clearly stained (Fig. 1A), although in this case there is more abundant PR2D3 staining, and the tight association with the tumor epithelial cells, counterstained with an anti-epithelial cell adhesion molecule (EpCAM) epithelialspecific mAb in red, is much less prominent. Western blotting of a human smooth muscle lysate with PR2D3 showed the expected band at about 150 kDa only under native and not under reducing conditions, as is consistent with previous published results (Fig. 1B) (7). A pull-down immunoprecipitation was used to purify and identify the protein bound to PR2D3 from a lysate of human smooth muscle. Fig. S1A shows a Coomassie blue-stained SDS-PAGE of the PR2D3-bound enriched material. Under reducing conditions, there are two specific bands of ∼100 kDa and 250 kDa, which are the putative PR2D3 target proteins. These bands were excised from a series of parallel gels, digested with trypsin, and submitted to MALDI-TOF mass spectrum peptide analysis that clearly identified the more prominent 100-kDa band as AOC3 and the 250-kDa band as myosin heavy chain 11 (MYH11) (Fig. S1A). The commercially available anti-AOC3 mAb TK8-14 (12) recognized a band of ∼150 kDa in a Western blot of a human smooth muscle lysate under nonreducing conditions, as would be expected if it and PR2D3 were detecting the same product (Fig. S1B). Confirmation that AOC3 was the target of PR2D3 was obtained by showing that the anti-AOC3 mAb TK8-14reacted in a Western blot with a PR2D3 immunoprecipitate from human smooth muscle (Fig. S1C). A commercial anti-AOC3 that can be used in paraffin-embedded sections (Clone 393112 from R&D Systems raised against recombinant human VAP-1/AOC3) was used for immunohistochemistry analysis to show that the tissue distribution of AOC3 substantially matched that determined for PR2D3. Fig. 2A clearly shows the expected pericryptal staining of MFs in normal colon, and Fig. 2B shows the expected overlap of staining of AOC3 and αSMA in the normal colon. Further examples of the distribution
A
Normal
PNAS PLUS
AOC3 Expression Clearly Distinguishes Cultured Colorectal-Derived MFs from Cultured Skin Fibroblasts. The CCD 18CO cell line
[American Type Culture Collection (ATCC): CRL-1459], originally derived from neonatal colonic mucosa, was defined as being an MF line by Valentich et al. (14). Newly derived putative MF cultures were established in the W.F.B. laboratory from normal and cancer-involved fresh colorectal tissue from a variety of sources, as described in Materials and Methods. Foreskin fibroblasts were obtained from the ATCC. Skin fibroblasts from two different adult individuals were previously established in the W.F.B. laboratory using then-conventional approaches. Fig. 3A shows that the MF line CCD 18CO clearly stains with both fluorescence-labeled AOC3 and PR2D3, whereas the foreskin fibroblasts were completely unstained. The presence of the AOC3 protein in a subset of MF lines and its absence in fibroblasts are shown in Fig. 3B. Once it was established that the surface expression of AOC3 is highly sensitive to proteolytic digestion (Fig. S3A), we proceeded to use EDTA-isolated single-cell suspensions for FACS analysis of AOC3 expression using both anti-AOC3 and PR2D3 mAbs. This analysis provided further evidence of the presence of clearly detectable AOC3 on the surface of MFs and its absence from fibroblasts and two representative epithelial cell lines derived from CRCs (Fig. 3C). Fig. S3B shows the similarity between the FACS profiles for anti-AOC3 and PR2D3 as detected in four
B
CRC
SM
:
Na tiv
5x
ed
Identification of AOC3 as the Primary Target of mAb PR2D3 and AOC3 Expression as a Potential MF Marker. As shown in Fig. 1A,
e : R ed uc
Results
of AOC3 in normal and cancer tissue in the gastrointestinal tract are shown in Fig. 2C, and examples in other cancer tissues are shown in Fig. S2. Fig. 2D shows that AOC3 also labels the presumed cancer-associated MFs in lymph node metastases of CRC and the presumed MFs surrounding the lymph node capsule (13). In contrast to these results, there is a notable absence of AOC3 staining of the cancer-associated fibroblasts in breast cancer (Fig. 2E). Tables S1 and S2 summarize the data obtained so far on the tissue distribution of AOC3 in a range of normal and tumor tissues. There generally is a strong correspondence between the AOC3 and αSMA staining wherever MFs are presumed to be present, but neither is expressed in normal skin. The notable exceptions, with αSMA staining but not AOC3, are breast carcinomas, squamous cell skin carcinomas, and salivary glands. Uterine cervical carcinomas, in contrast, stained for AOC3 but not for αSMA. This variation in staining patterns suggests further heterogeneity of MFs and fibroblasts in different tissues, in addition to that identified only by the presence or absence of AOC3.
SM
normal and cancerous colorectal tissues. Other markers, shown to be clearly associated with this AOC3-based distinction, provide new candidates for the identification of the complex of fibroblastrelated cell types found in many tissues and disease states.
