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Validation of COL11A1/procollagen 11A1 expression in TGF-β1-activated immortalised human mesenchymal cells and in stromal cells of human colon adenocarcinoma

  • José A Galván1, 2, 6,
  • Jorge García-Martínez1, 2,
  • Fernando Vázquez-Villa2,
  • Marcos García-Ocaña2, 3,
  • Carmen García-Pravia2, 4,
  • Primitiva Menéndez-Rodríguez4,
  • Carmen González-del Rey4,
  • Luis Barneo-Serra1, 2 and
  • Juan R de los Toyos2, 5Email author
BMC Cancer201414:867

DOI: 10.1186/1471-2407-14-867

Received: 29 April 2014

Accepted: 12 November 2014

Published: 23 November 2014

Abstract

Background

The human COL11A1 gene has been shown to be up-regulated in stromal cells of colorectal tumours, but, so far, the immunodetection of procollagen 11A1, the primary protein product of COL11A1, has not been studied in detail in human colon adenocarcinomas. Some cancer-associated stromal cells seem to be derived from bone marrow mesenchymal cells; the expression of the COL11A1 gene and the parallel immunodetection of procollagen 11A1 have not been evaluated in these latter cells, either.

Methods

We used quantitative RT-PCR and/or immunocytochemistry to study the expression of DES/desmin, VIM/vimentin, ACTA2/αSMA (alpha smooth muscle actin) and COL11A1/procollagen 11A1 in HCT 116 human colorectal adenocarcinoma cells, in immortalised human bone marrow mesenchymal cells and in human colon adenocarcinoma-derived cultured stromal cells. The immunodetection of procollagen 11A1 was performed with the new recently described DMTX1/1E8.33 mouse monoclonal antibody. Human colon adenocarcinomas and non-malignant colon tissues were evaluated by immunohistochemistry as well. Statistical associations were sought between anti-procollagen 11A1 immunoscoring and patient clinicopathological features.

Results

Procollagen 11A1 was immunodetected in human bone marrow mesenchymal cells and in human colon adenocarcinoma-associated spindle-shaped stromal cells but not in colon epithelial or stromal cells of the normal colon. This immunodetection paralleled, in both kinds of cells, that of the other mesenchymal-related biomarkers studied: vimentin and alpha smooth muscle actin, but not desmin. Thus, procollagen 11A1+ adenocarcinoma-associated stromal cells are similar to “activated myofibroblasts”. In the series of human colon adenocarcinomas here studied, a high procollagen 11A1 expression was associated with nodal involvement (p = 0.05), the development of distant metastases (p = 0.017), and advanced Dukes stages (p = 0.047).

Conclusion

The immunodetection of procollagen 11A1 in cancer-associated stromal cells could be a useful biomarker for human colon adenocarcinoma characterisation.

Keywords

Procollagen 11A1 Human bone marrow mesenchymal cells Cancer-associated stromal cells Human colon adenocarcinoma

Background

A wealth of studies have reported that the COL11A1 human gene, coding for the α1 chain of procollagen and mature collagen of type XI, which is an extracellular minor fibrillar collagen, is up-regulated in some human tumours and in mesenchymal-derived tumour cell lines [132], as well as in mesenchymal stem cells and osteoblasts [3335].

Collagen polypeptides are synthesized as procollagens, with the N- and C-propeptides at the ends of the prototypical collagen triple helix. Upon secretion, the propeptides are excised and then the mature collagen molecules assemble in fibrils.

In tumours, the expression of the COL11A1 gene is currently associated to a fibroblast-like stromal phenotype [12, 19] but the origin and nature of the cells which produce procollagen and collagen 11A1 remain controversial to some extent [26].

The so-called cancer-associated stromal cells, resulting from the desmoplastic reaction which accompanies the development of human invasive carcinomas, comprise cells of different types, and are at least in part derived from mesenchymal progenitors and local resident cells. It is also well-established that TGF-β1 in cancer promotes the activation of cancer-associated stromal cells [36].

For the present study, we set out to verify the expression of the COL11A1 gene, by quantitative RT-PCR in TGF-β1-exposed epithelial human colorectal HCT 116 cells and Immortalised Human Bone Marrow Mesenchymal Cells (hTERT-HMCs); and the expression of procollagen 11A1 by immunocytochemistry (ICC)/immunohistochemistry (IHC), using the DMTX1/1E8.33 monoclonal antibody (mAb) [37], on those cell cultures as well as on biopsies of human colon adenocarcinomas. Concurrently, we studied the expression of DES/desmin, VIM/vimentin and ACTA2/αSMA (alpha smooth muscle actin) as mesenchymal (myofibroblast)/stromal markers.

Within the N-propeptide of human procollagen 11A1, it is the so-called “variable region”, the most divergent amino acid sequence stretch among different procollagens. The DMTX1/1E8.33 mAb recognises an epitope in the YNYGTMESYQTEAPR amino acid stretch within the variable region of human procollagen 11A1 [37].

Methods

Cell cultures

Ascorbate is a well-known inducer of the synthesis of some collagens [38, 39]; thus, to favour the expression of procollagen 11A1, cells were habitually cultured with this supplement. Since TGF-β1 levels are increased in the serum of patients with invasive carcinomas [40], we chose to analyse its effects after continued and protracted exposure of cell cultures to this cytokine.

The human colorectal adenocarcinoma HCT 116 (CCL-247) cell line, derived from a primary tumour, was obtained from the American Type Culture Collection (ATCC) and cultured in DMEM, supplemented with 1 mM sodium pyruvate (Biochrom), 2 mM L-glutamine (Biochrom), 1X non-essential amino acids (Biochrom), 10% foetal bovine serum (Biochrom), and ascorbate 2-phosphate (37.5 μg/ml) (Wako Chemicals).

Immortalised Human Bone Marrow Mesenchymal Cells-hTERT (hTERT-HMCs) were obtained from Applied Biological Materials (ABM) Inc., Richmond, BC, Canada (Cat. No. T0523), and grown in T25 ECM-coated flasks in Prigrow II medium (ABM, Cat. No. TM002), with the addition of 10% foetal bovine serum, 1 μM hydrocortisone (Sigma) and ascorbate 2-phosphate (37.5 μg/ml) (Wako Chemicals).

