Accepted Manuscript Serum CD10 is associated with liver metastasis in colorectal cancer Takamitsu Sasaki , Hiroki Kuniyasu , Yi Luo , Rina Fujiwara , Misaho Kitayoshi , Eriko Tanabe , Daisuke Kato , Satoshi Shinya , Kiyomu Fujii , Hitoshi Ohmori , Yuichi Yamashita PII:
S0022-4804(14)00537-X
DOI:
10.1016/j.jss.2014.05.071
Reference:
YJSRE 12770
To appear in:
Journal of Surgical Research
Received Date: 26 February 2014 Revised Date:
15 April 2014
Accepted Date: 23 May 2014
Please cite this article as: Sasaki T, Kuniyasu H, Luo Y, Fujiwara R, Kitayoshi M, Tanabe E, Kato D, Shinya S, Fujii K, Ohmori H, Yamashita Y, Serum CD10 is associated with liver metastasis in colorectal cancer, Journal of Surgical Research (2014), doi: 10.1016/j.jss.2014.05.071. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sasaki et al.
ACCEPTED MANUSCRIPT
REVISED 2014.4.15 Serum CD10 is associated with liver metastasis in colorectal cancer
Takamitsu Sasaki1), Hiroki Kuniyasu2), Yi Luo2), Rina Fujiwara2), Misaho Kitayoshi2), Eriko
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Tanabe2), Daisuke Kato1), Satoshi Shinya1), Kiyomu Fujii2), Hitoshi Ohmori2), Yuichi Yamashita1)
Fukuoka, Japan
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1) Department of Gastroenterological Surgery, Fukuoka University School of Medicine,
2) Department of Molecular Pathology, Nara Medical University, Kashihara, Japan
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Short title: Serum CD10 predicts liver metastasis
Key words: colorectal cancer, CD10, liver metastasis, serum marker Correspondence: Hiroki Kuniyasu, at Department of Molecular Pathology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, 634-8521, Japan. Tel: +81-744-22-3051, FAX:
Author's contribution:
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+81-744-25-7308, e-mail: [email protected]
TS: data collection, writing the article, and obtaining funding
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HK: conception and design, and obtaining funding YL: data collection, and interpretation
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RF: data collection
MK: data collection ET: data collection DK: data collection SS: data collection, and interpretation KF: critical revision of the article HO: critical revision of the article YY: approving the final version of the manuscript 1
Sasaki et al.
ACCEPTED MANUSCRIPT Abstract Introduction
Colorectal cancer (CRC) is the third leading cause of cancer death in Japan. CD10 expression is closely associated with liver metastasis. In the present study, we explored the possibility of
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serum CD10 as a marker of liver metastasis in CRC. Methodology
BALB/c mouse with subcutaneous tumor of syngeneic CT26 CRC cells were examined serum
examined. CD10 concentration was measured by ELISA.
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Results
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CD10. In 84 CRC patients and patients undergoing hemodialysis, serum CD10 were
In a mouse subcutaneous tumor model, serum CD10 correlated with the weight of the tumors. Serum CD10 was examined in 84 patients with CRC. The serum levels of CD10 were higher in patients with more advanced cancer stages. Patients with liver metastasis showed the
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highest levels of serum CD10 among all patients. Importantly, patients with high serum CD10 levels had metachronous liver metastasis. Healthy volunteers showed low levels of CD10; however, serum CD10 levels in patients undergoing hemodialysis showed levels as high as
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those with stage II and III CRC. A cutoff of serum CD10 set to >1000 pg/mL showed 70% sensitivity and 93% specificity for liver metastasis in CRC. This cutoff included all cases of
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metachronous liver metastasis. Conclusion
With the exclusion of mimicking factors, serum CD10 levels might serve as a useful marker of synchronous and metachronous liver metastasis in CRC.
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ACCEPTED MANUSCRIPT Introduction
Colorectal cancer (CRC) is the third leading cause of cancer death in Japan (1). CRC incidence is on the rise with increasing western life styles, especially high fat and high glucose diets. The majority of CRC cases are managed through resection with endoscopy or
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surgery. However, the metastatic disease shows poor prognosis. One-fourth of advanced cases are associated with liver metastasis, which is a life-threatening event in 30% of CRC deaths (2, 3). The prompt adjuvant chemotherapy based on the evidence of the liver metastasis might
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improve patient prognosis.
