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

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 (, copper (8) using an anti-αSMA mAb that also showed very clear staining containing 3), a surface , as a new marker of myo- of pericryptal cells as well as smooth muscle. Following the fibroblasts by showing that it is the target 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 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 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- 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.

E2162–E2171 | PNAS | Published online March 28, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1603534113 Downloaded by guest on September 26, 2021 normal and cancerous colorectal tissues. Other markers, shown to of AOC3 in normal and cancer tissue in the gastrointestinal tract PNAS PLUS 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 from Cultured Skin Fibroblasts. The CCD 18CO cell line Fig. S1A shows a Coomassie blue-stained SDS-PAGE of the [American Type Culture Collection (ATCC): CRL-1459], origi- PR2D3-bound enriched material. Under reducing conditions, there nally derived from neonatal colonic mucosa, was defined as be- are two specific bands of ∼100 kDa and 250 kDa, which are the ing an MF line by Valentich et al. (14). Newly derived putative putative PR2D3 target . These bands were excised from MF cultures were established in the W.F.B. laboratory from a series of parallel gels, digested with trypsin, and submitted to normal and cancer-involved fresh colorectal tissue from a variety MALDI-TOF mass spectrum peptide analysis that clearly identified of sources, as described in Materials and Methods. Foreskin fi- themoreprominent100-kDabandasAOC3andthe250-kDaband broblasts were obtained from the ATCC. Skin fibroblasts from as myosin heavy chain 11 (MYH11) (Fig. S1A). The commer- two different adult individuals were previously established in the cially available anti-AOC3 mAb TK8-14 (12) recognized a band W.F.B. laboratory using then-conventional approaches. Fig. 3A of ∼150 kDa in a Western blot of a human smooth muscle lysate shows that the MF line CCD 18CO clearly stains with both under nonreducing conditions, as would be expected if it and PR2D3 fluorescence-labeled AOC3 and PR2D3, whereas the foreskin were detecting the same product (Fig. S1B). Confirmation that fibroblasts were completely unstained. The presence of the AOC3 was the target of PR2D3 was obtained by showing that the AOC3 protein in a subset of MF lines and its absence in fibro- anti-AOC3 mAb TK8-14reacted in a Western blot with a PR2D3 blasts are shown in Fig. 3B. immunoprecipitate from human smooth muscle (Fig. S1C). Once it was established that the surface expression of AOC3 is A commercial anti-AOC3 that can be used in paraffin-embedded highly sensitive to proteolytic digestion (Fig. S3A), we proceeded sections (Clone 393112 from R&D Systems raised against to use EDTA-isolated single-cell suspensions for FACS analysis recombinant human VAP-1/AOC3) was used for immunohisto- of AOC3 expression using both anti-AOC3 and PR2D3 mAbs. chemistry analysis to show that the tissue distribution of AOC3 This analysis provided further evidence of the presence of clearly substantially matched that determined for PR2D3. Fig. 2A clearly detectable AOC3 on the surface of MFs and its absence from shows the expected pericryptal staining of MFs in normal colon, fibroblasts and two representative epithelial cell lines derived and Fig. 2B shows the expected overlap of staining of AOC3 and from CRCs (Fig. 3C). Fig. S3B shows the similarity between the αSMA in the normal colon. Further examples of the distribution FACS profiles for anti-AOC3 and PR2D3 as detected in four

A Normal CRC B 5x

SM: NativeSM: Reduced

150 kDa 20x 102 kDa 76 kDa

WB: PR2D3

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. MEDICAL SCIENCES

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SMA AOC3 Merge Colon Duodenum adenocarcinoma

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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 an unambiguous separation of cultured MFs and epithelial cells these antibodies are detecting the same determinant. from a 1:1 mixture of primary Myo6544 cells and cells from Affymetrix microarray gene-expression data comparing the SW1222 (a CRC-derived cell line) is illustrated in Fig. 4A.Initial mRNA-expression levels of ACTA2 and AOC3 in panels of MF attempts to obtain such a separation from fresh colorectal tissue primary cultures and skin-derived fibroblast cultures strongly proved unsuccessful, because AOC3 apparently was not expressed supports the evidence that AOC3 is a specific marker for MFs. on the fresh tissue-derived MFs, notwithstanding its later expres- This specificity of AOC3 is in contrast to αSMA, which is clearly sion on the cultured explants. However, collagenase is commonly expressed in both colon-derived MFs and skin-derived fibroblasts used in the isolation of single cells from fresh colorectal and other (Fig. 3D). Further mRNA analysis by quantitative RT-PCR (RT- tissues, and it seemed possible that the AOC3 surface protein was qPCR) confirmed that AOC3 is expressed at the mRNA level in as sensitive to collagenase as it is to trypsin. This sensitivity to most MF lines and is absent from fibroblasts (Fig. S3C). collagenase is shown in Fig. S4. The sensitivity is explained by the The AOC3 protein belongs to the SSAO family (15, 16). When existence of two collagenase-target amino acid sequences in the AOC3 was knocked down in CCD 18CO cells by siRNA, the MF outer membrane proximal region of AOC3. AOC3 surface ex- SSAO enzyme activity was also eliminated (Fig. 3E). The in- pression is clearly observed when EDTA is used to obtain single- hibition of the enzyme activity by semicarbazide and the lack of cell suspensions and is, as expected, lost after treatment with inhibition by the selective monoamine oxidase inhibitor Clorgy- trypsin. The use of anti-AOC3 for MF separation from an EDTA line confirmed that the surface expression of AOC3 in the MF nonenzymatic digest of normal human colon tissue is shown in + CCD 18CO cell line is associated with the expected SSAO Fig. 4B.AOC3 cells sorted by FACS using fluorescence-labeled enzyme activity. AOC3 were uniformly positive not only for anti-AOC3 and PR2D3 but also for vimentin (the product of the VIM gene, a Surface Detection of AOC3 Enables FACS Separation of MFs from widely used fibroblast marker) (Fig. 4C). Epithelial Cells. To date there have been no surface markers that could distinguish MFs from fibroblasts or epithelial cells, but Analysis of the Differences in Expression in MF and Fibroblast Cell now, using either anti-AOC3 or PR2D3 mAbs, MFs can be Lines. Whole-genome microarray mRNA-expression profiles were separated from other cells using flow cytometry for analysis or obtained from four MF and four fibroblast cell lines. Partek Ge- sorting. The use of anti-AOC3 and anti-EpCAM mAbs to obtain nomics Suite software was used to identify significant differences