For TGF-β1 induction, media were further supplemented with 10 ng/ml of recombinant TGF-β1 (Peprotech). The medium was replaced every 3–4 days and the cells were cultured for at least 15 days.

All the cultures were carried out in a humidified atmosphere of 5% CO2 in air at 37°C.

Culture passages and cell collections were done with trypsin/EDTA 0.05%/0.02% (Biochrom). Three different harvests from each cell culture type were obtained; for Q-RT-PCR, fresh cell pellets were kept at -80°C.

Colon adenocarcinoma stromal cells isolation and culture

Fresh human tissue samples were procured after written informed consent of the patients and approval by the Principality of Asturias Ethics Committee of Clinical Research, Oviedo, Spain.

Short-term cultures of colon adenocarcinoma stromal cells were carried out, as previously described [41], from samples of tumoral sites, avoiding necrotic areas. A sample from the operating theatre was directly transferred to a sterile tube containing DMEM culture medium (Gibco, Invitrogen), supplemented with vancomycin (40 μg/ml) and amikacin (40 μg/ml) (Normon Laboratories, Madrid, Spain), and stored for 24 hours at 4°C.

After three washings with phosphate buffer saline (PBS), the sample was cut into several small fragments. These fragments were first incubated with collagenases (Type I 2 mg/ml, Sigma) for 1.5 hours and then centrifuged to eliminate supernatant; subsequently, the pellet was subjected to a second incubation in trypsin/EDTA for 30 min. After digestion, the cells were again collected in a pellet, resuspended in DMEM culture medium, supplemented with 10% foetal bovine serum, L-glutamine and penicillin/streptomycin, transferred to T-flasks and cultivated in 5% CO2 at 37°C.

Stromal cell cultures were stable up to 5–6 passages before going into senescence. The purity of these stromal cell cultures was assessed by morphology and by immunostaining for vimentin.

Q-RT-PCR

For normalisation of data, quantitative RT-PCR of DES, VIM, ACTA2 and COL11A1 mRNA, and PUM1, RPL10, and GAPDH mRNA was performed using the BioMark™ HD System of the Fluidigm technology (Fluidigm, San Francisco, USA).

Briefly, total RNA was isolated from pooled cell cultures, kept at -80°C, with the RNeasy Mini kit (Qiagen). cDNA was synthesized from 100 ng of RNA from each sample, using the AffinityScript Multiple Temperature cDNA Synthesis kit (Agilent Technologies). A pre-amplification was carried out, applying the QIAGEN® Multiplex PCR Kit and the pool of all the 20x TaqMan® Gene Expression Assays. Real time Q-PCR reactions were carried out with the TaqMan Universal PCR Master Mix kit (Applied Biosystems). Further details, according to Applied Biosystems’ recommendations, are in Table 1.
Table 1

Assays selected and PCR conditions for Q-RT-PCR of mRNA analysis

Gene

Assay ID

COL11A1

Hs01097664_m1

GAPDH

Hs02758991_g1

PUM1

Hs004472881_m1

RPL10

Hs00749196_s1

DES

Hs00157258_m1

ACTA2

Hs00426835_g1

VIM

Hs00185584_m1

PCR conditions were: 50°C – 2 min; 95°C – 10 min; and 40 amplification cycles:

95°C – 15 sec and 60°C – 1 min.

Data were normalised by applying the ΔCt method, after PCR efficiency corrections. These analyses were performed by Progenika Biopharma, S.A., Derio, Spain.

Three independent samples (n =3) of different cell harvests of each cell type were studied. Data are presented as mean and SEM. For each gene, differences between cell culture expressions were analysed by a two-tailed unpaired t-test. A P value <0.01 was considered statistically significant.

Immunohistochemistry (IHC)

For immunohistochemical techniques, a cohort of 51 patients with colon adenocarcinoma and 6 patients diagnosed with incipient bowel infarction were collected from the Archive of the Pathology Department, Asturias Central University Hospital, with the Principality of Asturias Ethics Committee of Clinical Research, Oviedo, Spain, approval for guidelines on ethical procedures. The samples had been fixed with 10% formaldehyde for 24 h and embedded in paraffin.

Three-μm thick tissue sections were stained with Hematoxylin and Eosin (H&E) for histological examination. Antigen retrieval was performed by heating in PTLink (DakoCytomation, Denmark) in buffer solution at high pH for 20 minutes. Endogenous peroxidase activity was blocked with Peroxidase Blocking Reagent (DakoCytomation, Denmark) for 5 minutes. After that, samples were first incubated at 37°C with the primary antibodies described in Table 2. Subsequently, the EnVision system (HRP Flex) (DakoCytomation) was applied for 30 minutes at room temperature. Then, the samples were stained with DAB (3-3′-Diaminobenzidine) (DakoCytomation, Denmark) for 10 minutes, counterstained for 10 minutes with hematoxylin (DakoCytomation), dehydrated and mounted in Entellan® (Merck, Germany). Finally, the stained tissue sections were studied and photographed (40× objective) under a light microscope (Nikon - Eclipse 80i).
Table 2

Antibodies used in IHC/ICC analysis

Primary antibodies (species)

Clone

Commercial reference

Dilution

Incubation time (min)

Procollagen 11A1 (mAb)

1E8.33

DMTX1/Oncomatrix, Spain

1:400

30

Desmin (mAb)

D33

Dako, Denmark

R-t-U

20

α-SMA (mAb)

1A4

Dako, Denmark

R-t-U

20

Vimentin (pAb)

C-20

Santa Cruz Biotech, Germany

1:600

10

mAb: Mouse monoclonal antibody.

pAb: Rabbit polyclonal antibodies.

R-t-U: Ready-to-Use.

Immunocytochemistry (ICC)

Cells were fixed in 10% formaldehyde for 10 minutes in the chamber slide (BD Falcon™, ref. 354114). Endogenous peroxidase activity was blocked with Peroxidase Blocking Reagent (DakoCytomation, Denmark) for 5 minutes. Permeabilization step was performed adding wash buffer 1× (DakoCytomation, Denmark) which contains 0.05 mol/L Tris/HCl, 0.15 mol/L NaCl, 0.05% Tween-20 [41]. Primary antibodies were applied, as described in Table 2, at room temperature. After that, slides were incubated with the EnVision system (HRP Flex) for 10 minutes at room temperature. Then, the samples were visualised with DAB for 5 minutes, and counterstained with hematoxylin for 5 minutes. Finally, the stained slides were dehydrated, mounted, studied and photographed as above.