CD10 (enkephalinase, or neprilysin) is a neutral endopeptidase (4), which is closely
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associated with liver metastasis in CRC (2, 5, 6). CD10 expression in CRC detected through immunohistochemistry is a good marker for the liver metastasis in CRC. CD10-positive CRC is associated with the liver metastasis in 60–70% of the cases (5, 7). The levels of CD10 mRNA are also associated with tumor invasion depth, lymph node status, and TNM stage (8).
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CD10 not only is a marker for liver metastasis but is also a pathognomonic factor for the process (7, 9). CD10 degrades enkephalins, which are secreted from CRC cells and are known to suppress CRC growth as well as promote apoptosis of T lymphocytes (10-12). Thus,
metastasize.
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CD10-positive CRC cells avoid pro-suppression signaling and thereby enhance their ability to
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CRC cell-inhibitory enkephalins are secreted from hepatocytes stressed by bile stasis, hepatitis, and cancer metastasis, and might have anti-inflammatory and anti-tumorigenic properties (7, 13-15). In such cases, CD10-positive CRC cells show an enhanced ability for establishing liver metastases. CD10 is localized mainly on the apical plasma membrane; however, we detected CD10 in the stromal space among cancer nests, which suggests that CD10 is secreted from CRC cells (7). Increased levels of CD10 are reported in allergic diseases such as bronchial asthma or hemodialysis conditions (16, 17). These findings suggest that serum CD10 might be 3
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a good marker for the liver metastasis in CRCs. In the present study, we explored the potential
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of serum CD10 as a marker for CRC liver metastasis.
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ACCEPTED MANUSCRIPT Materials and methods Cell culture and reagents
CT26 mouse colon cancer cell line were provided by Professor I. J. Fidler (M.D. Anderson Cancer Center) and were maintained in Dulbecco's modified essential medium (DMEM,
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Sigma Chemical Co., St. Louis, MO) containing 10% fetal bovine serum (FBS, Sigma Chemical Co.) under the conditions of 5% CO2 at 37°C.
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Animal model
BALB/c mice were purchased from Japan SLC Inc. (Shizuoka, Japan). The mice were
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maintained according to the institutional guidelines approved by the Committee for Animal Experimentation of Nara Medical University, in accordance with the current regulations and standards of the Ministry of Health, Labour and Welfare. The mice were used according to the institutional guidelines when they were 5 weeks old. CT26 cells were briefly trypsinized and
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washed thrice with Hank’s balanced saline solution (HBSS). The cells (1x107) were suspended in HBSS and injected into the subcutaneous region. The mice were euthanized by performing cervical dislocation at 2 weeks after inoculation of cancer cells and tumors were
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Serum specimens
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excised for measurements (size and weight).
Serum specimens from 8 patients with CRC and 4 patients with non-cancerous diseases examined in the first set of experiments were obtained at Fukuoka University Hospital in 2008. Serum specimens from 75 patients with CRC examined in the second set of experiments were obtained at Fukuoka University Hospital before 2008 with subsequent follow up of the disease recurrence. Serum specimens from 19 patients undergoing hemodialysis and 26 healthy volunteers examined in the second set of experiments were obtained at Tanaka Urology Clinic. The classification of the disease were according to TNM 5
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classification (20); stage II; tumor invades beyond the muscularis propria without lymph node metastasis, stage III; any cases with lymph node metastasis, and stage IV; any case with or without lymph node metastases but with distant metastases (all cases metastasized to the liver). All cases were negative for hepatitis virus type B or type C, and negative for
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inflammatory changes or renal dysfunction. Since written informed consent was not obtained, identifying information for all samples was removed before analysis for strict privacy protection; the procedure was in accordance with the Ethical Guidelines for Human
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Genome/Gene Research enacted by the Japanese Government.
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Enzyme-linked immunosorbent assay (ELISA)
Sera were used for ELISA. Concentrations of CD10 (Neprilysin) were detected by using Neprilysin Human Duo Kit (R&D Systems Inc., Minneapolis, MN) according to the
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provider’s instructions.
Statistical analysis
Statistical analyses of experimental data were performed by using the Mann-Whitney U test,
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and ANOVA test. Statistical significance was defined as a two-sided P value of less than 0.05.
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ACCEPTED MANUSCRIPT Results Serum CD10 concentration in tumor-burden mice
CT26 mouse colon cancer cells were inoculated in the subcutaneous tissues of syngeneic BALB/c mice. The tumor sizes were compared with mouse serum CD10 concentrations (Fig.