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AOC3-APC Fibroblasts F Fibro Skin Fibro 1 Skin Fibro 2 Count

AOC3-APC Epithelial cells SW1222 LS 174T Count

AOC3-APC

Fig. 3. AOC3 is expressed in primary MFs but not in fibroblasts. (A) Immunofluorescent staining of CCD 18CO cells and foreskin fibroblast cells with AOC3 mAb (TK8-14) and PR2D3. (Magnification: 20×.) (B) Western blot using AOC3 mAb (TK8-14) detected a 150-kDa protein in CCD 18CO, myo2020, myo1998, and myo6544 cells but not in foreskin fibroblasts (F. Fibro) or skin fibroblasts (Skin Fibro). Tubulin was used as the loading control. (C) Flow cytometric analysis of primary cell cultures (myo6544 and myo1998) and CCD 18CO cells. Fibroblasts (foreskin fibroblasts and two sets of adult skin fibroblasts) and epithelial cells + (SW1222 and LS174T) show that MFs are AOC3 , whereas fibroblasts and epithelial cells do not express AOC3. Isotype control is shown in red, and anti-AOC3 (TK8-14) is shown in green. (D) mRNA-expression levels of AOC3 and ACTA2 in MF and fibroblast cultures. AOC3 and ACTA2 mRNA expression in eight cell lines (four MF cultures and four fibroblast cultures) was measured by the Affymetrix U133 Plus 2.0 microarray. The mRNA-expression levels along the y axis are actual ΔCt fluorescence intensities. (E) AOC3 functions as an SSAO enzyme in MFs. The enzyme activity of MFs was determined by SSAO-

mediated H2O2 production. The siRNA for AOC3, the SSAO inhibitor semicarbazide (SEM) (1 mM), and the MAO-A inhibitor Clorgyline (1 mM) were added under serum-free conditions for 48 h. The enzyme activity in untreated CCD 18CO control cells was set to 100%. The P values are based on t tests for the difference between the treated and the serum-free control samples: **P < 0.001.

in mRNA expression between the MFs and the fibroblasts using Control of AOC3, αSMA, and SHOX2 Expression in MFs and Fibroblasts a Benjamini and Hochberg step-up false discovery rate-corrected by TGFβ. Rather surprisingly, it was found that incubating MF P value cutoff of 0.05 (Table 1 and Fig. 5A; the volcano plot in cultures in serum-free conditions led to a substantial increase in Fig. S5 provides the background for the selection of these four AOC3 expression at both the protein (Fig. 6A) and mRNA (Fig. A β genes for further analysis). The most striking differences in ex- S6 ) levels. When TGF was added to 10% (vol/vol) normal pression are the high levels of expression of the homeobox serum, the expression of AOC3 was completely inhibited (Fig. B β transcription factor SHOX2 in fibroblasts and its almost com- 6 ), suggesting that the levels of TGF found in normal serum can explain the effects of serum deprivation. The inhibitory ef- plete absence in MFs, and the reverse for the transcription factor β NKX2-3, which was highly expressed in MFs and not at all in fects of TGF on AOC3 expression in CCD 18CO cells are also TBX5 seen at the mRNA level, suggesting transcriptional control (Fig. fibroblasts. The differences in the expression of and B AOC3 LRCC17 A S6 ). This idea is supported by the increase in mRNA are also substantial (Fig. 5 ). The data shown in Fig. AOC3 B after serum starvation and the subsequent decrease in 5 , using RT-qPCR to measure mRNA levels, confirm the clear- β C NKX2-3 SHOX2 mRNA after TGF was added (Fig. 6 ). However, we did not cut differences in and mRNA-expression levels see a concomitant decrease in AOC3 protein levels (Fig. S6C). between MFs and fibroblasts. Western blotting of selected MF This result suggests that the AOC3 protein might have a long and fibroblast cell lines with an anti-NKX2-3 mAb shows that the turnover time during which the protein is degraded slowly, and NKX2-3 difference for at the mRNA level is also seen at the that the Western blot detects the accumulated protein. protein level (Fig. 5C). Fig. 5D confirms that these differences in Neither serum starvation nor TGFβ addition has any effect on expression are also seen in freshly isolated MFs and are not AOC3 expression in fibroblasts (Fig. 6D). However, as would be artifacts of the culture conditions. The complete lack of ex- expected from many previous studies, TGFβ dramatically in- pression of SHOX2 clearly indicates that these FACS-sorted creases the expression of ACTA2 in fibroblasts, even in the ab- + AOC3 cells do not include any fibroblasts analogous to those sence of serum, but apparently decreases the expression of found in skin. SHOX2 (Fig. 6E). The dramatic effect of TGFβ treatment on the MEDICAL SCIENCES