Immunohistochemistry assessment

Specimens were assessed by three observers (JAG, CGP and CGR), following these criteria: procollagen 11A1 immunostaining was evaluated according to the cytoplasmatic signal as the product of two parameters: extent of immunoreactivity, which was evaluated in the most densely stained area (hot spot) under the 20× objective and scored on a 0–3 scale, according to the proportion of positive fibroblasts: (0) 0%; (1) <10%; (2) 10-50% and (3) >50%; and granularity in the cytoplasm, evaluated as dispersed vs. confluent (1 and 2 points, respectively), with the 40× objective. Immunoscore values ranged from 0 to 6. Adjacent non-malignant tissue was used as a negative control.

Statistical analysis

The experimental results were tested for significance employing the χ2 test (with Yates’ correction, when appropriate). The statistical analysis was carried out with the IBM SPSS 20.0 software package (SPSS, Inc., Chicago, IL). All tests were two-sided and p < 0.05 values were considered statistically significant.

Results

Study of human cell cultures

So far, we are not aware of any human colorectal cell line in which the expression of the COL11A1 gene has been reported, but we had observed that primary cultures of bone marrow-derived human mesenchymal cells, expressed COL11A1/procollagen 11A1, especially after long exposure (≥15 days) to TGF-β1 (data not shown).

We have presently studied the well-known epithelial human colorectal HCT 116 cell line as a negative control for the expression of the mesenchymal DES, VIM, ACTA2 and COL11A1 genes in relation to their expression by cultured immortalised hTERT-HMCs. The expression of the DES, VIM, ACTA2 and COL11A1 genes was analysed by Q-RT-PCR; the immunodetection of desmin, vimentin, αSMA and procollagen 11A1 was performed by ICC.

According to the normalised Q-RT-PCR data we have obtained (Figure 1), TGF-β1-activated hTERT-HMCs did not express DES mRNA, but noticeable amounts of VIM, ACTA2 and COL11A1 mRNA. The corresponding protein expression was confirmed by ICC (Figure 2). An average of 20% of these cells in cultures exposed to TGF-β1 showed a granular pattern of intracytoplasmic immunostaining of procollagen 11A1. None of these markers was expressed by the HCT 116 cells, but certain levels of DES.
Figure 1

Q-RT-PCR data of DES , VIM , ACTA2 and COL11A1 mRNA expression in cell cultures of the HCT 116 cell line and in immortalised hTERT-HMCs, both after long exposure to ascorbate 2-phosphate and TGF-β1. The data were normalised in relation to PUM1, RPL10, and GAPDH mRNA expression (n =3; mean ± SEM; *P <0.05, **P <0.01).

Figure 2

Representative immunostaining of cultured immortalised hTERT-HMCs after long exposure to ascorbate 2-phosphate and TGF-β1. A) Procollagen 11A1 B) Desmin, C) αSMA and D) Vimentin. Scale bar 50 μm (400×).

Examination of human tissues

Fifty one paraffin-embedded archival samples of human colon adenocarcinomas were examined by IHC with the DMTX1/1E8.33 mAb; all cases presented adjacent non-malignant tissue as control. Six cases of incipient bowel infarction were similarly studied. Table 3 shows the characteristics of patients and samples, and their anti-procollagen 11A1 immunoscores, evaluated as described in Methods; as shown, these immunoscores ranged from 0 to 6. A more detailed description of these characteristics is in Additional file 1. In three of these 51 diagnosed adenocarcinoma cases, no procollagen 11A1 staining could be detected, in spite of extensive re-examination. Procollagen 11A1 was neither immunodetected in the adjacent non-malignant tissues nor in the infarction cases.Figure 3 shows representative procollagen 11A1 immunostaining patterns (panels A, B, C and D); only a granular cytoplasmic staining of peritumoral spindle-shaped fibroblast-like stromal cells was observed. This granularity was either dispersed, with a few granules in the cytoplasm of stromal cells (panel G) or frankly confluent (panel H). No staining was observed on specimens of bowel ischemia (panel E) or on non-malignant tissues (panel F).Figure 4 shows a representative immunostaining of an adenocarcinoma specimen with a procollagen 11A1 immunoscore of 6. As shown on panel A, only peritumoral stromal cells were stained with the anti-procollagen 11A1 mAb; besides, these cells seemed to be positive for αSMA (C) and vimentin (D), but negative for desmin (B). This immunostaining pattern was reproduced (panels E, F, G and H) in stromal cells cultured from fresh specimens of the same patient.
Table 3

Patient characteristics (N = 51)*

  

Frecuency N

(%)

Gender

Female

19

37.3

 

Male

32

62.7

Age (years)

Median (range)

70

(31–85)

Tumor size (cm)

Median (range)

3.7

(0.5 - 11)

Localization

Ascending colon

21

41.2

 

Descending colon

8

15.7

 

Sigmoid

22

43.1

Differentiation

Well differentiated

19

37.3

 

Moderately differentiated

28

54,9

 

Poorly differentiated

4

7.8

T

T1

3

5,9

 

T2

7

13.7

 

T3

28

54.9

 

T4

13

25.5

N

pN0

25

49.0

 

pN1

26

51.0

M

M0

39

76.5

 

M1

12

23.5

TNM staging

I

8

15,7

 

IIA

12

23,5

 

IIB

4

7,8

 

IIIA

1

2,0

 

IIIB

9

17,6

 

IIIC

5

9,8

 

IV

12

23,5

Dukes staging

A

8

15,7

 

B

16

31,4

 

C

15

29,4

 

D

12

23,5

Anti-procollagen 11A1 immunostaining by score

0

3

5.9

 

1

12

23.5

 

2

13

25.5

 

3

4

7.8

 

4

6

11.8

 

6

13

25.5

Anti-procollagen 11A1 immunostaining

Low (≤2)

28

54.9

(Median =2)

High (>2)

23

45.1

(*) Patients diagnosed with colon adenocarcinoma.