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1). Serum CD10 concentrations were significantly correlated with the tumor sizes.
Serum CD10 concentrations in patients with CRC
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The first set of the patients included 4 patients without CRC, 5 patients with pT3, and 3 patients with liver metastasis, which were not followed up after resection. Patients had no
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history of liver or renal diseases (Fig. 2). The serum levels of CD10 were higher in patients with more advanced stage cancer. Patients with liver metastasis, especially, showed highest CD10 concentrations.
The second set of patients included 29 stage II, 29 stage III, and 17 stage IV patients
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who were followed up for more than 5 years after resection of the primary tumors. Healthy volunteers and patients undergoing hemodialysis were also examined. Serum CD10 levels in patients with CRC were higher than those in healthy volunteers. Within patients with CRC,
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the CD10 levels were higher in patients with advanced stage of disease. Serum CD10 levels in dialysis patients were higher than those in healthy volunteers and patients with stage II and III
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CRC. Importantly, the three patients with the high serum CD10 levels showed metachronous liver metastasis. In the 29 cases of stage III, the 26 cases without metachronous liver metastasis showed decrease of serum CD10 levels less than 500 pg/ml after resection of the primary tumors. In contrast, 3 cases with metachronous liver metastasis, serum CD10 levels were not reduced after resection of primary tumors. The 2 cases out of the 3 cases with metachronous liver metastasis were received enucleation of the liver metastasis foci. After then serum CD10 level was decreased. From the results of the second set of examinations, we set two levels of cutoff values 7
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for prediction of liver metastasis (Table 1). The cutoff set to >1000 pg/mL showed 70% sensitivity and 93% specificity. In contrast, the cutoff set to >750 pg/mL showed 85% sensitivity and 76% specificity. The cutoff with >1000 pg/mL contained all 3 cases of the metachronous liver metastasis, and this cutoff might be very efficient in predicting liver
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metastasis.
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ACCEPTED MANUSCRIPT Discussion
Liver metastasis is one of the most important life-threatening conditions in patients with CRC (2). Early detection and treatment are essential in the management of liver metastasis (19). Early detection of liver metastasis involves detection of metastasis by using imaging or serum
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markers and using prediction tools to identify high risk of liver metastasis (22). For example, liver metastasis has been shown to have a high association with serum CEA levels (23). Overexpression of Sialyl Lewis X, c-erbB2 or c-met are associated with higher malignant
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potential of liver metastasis (24-26). However, these factors have not been shown to have high sensitivity and/or specificity to predict liver metastasis of CRC.
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CD10 is a neutral endopeptidase, which reacts with short peptides including enkephalins, substance P, angiotensin II, and some opioids as substrates (4). CD10 is expressed at the luminal surface of the endothelial cells, small intestine and other many organs (27, 28). CD10 has been suggested based on the common denominators of CD10
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substrates to possess anti-inflammatory and anti-narcotic properties. Since substance P and angiotensin are strong pro-inflammatory factors, CD10 abrogates inflammation caused by these factors. Met- and Leu-enkephalins, or a dietary opioid, beta-casomorphin are degraded
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in the intestines or vascular vessels, thereby inhibiting their narcotic effects. In contrast, CD10 has different roles in cancer-host interaction. Met-enkephalin is a
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pro-apoptotic factor to cancer cells and also T lymphocytes (9, 10, 29). Cancer metastasis associated stress leads to increase in Met-enkephalin production in the liver, which is potentially anti-tumorigenic and anti-inflammatory (7). CD10-producing CRC cells possess survival advantage in the enkephalin-rich environment. Therefore, CD10 expression levels might be a relevant and useful marker for liver metastasis in CRC. In the present study, we examined the usefulness of serum CD10 as a marker of liver metastasis in CRC. Serum CD10 levels were well correlated with tumor stage and progression, especially liver metastasis. The most impressive finding was that stage III patients with high 9
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serum CD10 showed disease recurrence through metachronous liver metastasis. These findings suggest that serum CD10 levels might predict liver metastasis in patients with CRC. This might also help in planning therapy in patients with CRC well ahead in time which may potentially prevent liver metastasis. In our small panel of patients, no patients have shown to
reveal the meaning of an increased value of CD10 but a lower stage.