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SW1222

Myo6544 AUA1-FITC Log AOC3-Alexa488 Log

AOC3-APC Log AOC3-Sorted cells C

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Fig. 4. FACS isolation of MFs. (A) Flow cytometry-based separation of a 1:1 mixture of epithelial cells (SW1222) and primary MFs (myo6544). MFs were labeled with anti–AOC3-APC (TK8-14), and epithelial cells were labeled with AUA1-FITC. (B) FACS isolation of MFs from fresh tissue. Cells expressing AOC3 (mAb TK8-14) were gated using the Alexa-488 fluorescent signals (y axis) with a cutoff based on unstained samples. The x axis is forward scatter. (C)Im- munofluorescent staining of AOC3 FACS-sorted cells with anti-vimentin (3B4; Dako) on fixed cells and anti-AOC3 (TK8-14) and PR2D3 on live cells. (Magni- fication: 20×.)

increased expression of αSMA at the protein level is shown in our finding of AOC3 on MFs. The apparent normal gut function Fig. S7. in the AOC3-knockout mice could easily be explained by an in- To explore the role of NKX2-3 in the control of MF functions, crease in the expression of AOC1 (18), which then would suggest we assessed the effect of knocking down NKX2-3 in MFs using that the SSAO activity of AOC3 might not be required for the siRNA. Knocking down the expression of NKX2-3 in both the normal gut function. Immunofluorescence using either PR2D3 or CCD 18CO and myo6526 MF lines dramatically increased the other anti-AOC3 mAbs provides a good marker for identifying expression of SHOX2 but decreased the expression of AOC3, MFs in fresh or frozen tissue, in -fixed, paraffin- ACTA2, and MYH11 (Fig. 6F), so that the expression profile of embedded (FFPE) sections, and in MFs in culture and for FACS these genes then closely resembled the profile we observed in the analysis of mixtures of MFs and other cell types, including epi- fibroblast cell lines. In contrast, knocking down AOC3 resulted in thelial cells. AOC3 FACS-purified MFs uniformly express AOC3, a significant decrease in NKX2-3 expression in CCD 18CO cells, as expected, but only a subset of the purified cells express αSMA but the expression of MYH11, ACTA2, and SHOX2 remained (Fig. S8). The heterogeneous staining of αSMA on AOC3-sorted unchanged (Fig. 6G). This result suggests a positive feedback MFs emphasizes the inadequacy of using this marker to identify loop between NKX2-3 and AOC3 expression in MFs (Fig. 6H). MFs. It seems likely that the presence of MYH11 (which is a These data show that MFs and fibroblasts not only have signif- major component of smooth muscle myosin) in the PR2D3 pre- icantly different expression profiles for a few key genes but also cipitate was the result of adventitious, nonspecific contamination, differ dramatically in their response to TGFβ, which inhibits perhaps because of the large amounts of MYH11 in smooth AOC3 expression in MFs without increasing ACTA2 expression muscle and its inherently “sticky” nature. but substantially increases αSMA in fibroblasts, as expected. The Through a genome-wide analysis of microarray-based differ- results clearly suggest that TGFβ-activated fibroblasts and MFs, ences in the mRNA-expression profiles of MF and fibroblast cell as defined by the expression of AOC3 and NKX2-3, are signifi- lines in culture, four additional markers that distinguish MFs and cantly different cell types. fibroblasts have been identified, including the homeobox tran- scriptionfactorNKX2-3onMFsand SHOX2, another transcrip- Discussion tion factor, on fibroblasts. While this paper was in preparation, Identification of AOC3 as the target of the PR2D3 mAb, by which Higuchi et al. (19), in an extensive study of fibroblast mRNA- colorectal pericryptal cells were first clearly shown to be MFs, has expression profiles, reported as incidental findings that NKX2-3 provided a new marker for the characterization and functional was expressed in a subset of human gastrointestinal MFs and that study of these important epithelial-associated mesenchymal cells. the expression of SHOX2 and TBX5 was up-regulated in non- Stolen et al. (17) showed that pericryptal cells in guts of AOC3- gastrointestinal fibroblasts. These findings are in agreement with knockout mice are not stained by anti-AOC3, as is consistent with our results for the colon but were not associated by Higuchi et al.

Table 1. The four genes most significantly differentially expressed in MFs and fibroblasts Step-up Fold-change, MFs vs. Fold-change, MFs vs. Gene symbol Gene name P value P value fibroblasts fibroblasts

SHOX2 Short stature homeobox 2 5.97E-10 0.0000327 −90.0583 Down in MFs vs. fibroblasts NKX2-3 NK2 transcription factor-related, locus 3 2.34E-06 0.0256099 35.0344 Up in MFs vs. fibroblasts TBX5 T-box 5 3.65E-06 0.033282 −9.89776 Down in MFs vs. fibroblasts LRRC17 Leucine-rich repeat-containing 17 6.50E-06 0.050798 57.6304 Up in MFs vs. fibroblasts

Expression levels are listed by rank order of corrected P values. Fold-changes are given relative to MFs; negative values are for fold-changes that are expressed at higher levels in fibroblasts than in MFs.