Patients diagnosed with ischemia were excluded.

Figure 3

Representative procollagen 11A1 immunostaining in colon adenocarcinoma. A) Score 1, B) Score 2; C) Score 4; D) Score 6. Arrow heads point to stained peritumoral stromal cells. E) Bowel ischemia; F) Non-malignant tissue. Scale bar 50 μm (400X). G) Dispersed granularity and H) Confluent granularity. Scale bar 20 μm (1000×).

Figure 4

Representative immunostaining of a colon adenocarcinoma: A) Procollagen 11A1 (immunoscore 6), B) Desmin, C) αSMA and D) Vimentin (these images were taken from the same area of serial sections; and of cultured stromal cells from the same case: E) Procollagen 11A1, F) Desmin, G) αSMA and H) Vimentin. Scale bar 50 μm (400×).

Association between procollagen 11A1 expression and clinicopathological features

For statistical purposes, some variables (age at diagnosis, tumour size, anti-procollagen 11A1 immunostaining) were divided into 2 groups, taking the median score value as a cut-off point (Table 4). Patients diagnosed with ischemia were excluded from this analysis.
Table 4

Association between procollagen 11A1 expression and clinicopathological features

 

Anti-procollagen 11A1 immunostaining (Median =2)

  

Low (≤2)

High (>2)

p

Age (Median 70 years)

≤ 70 years

13

14

0.304

> 70 years

15

9

Gender

Female

18

14

0.802

Male

10

9

Localization

Ascending colon

9

12

0.260

Descending colon

6

2

Sigmoid

13

9

0.200

Tumor size (Median = 3.7 cm)

Small ≤3.7 cm

12

14

Large >3.7 cm

16

9

0.370

Differentiation

Well differentiated

12

7

Moderately differentiated

15

13

 

Poorly differentiated

1

3

 

T

T1-T2

8

2

0.075

T3-T4

20

21

 

N

Absent

17

8

 

Present

11

15

0.059

M

Absent

25

14

 

Present

3

9

0.017

Stage grouping

I

7

1

0.141

IIA

7

5

 

IIB

2

2

 

IIIA

1

0

 

IIIB

6

3

 

IIIC

2

3

 

IV

3

9

 

Dukes staging

A

7

1

 

B

9

7

 

C

9

6

0.047

D

3

9

 

9/12 patients that had developed distant metastases at diagnosis and 15/27 patients with advanced Dukes stages were associated with high procollagen 11A1 expression (p = 0.017 and p = 0.047, respectively). The same can be observed for 15/26 patients with nodes affected, however, only with a trend towards significance (p = 0.059).

Discussion

We have presently shown, extending our previous observations [42], that procollagen 11A1, as a protein expression product of the COL11A1 gene, is immunodetected in stromal cells of human colon adenocarcinoma. By contrast, and contrary to a previous report [15], we have never observed immunodetection of procollagen 11A1 in epithelial cells of normal colon tissue or colon adenocarcinoma with the DMTX1/1E8.33 mAb. This immunodetection was observed in 48 of the 51 cases studied. Three cases (5.9%), which were classified under the same criteria as the rest of the cases examined as conventional adenocarcinomas with desmoplastic reaction, did not stain; so far, we have not identified in them any characteristics to which this negative immunostaining could be associated. Except for the report of Fischer et al. [1] who did not find either the expression of COL11A in 5 out of 15 (33.3%) colonic carcinomas analysed, we are not aware of any more studies reporting the percentage of colon adenocarcinomas expressing the COL11A1 gene; this aspect should be studied in detail.

We have very recently reported that procollagen 11A1+ cancer-associated stromal cells of pancreatic ductal adenocarcinoma co-express αSMA, and/or vimentin, and/or desmin in different proportions [41]. We have now confirmed, by Q-RT-PCR and IHC/ICC, the stromal expression of human procollagen 11A1 in colon adenocarcinoma. Although not formally proven, procollagen 11A1+ colon adenocarcinoma stromal cells, being spindle-shaped, seem to simultaneously express alpha smooth muscle actin and vimentin, but no desmin; these traits confer to them a myofibroblast-like phenotype rather than a pericyte one [43]. While in the desmoplastic component of hepatocellular carcinomas and pancreatic ductal adenocarcinomas there is a significant contribution of desmin + stellate cells, this is not the case in colon adenocarcinomas. As normal resident intestinal myofibroblasts are not immunoreactive to the DMTX1/1E8.33 mAb, these procollagen 11A1+ desmin- colon adenocarcinoma stromal cells could be a type of “activated myofibroblasts”.

We have also shown that a fraction of cultured immortalised HMCs, after long exposure to TGF-β1, exhibit a very similar phenotype to the described above for cancer-associated stromal cells. It is intriguing that only as much as 20% of these cultured cells express procollagen 11A1; this aspect warrants further analysis as well as the global genotype and phenotype of procollagen 11A1+ cells.

It has been reported that human bone marrow-derived mesenchymal cells may differentiate in vitro to fibroblast/myofibroblast-like cells under certain conditions, such as coculture with human colon carcinoma cells and TGF-β1, or prolonged exposure to conditioned medium from MDA-MB-231 breast cancer cells; these fibroblast/myofibroblast-like cells are able to promote tumour growth both in vitro and in vivo[36, 4447]. As the phenotype of procollagen 11A1+ “myofibroblasts” from colon adenocarcinoma resembles that of cultured TGF-β1-activated human bone marrow mesenchymal cells, all these observations add support to the tenet that at least some cancer-associated stromal cells, such as the procollagen 11A1+ ones, could be bone marrow-derived mesenchymal cells. Altogether, we may suggest that procollagen 11A1 could be expressed by a more specialized subpopulation among “activated myofibroblasts”.

Halsted et al.[14] reported the cytoplasmic, stromal and vascular immunostaining of both normal and malignant human breast tissues with polyclonal antisera to specific regions of N-terminal domains of human procollagen 11A1. Vargas et al. [30] immunodetected collagen 11A1 in the normal epithelium of human breast and Wu et al.[31] performed it in some human ovarian cancer cell lines, after applying another antibody preparation. Moreover, rather contradictory observations have been reported in relation to the kind of cells in which COL11A1 mRNA has been detected. While in situ hybridization studies have spotted its detection only in stromal cells [1, 17], another study, based on differentially expressed gene analyses by GeneChip hybridization, has pointed to the over-expression of COL11A1 mRNA in tumour epithelia [13]; very recently, Cheon et al.[48] have reported, also through in situ hybridization and immunohistochemistry with the DMTX1/1E8.33 mAb of serous ovarian cancer, that “COL11A1 expression was confined to intra/peritumoral stromal cells and rare foci of tumor epithelial cells”.