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hepatic recurrence by serum CD10-based adjuvant chemotherapy. The follow-up study will
Our results also showed the possibility of false positive cases. We designed
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examination of patients undergoing hemodialysis based on previous reports documenting that these patients have high levels of serum CD10 (17). Our results reconfirmed the high levels of
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CD10 in patients undergoing hemodialysis. In the literature, some allergic diseases such as bronchial asthma show increase in serum CD10 (16). In literature, activation of polymorphonuclear leukocytes by TNF-α or C5a is responsible for upregulation of CD10 (18, 19). In usage of CD10 as a metastasis marker, these mimicking factors should be
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differentiated and excluded.
Since CD10 plays a role in pro-metastatic mechanism by affecting the host anti-tumor defense by degradation of enkephalin or angiotensin, CD10 is a good candidate for
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molecular targeting therapy. CD10 expression in gastrointestinal cancer is thought to be one of a small intestine-related phenotype. Recently, in spite of still unclearness of CD10
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induction, CD10 might associated with cancer stemness through the enzymatic activity affecting stem niche and the intracellular signaling associated with AKT (30). CD10 increment in metastatic CRC might be newly translated as an increase of stemness in cancer cells. We have earlier reported that CD10 knockdown inhibits liver metastasis of CRC cells by using a mouse model (31). Racecadotril, an anti-relaxant drug, Thiorphan and AHU377 used as anti-hypertensives are anti-CD10 drugs (32-34). Application of these drugs to CRC treatment in large clinical trials might be needed to identify effective anti-metastatic strategies in CRC. 10
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Acknowledgment This work was supported in part by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, Japan
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. Conflict of Interest Statement
We declare that there is not any Financial Support or Relationships which may pose a conflict
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of interest in the contents of the submitted manuscript. All authors have approved the
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comments.
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24. Ono M, Sakamoto M, Ino Y, Moriya Y, Sugihara K, et al. Cancer cell morphology at the invasive front and expression of cell adhesion-related carbohydrate in the primary lesion
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28. Scholzen TE, Konig S, Fastrich M, Bohm M, Luger TA Terminating the stress: peripheral peptidolysis of proopiomelanocortin-derived regulatory hormones by the dermal
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and caspase pathway. Adv Exp Med Biol 2001:493:127-135.
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endopeptidase (CD10): a multifaceted environment actor in stem cells, physiological mechanisms, and cancer. Stem Cells 2011;29:389-396.
31. Luo Y, Fujii K, Ohmori H, Sasahira T, Moriwaka Y, et al. Antisense phosphorothioate oligodeoxynucleic acid for CD10 suppresses liver metastasis of colorectal cancer.
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Pathobiology 2009:78:285-290.
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33. Raasch W, Dominiak P, Dendorfer A Angiotensin I-converting enzyme-dependent and neutral endopeptidase-dependent generation and degradation of angiotensin II contrarily
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Table 1. Serum CD10 cutoff values for detecting liver metastasis of CRC ___________________________________________________ Serum CD10 cutoff ________________________ >1000 pg/mL
>750 pg/mL
26
0
0
Dialysis
19
2
6
Stage II
29
0
3
Stage III
26
1
Liver met*
20
14
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N
4
17
___________________________________________________ Sensitivity
Specificity
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Cutoff
___________________________________________________ >1000 pg/mL
93%
85%
76%
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>750 pg/mL
70%
___________________________________________________
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*Patients in stage IV with liver metastasis and patients with metachronous liver metastasis
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ACCEPTED MANUSCRIPT Figure legends
Figure 1. Relationship between serum CD10 levels and CT26 tumor weights (a) CD10 concentration determined by performing ELISA. (b) CD10 concentration and tumor
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weight.
Figure 2. Serum CD10 levels in patients with CRC
Serum CD10 concentration was examined by performing ELISA. Data is shown as Mean ±
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SD.
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Figure 3. Serum CD10 levels in patients with CRC and patients undergoing hemodialysis Serum CD10 concentration was examined by performing ELISA. Green circles in stage III
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indicate metachronous liver metastasis.
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S0022-4804(14)00537-X
DOI:
10.1016/j.jss.2014.05.071
Reference:
YJSRE 12770
To appear in:
Journal of Surgical Research
Received Date: 26 February 2014 Revised Date:
15 April 2014
Accepted Date: 23 May 2014
Please cite this article as: Sasaki T, Kuniyasu H, Luo Y, Fujiwara R, Kitayoshi M, Tanabe E, Kato D, Shinya S, Fujii K, Ohmori H, Yamashita Y, Serum CD10 is associated with liver metastasis in colorectal cancer, Journal of Surgical Research (2014), doi: 10.1016/j.jss.2014.05.071. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sasaki et al.