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C ro .Fibro CCD 18CO Myo 6769C Myo 1998 Myo 6526 Skin Fib F

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Fig. 5. Identification of candidate markers NKX2-3 for MFs and SHOX2 for fibroblasts. (A) NKX2-3, SHOX2, LRRC17, and TBX5 mRNA expression in MFs and fibroblasts measured by the Affymetrix Human Genome U133 Plus 2.0 microarray. NKX2-3 and LRRC17 are highly expressed in MFs; SHOX2 and TBX5 are highly expressed in fibroblasts. (B) RT-qPCR verification of NKX2-3 and SHOX2 mRNA expression in MF and fibroblast cultures. Expression levels are given as linearized ΔCt values generated as described in Materials and Methods.TheNKX2-3 mRNA levels in skin fibroblasts and foreskin fibroblasts were too low to be determined (ND: no data). Columns in red represent MF cultures; columns in blue represent fibroblast cultures. (C) NKX2-3 protein is expressed in MF cultures (CCD 18CO, myo6769C, myo1998, and myo6526 cells) but not in fibroblast cultures. Western blot with anti NKX2-3 (polyclonal; LSBio). Anti-tubulin was used as a control. (D) The mRNA-expression levels of AOC3 and NKX2-3 in AOC3-sorted MFs. Expression levels were determined by RT-qPCR and are given as converted linearized ΔCt values. Red columns represent AOC3-sorted cells; the blue column represents skin fibroblast cultures. The NKX2-3 mRNA levels of skin fibroblasts and SHOX2 mRNA levels of AOC3-sorted cells were not determined, because there was no measurable Ct for the sample, indicating a very low or absent amount of mRNA.

with the definition of MFs. By clustering the expression profiles AOC3 (25-28) similar to the positive feedback loop we have obtained from fibroblasts from different organs, they also reported observed in MFs. The key role for NKX2-5, a close homolog of that NKX2-3 is organ-specific and restricted to the NKX2-3 that recognizes the same consensus sequence, in the duodenum, ileum, and colon. regulation of heart development (20, 29) and the interrelation- Our preliminary results on the role of the homeobox tran- ship among NKX2-5, SHOX2, and TBX5 in the control of scription factor NKX2-3 in controlling MF function showed that pacemaker activity in myocytes (30) suggest that NKX2-3 may NKX2-3 knocking down in MF lines led to a substantial increase play a role in the gut analogous to that played by NKX2-5 in the SHOX2 AOC3 in expression associated with a decrease in ex- heart. This idea is consistent with the evidence that the normal pression. Although AOC3 is not a transcription factor, siRNA control of epithelial cell turnover is disrupted in the gut of Nkx2-3– knockdown of AOC3 had a profound negative effect on NKX2-3 knockout mice (20). expression, suggesting that AOC3 is able to mediate this tran- Our analysis of the effects of serum-free incubation and TGFβ scriptional regulation of NKX2-3 expression in MFs through an treatment of the MF and fibroblast cell lines has shown that MFs intermediate or indirect signaling mechanism. β It seems possible that NKX2-3 plays a wider role in controlling and TGF -activated skin fibroblasts are two quite distinct cell the determination of mesenchymal cell types than previously types, although they do share many common features. The understood. Thus, Bmp-2 and Bmp-4 have been identified as method most commonly used thus far to characterize presumed downstream targets of Nkx2-3 in regulating gut morphogenesis MFs has been the observation that TGFβ activates fibroblasts to in mice (20, 21). In endothelial cells, increased expression levels produce αSMA (31), but we now show that this activation is not a of AOC3 and NKX2-3 have been reported in inflammatory bowel distinctive property of MFs compared with fibroblasts. TGFβ, disease (22–24), and there are indications that lymphocyte traf- however, inhibits the expression of AOC3 in MFs but has no ficking could involve a feedback loop between NKX2-3 and effect on AOC3 expression in fibroblasts. MEDICAL SCIENCES