150 kDa 20x
102 kDa 76 kDa
Tubulin
PR2D3 / AUA1 Fig. 1. The MF marker mAb PR2D3 recognizes the AOC3 protein in human colonic lysates. (A) Immunofluorescence staining of cryostat sections of normal colon and CRC tissue using PR2D3 (green) for MFs and AUA-1 (red) to identify epithelial cells. (B) The immunoblot of human colonic smooth muscle (SM) lysate using mAb PR2D3 shows a band of about 150 kDa in a native (nonreduced) sample.
Hsia et al.
PNAS | Published online March 28, 2016 | E2163
MEDICAL SCIENCES
WB: PR2D3
A
AOC3
SMA
D
i
Normal
C
B
ii
Merge
Cancers
Colon
Duodenum adenocarcinoma
Rectum
Rectum adenocarcinoma
Stomach
Stomach adenocarcinoma
Prostate
Prostate adenocarcinoma
iii
E
Breast cancer, AOC3
Breast cancer, SMA
Fig. 2. AOC3 is expressed on MFs in human normal and cancer tissues. (A) Confocal immunofluorescence of pericryptal MFs in a normal colon cryostat section. AOC3 (TK8-14) is shown in green and DAPI in blue in all cases where not otherwise mentioned. (Magnification: 100×.) The red arrow identifies the MFs. (B) Paraffin-embedded sections of normal colon were double-stained using AOC3 antibody (green) (393112; R&D Systems) and anti-αSMA (red) (1A4; Sigma). DAPI staining is shown in blue. The yellow staining in the merged image shows that the expression of AOC3 colocalizes with αSMA on MFs. (C) Anti-AOC3 (red) (393112; R&D Systems) is expressed on MFs in various FFPE normal and cancerous human tissues (colon, rectum, stomach, and prostate). (Magnification: 20×.) (D) AOC3 expression on MFs in FFPE CRC lymph node metastases. (i) Micrometastasis. (ii) Macrometastasis. (Magnification: 5×.) (iii) Macrometastasis. (Magnification: 20×.) AOC3 (393112; R&D) is shown in green, and AUA-1 is shown in red. (E) FFPE MFs in breast cancer are AOC3− but αSMA+. Several different cases were tested; one representative example is shown. (Magnification: 20×.) AOC3 (393112; R&D) is shown in red, and anti-αSMA (1A4; Sigma) is shown in green.
different MF cultures; this similarity supports the evidence that these antibodies are detecting the same determinant. Affymetrix microarray gene-expression data comparing the mRNA-expression levels of ACTA2 and AOC3 in panels of MF primary cultures and skin-derived fibroblast cultures strongly supports the evidence that AOC3 is a specific marker for MFs. This specificity of AOC3 is in contrast to αSMA, which is clearly expressed in both colon-derived MFs and skin-derived fibroblasts (Fig. 3D). Further mRNA analysis by quantitative RT-PCR (RTqPCR) confirmed that AOC3 is expressed at the mRNA level in most MF lines and is absent from fibroblasts (Fig. S3C). The AOC3 protein belongs to the SSAO family (15, 16). When AOC3 was knocked down in CCD 18CO cells by siRNA, the MF SSAO enzyme activity was also eliminated (Fig. 3E). The inhibition of the enzyme activity by semicarbazide and the lack of inhibition by the selective monoamine oxidase inhibitor Clorgyline confirmed that the surface expression of AOC3 in the MF CCD 18CO cell line is associated with the expected SSAO enzyme activity. Surface Detection of AOC3 Enables FACS Separation of MFs from Epithelial Cells. To date there have been no surface markers
that could distinguish MFs from fibroblasts or epithelial cells, but now, using either anti-AOC3 or PR2D3 mAbs, MFs can be separated from other cells using flow cytometry for analysis or sorting. The use of anti-AOC3 and anti-EpCAM mAbs to obtain E2164 | www.pnas.org/cgi/doi/10.1073/pnas.1603534113
an unambiguous separation of cultured MFs and epithelial cells from a 1:1 mixture of primary Myo6544 cells and cells from SW1222 (a CRC-derived cell line) is illustrated in Fig. 4A. Initial attempts to obtain such a separation from fresh colorectal tissue proved unsuccessful, because AOC3 apparently was not expressed on the fresh tissue-derived MFs, notwithstanding its later expression on the cultured explants. However, collagenase is commonly used in the isolation of single cells from fresh colorectal and other tissues, and it seemed possible that the AOC3 surface protein was as sensitive to collagenase as it is to trypsin. This sensitivity to collagenase is shown in Fig. S4. The sensitivity is explained by the existence of two collagenase-target amino acid sequences in the outer membrane proximal region of AOC3. AOC3 surface expression is clearly observed when EDTA is used to obtain singlecell suspensions and is, as expected, lost after treatment with trypsin. The use of anti-AOC3 for MF separation from an EDTA nonenzymatic digest of normal human colon tissue is shown in Fig. 4B. AOC3+ cells sorted by FACS using fluorescence-labeled AOC3 were uniformly positive not only for anti-AOC3 and PR2D3 but also for vimentin (the product of the VIM gene, a widely used fibroblast marker) (Fig. 4C). Analysis of the Differences in Expression in MF and Fibroblast Cell Lines. Whole-genome microarray mRNA-expression profiles were
obtained from four MF and four fibroblast cell lines. Partek Genomics Suite software was used to identify significant differences Hsia et al.
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