ACCEPTED MANUSCRIPT
REVISED 2014.4.15 Serum CD10 is associated with liver metastasis in colorectal cancer
Takamitsu Sasaki1), Hiroki Kuniyasu2), Yi Luo2), Rina Fujiwara2), Misaho Kitayoshi2), Eriko
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Tanabe2), Daisuke Kato1), Satoshi Shinya1), Kiyomu Fujii2), Hitoshi Ohmori2), Yuichi Yamashita1)
Fukuoka, Japan
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1) Department of Gastroenterological Surgery, Fukuoka University School of Medicine,
2) Department of Molecular Pathology, Nara Medical University, Kashihara, Japan
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Short title: Serum CD10 predicts liver metastasis
Key words: colorectal cancer, CD10, liver metastasis, serum marker Correspondence: Hiroki Kuniyasu, at Department of Molecular Pathology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, 634-8521, Japan. Tel: +81-744-22-3051, FAX:
Author's contribution:
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+81-744-25-7308, e-mail: [email protected]
TS: data collection, writing the article, and obtaining funding
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HK: conception and design, and obtaining funding YL: data collection, and interpretation
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RF: data collection
MK: data collection ET: data collection DK: data collection SS: data collection, and interpretation KF: critical revision of the article HO: critical revision of the article YY: approving the final version of the manuscript 1
Sasaki et al.
ACCEPTED MANUSCRIPT Abstract Introduction
Colorectal cancer (CRC) is the third leading cause of cancer death in Japan. CD10 expression is closely associated with liver metastasis. In the present study, we explored the possibility of
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serum CD10 as a marker of liver metastasis in CRC. Methodology
BALB/c mouse with subcutaneous tumor of syngeneic CT26 CRC cells were examined serum
examined. CD10 concentration was measured by ELISA.
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Results
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CD10. In 84 CRC patients and patients undergoing hemodialysis, serum CD10 were
In a mouse subcutaneous tumor model, serum CD10 correlated with the weight of the tumors. Serum CD10 was examined in 84 patients with CRC. The serum levels of CD10 were higher in patients with more advanced cancer stages. Patients with liver metastasis showed the
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highest levels of serum CD10 among all patients. Importantly, patients with high serum CD10 levels had metachronous liver metastasis. Healthy volunteers showed low levels of CD10; however, serum CD10 levels in patients undergoing hemodialysis showed levels as high as
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those with stage II and III CRC. A cutoff of serum CD10 set to >1000 pg/mL showed 70% sensitivity and 93% specificity for liver metastasis in CRC. This cutoff included all cases of
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metachronous liver metastasis. Conclusion
With the exclusion of mimicking factors, serum CD10 levels might serve as a useful marker of synchronous and metachronous liver metastasis in CRC.
2
Sasaki et al.
ACCEPTED MANUSCRIPT Introduction
Colorectal cancer (CRC) is the third leading cause of cancer death in Japan (1). CRC incidence is on the rise with increasing western life styles, especially high fat and high glucose diets. The majority of CRC cases are managed through resection with endoscopy or
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surgery. However, the metastatic disease shows poor prognosis. One-fourth of advanced cases are associated with liver metastasis, which is a life-threatening event in 30% of CRC deaths (2, 3). The prompt adjuvant chemotherapy based on the evidence of the liver metastasis might
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improve patient prognosis.
CD10 (enkephalinase, or neprilysin) is a neutral endopeptidase (4), which is closely
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associated with liver metastasis in CRC (2, 5, 6). CD10 expression in CRC detected through immunohistochemistry is a good marker for the liver metastasis in CRC. CD10-positive CRC is associated with the liver metastasis in 60–70% of the cases (5, 7). The levels of CD10 mRNA are also associated with tumor invasion depth, lymph node status, and TNM stage (8).
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CD10 not only is a marker for liver metastasis but is also a pathognomonic factor for the process (7, 9). CD10 degrades enkephalins, which are secreted from CRC cells and are known to suppress CRC growth as well as promote apoptosis of T lymphocytes (10-12). Thus,
metastasize.