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Fig. 6. Regulation of AOC3, NKX2-3, and SHOX2 expression in MFs. (A) Serum starvation increased AOC3 expression in CCD 18CO cells. Cells were cultured with 10% FBS or without serum for 24, 48, 72, or 96 h. AOC3 protein expression was measured using Western blots with anti-AOC3 (TK8-14) and with anti- tubulin as the control. (B) AOC3 expression is down-regulated by TGFβ treatment. Cells were incubated with or without 10 ng/mL TGFβ in serum-free medium or in medium containing 10% normal serum for 24, 48, 72, or 96 h, and AOC3 levels were determined by Western blots as previously described. (C)The inhibitory effect of TGFβ on AOC3 expression in CCD 18CO cells. The AOC3 mRNA levels were determined using RT-qPCR. Cells were serum starved and then were treated with TGFβ (10 ng/mL) at the indicated times. The blue line indicates serum-starved levels; the red line indicates the levels after the addition of TGFβ. The Ct values were normalized to UBC, and the fold-change was calculated relative to cells in serum-free conditions for 24 h (y axis). (D) AOC3 ex- pression is not inducible in foreskin fibroblast cells. Foreskin fibroblasts were serum starved (SF) and treated with TGFβ for 72 h. Protein expression was measured using Western blots with anti-AOC3 (TK8-14) and anti-tubulin. CON, control. (E) Skin fibroblast mRNA expression of AOC3, ACTA2, and SHOX2 following TGFβ treatment. Cells were grown in normal 10% serum (NS) or under serum-free conditions with (SF/TGFβ) or without TGFβ (10 ng/mL) treatment for 48 h. Expression levels were determined using RT-qPCR and are given as linearized ΔCt values. The NKX2-3 mRNA level was undetectable. The ΔCt values for NKX2-3, AOC3, and SHOX2 are given on the left y axis, and the ΔCt values for ACTA2 are given on the right y axis. (F) The expression of ACTA2, MYH11, and SHOX2 is regulated by NKX2-3 in MFs. Relative mRNA-expression levels were determined using RT-qPCR following transfection of CCD 18CO and Myo6526 cells with siNKX2-3 or with the scrambled sequence (siCON) as control. Raw Ct values were normalized to UBC, and the fold-changes relative to siCON were calculated using the ΔΔCt method. (G) AOC3 silencing decreases NKX2-3 expression in CCD 18CO cells. The expression levels of NKX2-3, SHOX2, MYH11, and ACTA2 in siAOC3-transfected relative to siCON-transfected CCD 18CO cells were determined using RT-qPCR. Raw Ct values were normalized to UBC, and the fold-changes relative to siCON were calculated using the ΔΔCt method. The P values are based on t tests for the difference between the treated and the serum-free control samples: **P < 0.001. (H) Schematic model for feedback gene regulation in MFs and fibroblasts.

The fibroblast activation protein (FAP) has been shown to be (13), possibly involving hepatocyte and other growth factors. As specific for activated fibroblasts (aFs) in wounds, and antibodies to has often been suggested, blocking this interaction may prove to FAP react strongly with the stromal fibroblast-like cells present in be an effective form of treatment even for advanced CRCs. many epithelial cancers, including CRC. FAP reacts poorly with However, for this treatment to be developed it is essential to be normal skin fibroblasts in tissue but does react with fibroblasts sure of the cell type involved, because MFs are likely to have a in culture (32–34), probably because of activation by the TGFβ profile of secreted growth factors quite different from that of aFs present in FBS. FAP does not react with smooth muscle and has and a different form of interaction with epithelial cells as well. been reported to be absent in MFs defined as pericryptal cells. These differences emphasize the importance of being able to However, its extensive reactivity with stromal fibroblast-like cells, distinguish aFs from MFs in cancer tissues. which we identify as MFs using AOC3 expression, suggests that The combination of the markers AOC3, NKX2-3, and SHOX2 FAP is activated in MFs in cancers and cannot be used as a dis- with LRRC17 and TBX5, as revealed by the analysis of AOC3, tinctive marker to distinguish aFs from activated MFs. now makes it possible to identify the presence of MFs un- Yeung et al. (13) showed very substantial activation of pre- equivocally in colorectal and, possibly, many other adenocarci- sumed MFs in lymph nodes that had large CRC metastatic de- nomas. Smoothelin (35), which is present on smooth muscle but posits. The larger the metastasis in the lymph node, the greater not the fibroblast lineage, and desmin can be used to distinguish was the extent of MF activation. That this activation really did aFs and MFs from smooth muscle (Table 2). Thus, fibroblasts, involve MFs as we define them is confirmed by their reactivity aFs, MFs, and smooth muscle can now be clearly differentiated with anti-AOC3, as shown in Fig. 2D. A striking feature of this using currently available mAbs with many other mAb-based activation is that the MFs are no longer closely associated with markers available for the identification of epithelial cells and the epithelial cells as they are in well-differentiated CRCs and in cells of the hematopoietic system. A number of other proteins, normal colorectal tissue (see also Fig. 1A). This finding suggests including THY1, FSP1 (S100A4), Palladin 4Ig (PALLD), PDGF a strong exocrine effect of the MFs, as discussed by Yeung et al. receptor β, and Endosialin (CD 248), have been suggested

E2168 | www.pnas.org/cgi/doi/10.1073/pnas.1603534113 Hsia et al. Downloaded by guest on September 26, 2021 Table 2. Molecular markers for distinguishing among MFs, fibroblasts, aFs, and smooth muscle cells PNAS PLUS Cell type AOC3 NKX2-3 SHOX2 αSMA* Desmin Vimentin Smoothelin THY-1 CD90

MFs ++ − +, ± − + − + Fibroblasts −−++ ± − + − + aFs −− +++− + − + Vascular smooth muscle cells ++ − ++ − + −

Anti-αSMA stains MFs and fibroblasts heterogeneously. *+, ±: the majority of the MFs were stained by αSMA; ±: only a subset of fibroblasts were positive for αSMA.