In our experience, the immunodetection of procollagen 11A1 with the DMTX1/1E8.33 mAb has never been observed in normal epithelial, vascular or stromal cells but in cancer-associated stromal cells; immunochemistry discrepancies between our observations and those above mentioned may be attributed to the different fine specificity of the applied antibody preparations. Besides this, transcription profiling studies of human colon biopsies obtained from active and inactive areas of ulcerative colitis and Crohn’s disease, compared with samples from infectious colitis and healthy controls, have shown that there are no differences in the expression levels of the COL11A1 gene between any of the above referred to conditions [4953]. COL11A1/procollagen 11A1 expression is mostly absent in benign inflammatory processes such as breast sclerosing adenosis [16, 54], chronic pancreatitis [41], and diverticulitis (our own observations; data not shown), and is rather low in familial adenomatosis polyposis adenomas [1, 2]. Thus, the in vivo up-regulation of the COL11A1 gene may be considered as a biomarker of cancer-associated stromal cells.

In this study, high procollagen 11A1 immunostaining was associated with clinicopathological variables such as lymph node involvement, advanced Dukes stages and presence of distant metastases. These results go according to the role of COL11A1 in promoting carcinoma aggressiveness and progression [11, 17, 20, 30, 31, 48, 5557].

Conclusions

Based on its high specificity, our observations stress once more the usefulness of the DMTX1/1E8.33 mAb for cancer research, and the clinical significance of procollagen 11A1 as a very valuable biomarker to characterise cancer-associated stromal cells and to evaluate human colon adenocarcinomas.

Abbreviations

HMCs: 

Human mesenchymal cells

hTERT-HMCs: 

Immortalised hTERT- human bone marrow mesenchymal cells

ICC: 

Immunocytochemistry

IHC: 

Immunohistochemistry

mAb: 

Monoclonal antibody

pAb: 

Polyclonal antibodies

H&E: 

Hematoxylin and eosin

PBS: 

Phosphate-buffered saline

DAB: 

3-3′-Diaminobenzidine.

Declarations

Acknowledgements

The authors thank Inti Zlobec for the critical reading of the manuscript and helpful comments. The excellent technical assistance of Laura Suárez-Fernández is greatly acknowledged.

This research has been co-financed by European Union ERDF Funds; by the INNPACTO-ONCOPAN IPT-010000-2010-31 Project; by the FISS-09-PS09/01911 Project, Ministry of Science and Innovation, Spain; by the FC-11-PC10-23, FICYT Project, Axe 1 of the 2007–2013 ERDF Operational Framework Programme of the Principality of Asturias, Spain; and by Oncomatrix, S.L. Derio, Spain.

Authors’ Affiliations

(1)
Surgery Department, School of Medicine and Health Sciences, University of Oviedo
(2)
Oncology University Institute of the Principality of Asturias (IUOPA)
(3)
Preparative Biotechnology Unit, Technical-Scientific Services, University of Oviedo
(4)
Pathological Anatomy Service, Asturias Central University Hospital (HUCA)
(5)
Immunology Department, School of Medicine and Health Sciences, University of Oviedo
(6)
Translational Research Unit (TRU), Institute of Pathology, University of Bern