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CD10-positive CRC cells avoid pro-suppression signaling and thereby enhance their ability to
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CRC cell-inhibitory enkephalins are secreted from hepatocytes stressed by bile stasis, hepatitis, and cancer metastasis, and might have anti-inflammatory and anti-tumorigenic properties (7, 13-15). In such cases, CD10-positive CRC cells show an enhanced ability for establishing liver metastases. CD10 is localized mainly on the apical plasma membrane; however, we detected CD10 in the stromal space among cancer nests, which suggests that CD10 is secreted from CRC cells (7). Increased levels of CD10 are reported in allergic diseases such as bronchial asthma or hemodialysis conditions (16, 17). These findings suggest that serum CD10 might be 3
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a good marker for the liver metastasis in CRCs. In the present study, we explored the potential
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of serum CD10 as a marker for CRC liver metastasis.
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ACCEPTED MANUSCRIPT Materials and methods Cell culture and reagents
CT26 mouse colon cancer cell line were provided by Professor I. J. Fidler (M.D. Anderson Cancer Center) and were maintained in Dulbecco's modified essential medium (DMEM,
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Sigma Chemical Co., St. Louis, MO) containing 10% fetal bovine serum (FBS, Sigma Chemical Co.) under the conditions of 5% CO2 at 37°C.
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Animal model
BALB/c mice were purchased from Japan SLC Inc. (Shizuoka, Japan). The mice were
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maintained according to the institutional guidelines approved by the Committee for Animal Experimentation of Nara Medical University, in accordance with the current regulations and standards of the Ministry of Health, Labour and Welfare. The mice were used according to the institutional guidelines when they were 5 weeks old. CT26 cells were briefly trypsinized and
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washed thrice with Hank’s balanced saline solution (HBSS). The cells (1x107) were suspended in HBSS and injected into the subcutaneous region. The mice were euthanized by performing cervical dislocation at 2 weeks after inoculation of cancer cells and tumors were
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Serum specimens
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excised for measurements (size and weight).
Serum specimens from 8 patients with CRC and 4 patients with non-cancerous diseases examined in the first set of experiments were obtained at Fukuoka University Hospital in 2008. Serum specimens from 75 patients with CRC examined in the second set of experiments were obtained at Fukuoka University Hospital before 2008 with subsequent follow up of the disease recurrence. Serum specimens from 19 patients undergoing hemodialysis and 26 healthy volunteers examined in the second set of experiments were obtained at Tanaka Urology Clinic. The classification of the disease were according to TNM 5
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classification (20); stage II; tumor invades beyond the muscularis propria without lymph node metastasis, stage III; any cases with lymph node metastasis, and stage IV; any case with or without lymph node metastases but with distant metastases (all cases metastasized to the liver). All cases were negative for hepatitis virus type B or type C, and negative for
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inflammatory changes or renal dysfunction. Since written informed consent was not obtained, identifying information for all samples was removed before analysis for strict privacy protection; the procedure was in accordance with the Ethical Guidelines for Human
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Genome/Gene Research enacted by the Japanese Government.
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Enzyme-linked immunosorbent assay (ELISA)
Sera were used for ELISA. Concentrations of CD10 (Neprilysin) were detected by using Neprilysin Human Duo Kit (R&D Systems Inc., Minneapolis, MN) according to the
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provider’s instructions.
Statistical analysis
Statistical analyses of experimental data were performed by using the Mann-Whitney U test,
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and ANOVA test. Statistical significance was defined as a two-sided P value of less than 0.05.
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ACCEPTED MANUSCRIPT Results Serum CD10 concentration in tumor-burden mice
CT26 mouse colon cancer cells were inoculated in the subcutaneous tissues of syngeneic BALB/c mice. The tumor sizes were compared with mouse serum CD10 concentrations (Fig.
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1). Serum CD10 concentrations were significantly correlated with the tumor sizes.
Serum CD10 concentrations in patients with CRC
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The first set of the patients included 4 patients without CRC, 5 patients with pT3, and 3 patients with liver metastasis, which were not followed up after resection. Patients had no
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history of liver or renal diseases (Fig. 2). The serum levels of CD10 were higher in patients with more advanced stage cancer. Patients with liver metastasis, especially, showed highest CD10 concentrations.