previously as possible markers for MFs (36), but our mRNA- FOXL1 and so are a different cell type. This difference in ex- expression profiles for the corresponding genes do not suggest pression emphasizes the heterogeneity of fibroblastic cell types that these proteins are useful for distinguishing between MFs that may exist in both the small and large intestine. and aFs as we have defined them. At the mRNA level, the availability of a well-defined clear-cut There is a large body of literature on what have been called gene-expression profile for distinguishing these various cell types “cancer (or carcinoma) associated fibroblasts” (CAFs). Olumi will become very valuable for the analysis of complex mixtures of et al. (37) were the first to use a functionally based definition of cells, such as those found in fresh tumor tissue, using the now in- CAFs to distinguish them from conventional fibroblasts. The creasingly available techniques for single-cell mRNA-expression simplest conclusion from their work and later reports (38) is that analysis (52). We suggest that the markers we have described for the CAFs represent a form of activated MFs that have much distinguishing MFs from aFs will make an important contribution to increased expression of αSMA compared with fibroblasts in sorting out the definitions of the various fibroblast-related cell types, normal tissue. which are likely to be different in different tissues and which play Pericytes, which express αSMA, are described as fibroblast- significant roles in a wide variety of diseases in addition to cancer. like cells that surround endothelial blood vessels. They appear to be multipotential and in this respect are similar to the mesen- Materials and Methods chymal stem cells (MSCs) that are found in the bone marrow. Antibodies and Reagents. Chemicals were from Sigma-Aldrich. Antibodies included MSCs have been shown to have the potential to give rise to a two monoclonal antibodies, Mouse anti-AOC3 (clone TK8-14; 1:50; Santa Cruz Biotechnology) and (clone 393112; 1:250; R&D Systems), mouse anti–α-smooth variety of cell types, including adipogenic, osteogenic, and muscle actin (clone 1A4; 1:200; Sigma-Aldrich), anti-vimentin (clone 3B4; Dako), chondrogenic lineages and probably also MFs (39, 40). Pericytes CD31 mAb (1:50; developed in house, Department of Cellular Pathology Oxford also have been identified as a possible source of MFs (41). University Hospitals), and anti-MYH11 [clone EPR5336(B); 1:200; Abcam]. Mouse A variety of labeling techniques, including the use of male-to- anti-EpCAM (AUA1) and anti-MF (PR2D3) were raised in the W.F.B. laboratory. female bone marrow transplants in mice and humans, have been Anti-mouse Alexa Fluor 555 and Alexa Fluor 488 were from Vector Laboratories. used to show that MFs (and most probably pericytes also) arise All antibodies were used at 1:100 dilutions unless specifically noted otherwise. from cells in the bone marrow (42, 43). It thus seems that MFs may arise either directly from bone marrow MSCs or more lo- Primary Cells and Cell Lines. The CCD 18CO MF line was originally obtained cally from pericytes. from ATCC, and the LS174T CRC cell line was obtained from the originator of the cell line, B. H. Tom, Northwestern University Medical Center, Chicago. The Many different terms have been used to describe MF and fi- α SW1222 CRC cell line was a generous gift from Meenhard Herlyn, Wistar broblast-associated cell types in relation to SMA expression. In Institute, Philadelphia. Three human normal skin fibroblast and foreskin fi- addition to CAFs, pericytes, and MSCs, there are fibrocytes, in- broblast cultures were established in the W.F.B. laboratory (now the Cancer testinal subepithelial MFs (ISEMFs), and interstitial cells of Cajal and Immunogenetics Laboratory, Oxford, UK). All cell lines and primary (ICCs). The ICCs seem to be most clearly distinguishable from the cultures were cultured in complete DMEM supplemented with 10% (vol/vol) others, because they have been shown to be pacemakers for the FBS and 1% penicillin/streptomycin (Invitrogen). Cells were incubated at – gut (44). The term “fibrocyte” was originally used by Kaye et al. 37 °C in a humidified 10% CO2 environment and were grown to 50 80% (45) to describe the fibroblast-like cells they saw at the top of the confluence before the next passage or further experiments. Cell cultures “ were regularly tested for Mycoplasma contamination using the MycoAlert crypt, which they described as differentiated and functioning fi- Assay kit (Lonza). broblasts” that could not divide and produce collagen at the same time. More recently, the term has been used for a quite different Cell Storage and Retrieval. To prepare cells for vapor phase of liquid nitrogen type of cell that combines fibroblast or MF-like characteristics storage, MFs from 80% confluent tissue-culture flasks were detached using with hematopoietic properties, including the expression of CD45 trypsin and centrifuged at 250 × g for 5 min. Cell pellets then were resuspended (46). These cells may play an important role in immune responses in 1 mL of FBS containing 10% (vol/vol) DMSO, were transferred into sterile in the tumor microenvironment (47). “ISEMF” is a term used by cryogenic vials (Corning), and were placed on dry ice for 90 min before they Powell et al. (48) effectively to describe pericryptal MFs in the gut. were transferred into cryogenic tanks with liquid nitrogen at −150 °C for long- That MFs may be different in different tissues is emphasized term storage. To thaw the cells, frozen cryogenic vials were incubated in a 37 °C water bath for about 1 min, resuspended in 10 mL of serum-free medium, and by the lack of AOC3 staining in breast tissue and breast carci- then transferred into a 75-cm2 cell-culture flask for incubation at 37 °C. nomas. A distinction also must be made between cells that are activated and those that are not, as is clear from the difference Establishment of Primary Colon MF Cultures. Fresh tissues were from eight between fibroblasts and aFs and the activation of MFs by TGFβ independent patients operated on for primary colorectal tumors at the to produce αSMA. Fine definitions of fibroblasts have been Oxford University Hospitals. Project approval was obtained from the local described in the skin (49) and lung (50), and a similar complexity Research Ethics Board, National Health Service (NHS) National Research might well exist in the gut. While this paper was under review, Ethics Service (NRES) Committee South Central - Oxford C, and informed Aoki et al. (51) described a small, Foxl1-expressing subset of consent was obtained from each patient. MFs were isolated from tumor and subepithelial fibroblasts that appear to play an important role in nontumor regions of whole colon tissues as determined by gross examina- tion at the time of surgical excision and subsequent histological analysis. the maintenance of the villus structure in the mouse small in- Tissues were dissected and then digested with collagenase type IV (1 mg/mL; testine. Although these cells share some expression features with Worthington Biochemical Corporation) at 37 °C for 3 h in DMEM. The tissue MFs as we have defined them, such as high levels of FGF2, suspension was filtered through a 250-μM nylon mesh (Pierce Biotechnology WNT5a,andGREM1 expression, our MFs do not express Inc.). The filtered suspension was centrifuged at 250 × g for 5 min, the tissue MEDICAL SCIENCES