References

  1. Fischer H, Stenling R, Rubio C, Lindblom A: Colorectal carcinogenesis is associated with stromal expression of COL11A1 and COL5A2. Carcinogenesis. 2001, 22: 875-878. 10.1093/carcin/22.6.875. doi:10.1093/carcin/22.6.875View ArticlePubMedGoogle Scholar
  2. Fischer H, Salahshor S, Stenling R, Björk J, Lindmark G, Iselius L, Rubio C, Lindblom A: COL11A1 in FAP polyps and in sporadic colorectal tumors. BMC Cancer. 2001, 1: 17-10.1186/1471-2407-1-17. [http://www.biomedcentral.com/1471-2407/1/17]View ArticlePubMedPubMed CentralGoogle Scholar
  3. Wang KK, Liu N, Radulovich N, Wigle DA, Johnston MR, Shepherd FA, Minden MD, Tsao MS: Novel candidate tumor marker genes for lung adenocarcinoma. Oncogene. 2002, 21: 7598-7604. 10.1038/sj.onc.1205953.View ArticlePubMedGoogle Scholar
  4. Xu SH, Qian LJ, Mou HZ, Zhu CH, Zhou XM, Liu XL, Chen Y, Bao WY: Difference of gene expression profiles between esophageal carcinoma and its pericancerous epithelium by gene chip. World J Gastroenterol. 2003, 9: 417-422.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Sok JC, Kuriakose MA, Mahajan VB, Pearlman AN, DeLacure MD, Chen FA: Tissue-specific gene expression of head and neck squamous cell carcinoma in vivo by complementary DNA microarray analysis. Arch Otolaryngol Head Neck Surg. 2003, 129: 760-770. 10.1001/archotol.129.7.760.View ArticlePubMedGoogle Scholar
  6. Schmalbach CE, Chepeha DB, Giordano TJ, Rubin MA, Teknos TN, Bradford CR, Wolf GT, Kuick R, Misek DE, Trask DK, Hanash S: Molecular profiling and the identification of genes associated with metastatic oral cavity/pharynx squamous cell carcinoma. Arch Otolaryngol Head Neck Surg. 2004, 130: 295-302. 10.1001/archotol.130.3.295.View ArticlePubMedGoogle Scholar
  7. Croner RS, Foertsch T, Brueckl WM, Guenther K, Siebenhaar R, Stremmel C, Matzel KE, Papadopoulos T, Kirchner T, Behrens J, Klein-Hitpass L, Stuerzl M, Hohenberger W, Reingruber B: Common denominator genes that distinguish colorectal carcinoma from normal mucosa. Int J Colorectal Dis. 2005, 20: 353-362. 10.1007/s00384-004-0664-7.View ArticlePubMedGoogle Scholar
  8. Barneo L, del Amo J, García-Pravia C, de los Toyos JR, Pérez-Basterrechea M, González-Pinto I, Vazquez L, Miyar A, Simón L: Identification of specific genes by microarrays, validation and use of polyclonal antibodies in pancreatic cancer: preliminary results. 41st Congress of the European Society for Surgical Research-ESSR 2006. Edited by: Vollmar B. 2006, Bologna, Italy: Medimond, International Proceedings, 27-35.Google Scholar
  9. del Amo-Iribarren J: PhD thesis. Identificación de marcadores para diagnóstico diferencial y potenciales dianas terapéuticas en adenocarcinoma ductal de páncreas mediante herramientas genómicas. 2006, Universidad del País Vasco, Genetics, Physical Anthropology and Animal Physiology DepartmentGoogle Scholar
  10. Chong IW, Chang MY, Chang HC, Yu YP, Sheu CC, Tsai JR, Hung JY, Chou SH, Tsai MS, Hwang JJ, Lin SR: Great potential of a panel of multiple hMTH1, SPD, ITGA11 and COL11A1 markers for diagnosis of patients with non-small cell lung cancer. Oncol Rep. 2006, 16: 981-988.PubMedGoogle Scholar
  11. Vecchi M, Nuciforo P, Romagnoli S, Confalonieri S, Pellegrini C, Serio G, Quarto M, Capra M, Roviaro GC, Contessini Avesani E, Corsi C, Coggi G, Di Fiore PP, Bosari S: Gene expression of early and advanced gastric cancer. Oncogene. 2007, 26: 4284-4294. 10.1038/sj.onc.1210208.View ArticlePubMedGoogle Scholar
  12. Pilarsky C, Ammerpohl O, Sipos B, Dahl E, Hartmann A, Wellmann A, Braunschweig T, Löhr M, Jesenofsky R, Friess H, Wente MN, Kristiansen G, Jahnke B, Denz A, Rückert F, Schackert HK, Klöppel G, Kalthoff H, Saeger HD, Grützmann R: Activation of Wnt signalling in stroma from pancreatic cancer identified by gene expression profiling. J Cell Mol Med. 2008, 12: 2823-2835. 10.1111/j.1582-4934.2008.00289.x.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Badea L, Herlea V, Dima SO, Dumitrascu T, Popescu I: Combined gene expression analysis of whole-tissue and microdissected pancreatic ductal adenocarcinoma identifies genes specifically overexpressed in tumor epithelia. Hepatogastroenterology. 2008, 55: 2016-2027.PubMedGoogle Scholar
  14. Halsted KC, Bowen KB, Bond L, Luman SE, Jorcyk CL, Fyffe WE, Kronz JD, Oxford JT: Collagen alpha1(XI) in normal and malignant breast tissue. Mod Pathol. 2008, 21: 1246-1254. 10.1038/modpathol.2008.129.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Bowen KB, Reimers AP, Luman S, Kronz JD, Fyffe WE, Oxford JT: Immunohistochemical localization of collagen type XI alpha1 and alpha2 chains in human colon tissue. J Histochem Cytochem. 2008, 56: 275-283.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Fuentes-Martínez N: PhD thesis. Colágeno 11: Nuevo marcador en el cáncer de mama. 2009, Universidad de Oviedo, Surgery and Medical Surgical Specialities DepartmentGoogle Scholar
  17. Zhao Y, Zhou T, Li A, Yao H, He F, Wang L, Si J: A potential role of collagens expression in distinguishing between premalignant and malignant lesions in stomach. Anat Rec. 2009, 292: 692-700. 10.1002/ar.20874.View ArticleGoogle Scholar
  18. An JH, Lee SY, Jeon JY, Cho KG, Kim SU, Lee MA: Identification of gliotropic factors that induce human stem cell migration to malignant tumor. J Proteome Res. 2009, 8: 2873-2881. 10.1021/pr900020q.View ArticlePubMedGoogle Scholar
  19. Erkan M, Weis N, Pan Z, Schwager C, Samkharadze T, Jiang X, Wirkner U, Giese NA, Ansorge W, Debus J, Huber PE, Friess H, Abdollahi A, Kleeff J: Organ-, inflammation- and cancer specific transcriptional fingerprints of pancreatic and hepatic stellate cells. Mol Cancer. 2010, 9: 88-10.1186/1476-4598-9-88. [http://www.molecular-cancer.com/content/9/1/88]View ArticlePubMedPubMed CentralGoogle Scholar
  20. Kim H, Watkinson J, Varadan V, Anastassiou D: Multi-cancer computational analysis reveals invasion-associated variant of desmoplastic reaction involving INHBA, THBS2 and COL11A1. BMC Med Genomics. 2010, 3: 51-10.1186/1755-8794-3-51. [http://www.biomedcentral.com/1755-8794/3/51]View ArticlePubMedPubMed CentralGoogle Scholar
  21. Pavlides S, Tsirigos A, Vera I, Flomenberg N, Frank PG, Casimiro MC, Wang C, Pestell RG, Martinez-Outschoorn UE, Howell A, Sotgia F, Lisanti MP: Transcriptional evidence for the “Reverse Warburg Effect” in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer’s disease, and “Neuron-Glia Metabolic Coupling”. Aging (Albany NY). 2010, 2: 185-199.View ArticleGoogle Scholar
  22. Chernov AV, Baranovskaya S, Golubkov VS, Wakeman DR, Snyder EY, Williams R, Strongin AY: Microarray-based transcriptional and epigenetic profiling of matrix metalloproteinases, collagens, and related genes in cancer. J Biol Chem. 2010, 285: 19647-19659. 10.1074/jbc.M109.088153.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Wilkerson MD, Yin X, Hoadley KA, Liu Y, Hayward MC, Cabanski CR, Muldrew K, Miller CR, Randell SH, Socinski MA, Parsons AM, Funkhouser WK, Lee CB, Roberts PJ, Thorne L, Bernard PS, Perou CM, Hayes DN: Lung squamous cell carcinoma mRNA expression subtypes are reproducible, clinically important and correspond to different normal cell types. Clin Cancer Res. 2010, 16: 4864-4875. 10.1158/1078-0432.CCR-10-0199. doi:10.1158/1078-0432.CCR-10-0199View ArticlePubMedPubMed CentralGoogle Scholar
  24. Hajdu M, Singer S, Maki RG, Schwartz GK, Keohan ML, Antonescu CR: IGF2 over-expression in solitary fibrous tumours is independent of anatomical location and is related to loss of imprinting. J Pathol. 2010, 221: 300-307. 10.1002/path.2715.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Planche A, Bacac M, Provero P, Fusco C, Delorenzi M, Stehle JC, Stamenkovic I: Identification of prognostic molecular features in the reactive stroma of human breast and prostate cancer. PLoS One. 2011, 6: e18640-10.1371/journal.pone.0018640. doi:10.1371/journal.pone.0018640View ArticlePubMedPubMed CentralGoogle Scholar
  26. Anastassiou D, Rumjantseva V, Cheng W, Huang J, Canoll PD, Yamashiro DJ, Kandel JJ: Human cancer cells express slug-based epithelial-mesenchymal transition gene expression signature obtained in vivo. BMC Cancer. 2011, 11: 529-10.1186/1471-2407-11-529. [http://www.biomedcentral.com/1471-2407/11/529]View ArticlePubMedPubMed CentralGoogle Scholar
  27. Navab R, Strumpf D, Bandarchi B, Zhu CQ, Pintilie M, Ramnarine VR, Ibrahimov E, Radulovich N, Leung L, Barczyk M, Panchal D, To C, Yun JJ, Der S, Shepherd FA, Jurisica I, Tsao MS: Prognostic gene-expression signature of carcinoma-associated fibroblasts in non-small cell lung cancer. Proc Natl Acad Sci U S A. 2011, 108: 7160-7165. 10.1073/pnas.1014506108. doi:10.1073/pnas.1014506108View ArticlePubMedPubMed CentralGoogle Scholar
  28. Lascorz J, Hemminki K, Försti A: Systematic enrichment analysis of gene expression profiling studies identifies consensus pathways implicated in colorectal cancer development. J Carcinog. 2011, 10: 7-10.4103/1477-3163.78268. doi:10.4103/1477-3163.78268View ArticlePubMedPubMed CentralGoogle Scholar
  29. Seemann L, Shulman J, Gunaratne GH: A robust topology-based algorithm for gene expression profiling. ISRN Bioinformatics. 2012, Article ID 381023. doi:10.5402/2012/381023Google Scholar
  30. Vargas AC, McCart Reed AE, Waddell N, Lane A, Reid LE, Smart CE, Cocciardi S, da Silva L, Song S, Chenevix-Trench G, Simpson PT, Lakhani SR: Gene expression profiling of tumour epithelial and stromal compartments during breast cancer progression. Breast Cancer Res Treat. 2012, 135: 153-165. 10.1007/s10549-012-2123-4. doi:10.1007/s10549-012-2123-4View ArticlePubMedGoogle Scholar
  31. Wu YH, Chang TH, Huang YF, Huang HD, Chou CY: COL11A1 promotes tumor progression and predicts poor clinical outcome in ovarian cancer. Oncogene. 2014, 33: 3432-3440. 10.1038/onc.2013.307. doi:10.1038/onc.2013.307View ArticlePubMedGoogle Scholar
  32. Gene expression atlas- summary for COL11A1 (Homo sapiens). [http://www.ebi.ac.uk/gxa/gene/ENSG00000060718]
  33. Boshoff C, Bryson K, Clements MO, Trotter MW, Cellek S, Elliman SJ: Transcription profiling of two populations of non-hematopoietic stem cells (MSC and MAPC) isolated from human bone marrow. ArrayExpress: E-MEXP-466. [http://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-466/]
  34. Grundberg E, Brändström H, Lam KC, Gurd S, Ge B, Harmsen E, Kindmark A, Ljunggren O, Mallmin H, Nilsson O, Pastinen T: Systematic assessment of the human osteoblast transcriptome in resting and induced primary cells. Physiol Genomics. 2008, 33: 301-311. 10.1152/physiolgenomics.00028.2008.View ArticlePubMedGoogle Scholar
  35. Kao L-P, Yu S-L, Singh S, Wang K-H, Kao A-P, Li SS: Comparative profiling of mRNA and microRNA expression in human mesenchymal stem cells derived from adult adipose and lipoma tissues. Open Stem Cell J. 2009, 1: 1-9. 10.2174/1876893800901010001. doi:10.2174/1876893800901010001View ArticleGoogle Scholar
  36. Polanska UM, Orimo A: Carcinoma-associated fibroblasts: non-neoplastic tumour-promoting mesenchymal cells. J Cell Physiol. 2013, 228: 1651-1657. 10.1002/jcp.24347. doi:10.1002/jcp.24347View ArticlePubMedGoogle Scholar
  37. García-Ocaña M, Vázquez F, García-Pravia C, Fuentes-Martínez N, Menéndez-Rodríguez P, Fresno-Forcelledo F, Barneo-Serra L, Del Amo-Iribarren J, Simón-Buela L, De Los Toyos JR: Characterization of a novel mouse monoclonal antibody, clone 1E8.33, highly specific for human procollagen 11A1, a tumor-associated stromal component. Int J Oncol. 2012, 40: 1447-1454. doi:10.3892/ijo.2012.1360PubMedGoogle Scholar
  38. Hering TM, Kollar J, Huynh TD, Varelas JB, Sandell LJ: Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension. Arch Biochem Biophys. 1994, 314: 90-98. 10.1006/abbi.1994.1415.View ArticlePubMedGoogle Scholar