Hsia et al. PNAS | Published online March 28, 2016 | E2169 Downloaded by guest on September 26, 2021 pellet was resuspended in DMEM with 10% FBS, and the cells were cultured Flow Cytometry and FACS. CCD 18CO MFs, normal skin fibroblasts, and on standard plastic tissue-culture plates. The successful primary MF cultures SW1222 cells were assessed by flow cytometry on a CyAn ADP analyzer using were labeled myo1998 (normal), myo2020 (cancer), myo6024 (normal), the integrated Summit data collection software. Fluorescence-activated cell myo6544 (normal), myo6769 (normal), myo6769C (cancer, from the same sorting was performed using standard protocols. Cells were labeled with patient as myo6769), myo6539 normal), myo6550 (normal), myo6550C primary antibody mouse anti-AOC3 (TK8-14) and mouse PR2D3 followed by (cancer, from the same patient as myo6550), and myo7659 (normal). We APC (Allophycocyanin)-conjugated goat anti-mouse IgG secondary antibody used MFs passaged for only up to 10 passages for subsequent experiments to (BD Pharmingen). Cells were analyzed using a Dako Cytomation Cyan ma- avoid replicative senescence. There was no evidence of outgrowth of any chine or were sorted using a MoFlo cell sorter (Beckman Coulter). fibroblast-like cells other than the described isolated MF cultures. Nonenzymatic tissue extraction was carried out using a modification of the Gene-Expression Analysis. Total RNA from cells was extracted using the method described by Ashley et al. (53). Briefly, tissues were mechanically dis- RNeasy kit (Qiagen) according to the manufacturer’s instructions. All samples rupted in a proprietary nonenzymatic EDTA-based cell-dissociating solution were processed in accord with the Affymetrix protocol, and 2 μg of frag- (Sigma) with agitation with 4-mm, undrilled glass beads (Fisher Scientific) for mented and labeled cDNA was hybridized to the Affymetrix GeneChip U133 1 h. This process enabled cell dissociation without excessive tissue disruption. Plus 2 arrays. Partek Genomic Suite software was used to compare expres- sion profiles of the fibroblast and MF cultures. For genes with significant Immunocytochemistry and Immunohistochemistry. Archived paraffin-embedded differential expression, step-up correction for multiple testing was used cryostat sections were obtained from noncancer and cancer patients from (with a Benjamini and Hochberg step-up false discovery rate-corrected P Oxford University Hospitals, Oxford, UK, with the approval from the local re- value cutoff of 0.05). search ethics committee, NHS NRES Committee South Central - Oxford C. The The data reported in this article have been deposited in the Gene Ex- commercial tissue array was purchased from SuperBioChips Laboratories. pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession Immunohistochemistry and immunofluorescence were performed accord- no. GSE77474). ing to standard protocols. Paraffin-embedded sections were deparaffinized in Histo-Clear (National Diagnostics) twice for 3 min and were hydrated in RNA Isolation and RT-qPCR. TaqMan (Applied Biosystems) RT-qPCR gene- 100% ethanol, 100% Industrial Methylated Spirits (IMS), 70% IMS, and 35% expression assays were used to confirm gene expression. Total RNA was IMS for 2 min each. Slides were rinsed in PBS. Cryostat tissue sections were extracted from primary cultures using Qiagen RNeasy Spin Columns according fixed in cold acetone (−20 °C) for 10 min at room temperature. Antigen to the manufacturer’s instructions. RNA quality was determined by analysis retrieval was performed using Target Retrieval solution (pH 6.1) (Dako) at with a NanoDrop spectrophotometer (Thermo Fisher). All reverse transcription 100 °C for 20 min, followed by cooling to room temperature for 1 h. Slides reactions were prepared using the Life Technologies High Capacity cDNA were blocked at room temperature for 30 min using Invitrogen blocking Synthesis kit with random primers according to the manufacturer’sinstruc- reagent [1% (wt/vol) in PBS-Tween 0.05%]. Then slides were incubated at tions. Comparative real-time PCR was performed in triplicate. cDNA samples room temperature for 1 h with the appropriate primary antibody in were used as input for TaqMan qPCR using predesigned, manufacturer-vali- blocking solution. The secondary antibody used was HRP-conjugated goat dated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C anti-mouse or anti-rabbit (1:100; Invitrogen) labeled with a fluorochrome (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Alexa Fluor 488 Tyramide; Invitrogen). (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies. Immunofluorescence staining on cells was done in fresh unfixed condition for anti-AOC3 (TK8-14). Cells were washed with PBS and blocked with 10% Mass Spectrometry and Database Analysis. The tryptic-digested samples were FBS for 30 min and were incubated for 1 h with mouse anti-AOC3 mAb (TK8- analyzed by MALDI-TOF mass spectrometry, which was performed on an 14). Following several subsequent washes, cells were incubated for 1 h with Ultraflex TOF mass spectrometer (Bruker Daltonics) in the Central Proteomics the appropriate secondary antibody labeled with a fluorochrome (Alexa Facilities, Weatherall Institute of Molecular Medicine, Oxford, UK. In brief, the Fluor; Invitrogen). Nuclei were counterstained with DAPI (Dako) for 15 min spectra generated with a unique set of masses are called “peptide-mass fin- before microscopy. Images were obtained using a Zeiss Axioskop 2 Plus gerprints” (PMF). These can be matched with entries in protein databases microscope or a Zeiss confocal laser scanning microscope (Zeiss). using search engines (54). In this study, the SWISS-PORT database was used, and the Mascot search engine (Matrix Science, London) was used for matching Antigen Preparation. Protein samples were prepared from either fresh tissue sample data with this database. Mascot is a fully automated search engine lysates or cell-culture lysates. Fresh samples of normal colon were obtained with probability-based algorithms. It also indicates whether a score is signifi- from the Bio-Bank, John Radcliffe Hospital, Oxford, UK. Smooth muscle cant; significance is determined according to individual cases. The resulting samples were dissected from the muscularis layer of the colon tissue, finely mass spectra of trypsin peptide digests from all the gel spots were matched minced with scalpels, homogenized in lysis buffer [1% Nonidet P-40, 0.5% against theoretical trypsin peptide digests in the SWISS-PORT 50.8 protein nordeoxycholate, 150 mM sodium chloride, 50 mM Tris base (pH 8.3)] con- database using the Mascot search engine. The Mascot default significance taining a protease inhibitor mixture (Roche), and then processed with an threshold of P < 0.05 for assignments was used in the searches, and a minimum ultrasonic mixer for 10 min on ice. These lysates were filtered using 250-μm of two unique peptides was used as a criterion for a match. cell strainers (Thermo Scientific) and were centrifuged at 21,000 × g for 30 min, after which the supernatant was collected and the protein con- Statistical Methods. RT-qPCR–based expression levels are given as ΔCt values centration was determined using the Bio-Rad protein assay. Cell lysates were converted to actual numbers by appropriate factors of 2, using ubiquitin c prepared similarly. Cells were harvested from 10-cm tissue-culture dishes in (UBC) as the endogenous control. The significance of differences was complete medium after growth to 80% confluence. After a brief rinse with assessed by a t test on the replicate converted ΔCt values. Fold-change val- ice-cold PBS, the cells were scraped into tubes on ice in 100–400 μL lysis ues for RT-qPCR were calculated using the ΔΔCt method relative to the buffer (for native protein) or RIPA buffer (for denatured protein) and were average Ct across all samples. Because the fold-change for the control is left on ice for a further 30 min. Cleared whole-cell extracts were prepared by always defined as 1, we did not plot control data points in RT-qPCR figures; centrifugation at maximum speed (21,000 × g) for 30 min at 4 °C. The instead we show a dotted line at 1. Error bars are not shown for RT-qPCR cleared extracts were immunoprecipitated for 1 h on ice as described below. data, because the exponential transformation involved in calculating fold- change does not preserve the variance in the primary data. Because the ΔCt Immunoprecipitation and Western Blot Analysis. Tissue lysates and cell lysates is actually inversely proportional to the starting amount of mRNA, we con- were immunoprecipitated using Dynabeads protein G (Invitrogen) according to verted the ΔCt values by subtracting them from the highest ΔCt value the manufacturer’s instructions. Anti-AOC3 (TK8-14; Santa Cruz Biotechnology, obtained within any single experiment and converted the resulting values Inc.), PR2D3 (prepared in house), and anti-α tubulin mAb (Abcam) were used from a logarithmic to a linear scale. for immunoprecipitation and immunoblotting. After immunoprecipitation, Statistically significant differences are indicated by asterisks denoting the P proteins were Western blotted and probed with monoclonal antibody as de- values based on t tests for differences between converted ΔCt values. scribed above. Blots were blocked for 1 h at room temperature with Tris- buffered saline (TBS) containing 0.1% Tween 20 and 1% BSA. The membranes Transfections. MFs were forward transfected by siRNA (OriGene) in 24-well were incubated at room temperature for 1 h with appropriate antibodies in plates at a final concentration of 20 nM using Oligofectamine transfection TBS containing 0.1% Tween 20 and 1% BSA buffer. The blots then were in- reagent (Life Technologies) following the manufacturer’s suggestions. cubated for 1 h at room temperature with secondary antibody and washed in TBS containing 0.1% Tween 20. Immunoreactive bands were revealed using an ACKNOWLEDGMENTS. We thank staff at the Oxford BioBank and donor pa- ECL Kit (GE Healthcare) according to the manufacturer’sinstructions. tients for the samples, Kevin Clark for his FACS expertise, Dr. Matteo Morotti

E2170 | www.pnas.org/cgi/doi/10.1073/pnas.1603534113 Hsia et al. Downloaded by guest on September 26, 2021 for help with confocal microscopy, and Dr. Trevor Yeung for sharing the tissue Framework Programme (FP7/2007–2013) under Grant Agreement 278204 PNAS PLUS sections of lymph node metastases of colorectal cancer. This research was (Cell-o-matic), by funds from the Department of Oncology, University of Ox- supported by funding from the European Commission under the Seventh ford, and by a Clarendon Award from the University of Oxford (to L.-t.H.).

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