  39. Ronzière MC, Farjanel J, Freyria AM, Hartmann DJ, Herbage D: Analysis of types I, II, III, IX and XI collagens synthesized by fetal bovine chondrocytes in high-density culture. Osteoarthritis Cartilage. 1997, 5: 205-214. 10.1016/S1063-4584(97)80015-5.View ArticlePubMedGoogle Scholar
  40. Elliott RL, Blobe GC: Role of transforming growth factor Beta in human cancer. J Clin Oncol. 2005, 23: 2078-2093. 10.1200/JCO.2005.02.047.View ArticlePubMedGoogle Scholar
  41. García-Pravia C, Galván JA, Gutiérrez-Corral N, Solar-García L, García-Pérez E, García-Ocaña M, Del Amo-Iribarren J, Menéndez-Rodríguez P, García-García J, de los Toyos JR, Simón-Buela L, Barneo L: Overexpression of COL11A1 by cancer-associated fibroblasts: clinical relevance of a stromal marker in pancreatic cancer. PLoS One. 2013, 8: e78327-10.1371/journal.pone.0078327. doi:10.1371/journal.pone.0078327View ArticlePubMedPubMed CentralGoogle Scholar
  42. Cueva-Cayetano R, Galvan-Hernandez JÁ, Suarez-Fernandez L, Menendez-Rodriguez MP, Garcia-Pravia C, Barneo L: Preliminary analysis of collagen, type XI, alpha 1 (COL11A1), inhibin alpha (INHBA) and secreted protein acidic and rich in cysteine (SPARC, osteonectin) as potential markers of colon cancer [abstract]. Brit J Surg. 2013, 100 (Suppl. 1): 7-Google Scholar
  43. Mifflin RC, Pinchuk IV, Saada JI, Powell DW: Intestinal myofibroblasts: targets for stem cell therapy. Am J Physiol Gastrointest Liver Physiol. 2011, 300: G684-G696. 10.1152/ajpgi.00474.2010. doi:10.1152/ajpgi.00474.2010View ArticlePubMedPubMed CentralGoogle Scholar
  44. Emura M, Ochiai A, Horino M, Arndt W, Kamino K, Hirohashi S: Development of myofibroblasts from human bone marrow mesenchymal stem cells cocultured with human colon carcinoma cells and TGF beta 1. In Vitro Cell Dev Biol Anim. 2000, 36: 77-80.View ArticlePubMedGoogle Scholar
  45. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, Richardson AL, Polyak K, Tubo R, Weinberg RA: Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007, 449: 557-563. 10.1038/nature06188.View ArticlePubMedGoogle Scholar
  46. ArrayExpress Experiment E-GEOD-9764: Transcription profiling of human mesenchymal stem cells reveals carcinoma associated fibroblast like differentiation. [http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-9764]
  47. Mishra PJ, Mishra PJ, Humeniuk R, Medina DJ, Alexe G, Mesirov JP, Ganesan S, Glod JW, Banerjee D: Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells. Cancer Res. 2008, 68: 4331-4339. 10.1158/0008-5472.CAN-08-0943. doi:10.1158/0008-5472.CAN-08-0943View ArticlePubMedPubMed CentralGoogle Scholar
  48. Cheon DJ, Tong Y, Sim MS, Dering J, Berel D, Cui X, Lester J, Beach JA, Tighiouart M, Walts AE, Karlan BY, Orsulic S: A collagen-remodeling gene signature regulated by TGF-β signaling is associated with metastasis and poor survival in serous ovarian cancer. Clin Cancer Res. 2014, 20: 711-723. 10.1158/1078-0432.CCR-13-1256. doi:10.1158/1078-0432.CCR-13-1256View ArticlePubMedGoogle Scholar
  49. ArrayExpress Experiment E-GEOD-6731: Transcription profiling of human colon biopsies obtained from patients with ulcerative colitis, Crohn’s disease vs. normal to identify pathogenic processes underlying these disease subtypes. [http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-6731]
  50. Wu F, Dassopoulos T, Cope L, Maitra A, Brant SR, Harris ML, Bayless TM, Parmigiani G, Chakravarti S: Genome-wide gene expression differences in Crohn’s disease and ulcerative colitis from endoscopic pinch biopsies: insights into distinctive pathogenesis. Inflamm Bowel Dis. 2007, 13: 807-821. 10.1002/ibd.20110.View ArticlePubMedGoogle Scholar
  51. ArrayExpress Experiment E-TABM-118: Transcription profiling of biopsies from the descending colon of Crohn’s disease patients. [http://www.ebi.ac.uk/arrayexpress/experiments/E-TABM-118/]
  52. Csillag C, Nielsen OH, Borup R, Nielsen FC, Olsen J: Clinical phenotype and gene expression profile in Crohn’s disease. Am J Physiol Gastrointest Liver Physiol. 2007, 292: G298-G304.View ArticlePubMedGoogle Scholar
  53. ArrayExpress Experiment E-GEOD-1152: Transcription profiling of human ileum and colonic tissues from patients with Crohns disease or ulcerative colitis. [http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-1152]
  54. Fuentes-Martínez N, García-Pravia C, García-Ocaña M, Menéndez-Rodríguez P, Del Amo J, Suárez-Fernández L, Galván JA, De Los Toyos JR, Barneo L: Overexpression of proCOL11A1 as a stromal marker of breast cancer. Histol Histopathol. 2014, [Epub ahead of print]Google Scholar
  55. Schuetz CS, Bonin M, Clare SE, Nieselt K, Sotlar K, Walter M, Fehm T, Solomayer E, Riess O, Wallwiener D, Kurek R, Neubauer HJ: Progression-specific genes identified by expression profiling of matched ductal carcinomas in situ and invasive breast tumors, combining laser capture microdissection and oligonucleotide microarray analysis. Cancer Res. 2006, 66: 5278-5286. 10.1158/0008-5472.CAN-05-4610.View ArticlePubMedGoogle Scholar
  56. Lee S, Stewart S, Nagtegaal I, Luo J, Wu Y, Colditz G, Medina D, Allred DC: Differentially expressed genes regulating the progression of ductal carcinoma in situ to invasive breast cancer. Cancer Res. 2012, 72: 4574-4586. 10.1158/0008-5472.CAN-12-0636. doi:10.1158/0008-5472.CAN-12-0636View ArticlePubMedPubMed CentralGoogle Scholar
  57. Castellana B, Escuin D, Peiró G, Garcia-Valdecasas B, Vázquez T, Pons C, Pérez-Olabarria M, Barnadas A, Lerma E: ASPN and GJB2 are implicated in the mechanisms of invasion of ductal breast carcinomas. J Cancer Educ. 2012, 3: 175-183. doi:10.7150/jca.4120View ArticleGoogle Scholar
  58. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/867/prepub

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