The second set of patients included 29 stage II, 29 stage III, and 17 stage IV patients
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who were followed up for more than 5 years after resection of the primary tumors. Healthy volunteers and patients undergoing hemodialysis were also examined. Serum CD10 levels in patients with CRC were higher than those in healthy volunteers. Within patients with CRC,
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the CD10 levels were higher in patients with advanced stage of disease. Serum CD10 levels in dialysis patients were higher than those in healthy volunteers and patients with stage II and III
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CRC. Importantly, the three patients with the high serum CD10 levels showed metachronous liver metastasis. In the 29 cases of stage III, the 26 cases without metachronous liver metastasis showed decrease of serum CD10 levels less than 500 pg/ml after resection of the primary tumors. In contrast, 3 cases with metachronous liver metastasis, serum CD10 levels were not reduced after resection of primary tumors. The 2 cases out of the 3 cases with metachronous liver metastasis were received enucleation of the liver metastasis foci. After then serum CD10 level was decreased. From the results of the second set of examinations, we set two levels of cutoff values 7
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for prediction of liver metastasis (Table 1). The cutoff set to >1000 pg/mL showed 70% sensitivity and 93% specificity. In contrast, the cutoff set to >750 pg/mL showed 85% sensitivity and 76% specificity. The cutoff with >1000 pg/mL contained all 3 cases of the metachronous liver metastasis, and this cutoff might be very efficient in predicting liver
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metastasis.
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ACCEPTED MANUSCRIPT Discussion
Liver metastasis is one of the most important life-threatening conditions in patients with CRC (2). Early detection and treatment are essential in the management of liver metastasis (19). Early detection of liver metastasis involves detection of metastasis by using imaging or serum
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markers and using prediction tools to identify high risk of liver metastasis (22). For example, liver metastasis has been shown to have a high association with serum CEA levels (23). Overexpression of Sialyl Lewis X, c-erbB2 or c-met are associated with higher malignant
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potential of liver metastasis (24-26). However, these factors have not been shown to have high sensitivity and/or specificity to predict liver metastasis of CRC.
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CD10 is a neutral endopeptidase, which reacts with short peptides including enkephalins, substance P, angiotensin II, and some opioids as substrates (4). CD10 is expressed at the luminal surface of the endothelial cells, small intestine and other many organs (27, 28). CD10 has been suggested based on the common denominators of CD10
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substrates to possess anti-inflammatory and anti-narcotic properties. Since substance P and angiotensin are strong pro-inflammatory factors, CD10 abrogates inflammation caused by these factors. Met- and Leu-enkephalins, or a dietary opioid, beta-casomorphin are degraded
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in the intestines or vascular vessels, thereby inhibiting their narcotic effects. In contrast, CD10 has different roles in cancer-host interaction. Met-enkephalin is a
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pro-apoptotic factor to cancer cells and also T lymphocytes (9, 10, 29). Cancer metastasis associated stress leads to increase in Met-enkephalin production in the liver, which is potentially anti-tumorigenic and anti-inflammatory (7). CD10-producing CRC cells possess survival advantage in the enkephalin-rich environment. Therefore, CD10 expression levels might be a relevant and useful marker for liver metastasis in CRC. In the present study, we examined the usefulness of serum CD10 as a marker of liver metastasis in CRC. Serum CD10 levels were well correlated with tumor stage and progression, especially liver metastasis. The most impressive finding was that stage III patients with high 9
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serum CD10 showed disease recurrence through metachronous liver metastasis. These findings suggest that serum CD10 levels might predict liver metastasis in patients with CRC. This might also help in planning therapy in patients with CRC well ahead in time which may potentially prevent liver metastasis. In our small panel of patients, no patients have shown to
reveal the meaning of an increased value of CD10 but a lower stage.
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hepatic recurrence by serum CD10-based adjuvant chemotherapy. The follow-up study will
Our results also showed the possibility of false positive cases. We designed
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examination of patients undergoing hemodialysis based on previous reports documenting that these patients have high levels of serum CD10 (17). Our results reconfirmed the high levels of
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CD10 in patients undergoing hemodialysis. In the literature, some allergic diseases such as bronchial asthma show increase in serum CD10 (16). In literature, activation of polymorphonuclear leukocytes by TNF-α or C5a is responsible for upregulation of CD10 (18, 19). In usage of CD10 as a metastasis marker, these mimicking factors should be
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differentiated and excluded.
Since CD10 plays a role in pro-metastatic mechanism by affecting the host anti-tumor defense by degradation of enkephalin or angiotensin, CD10 is a good candidate for
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molecular targeting therapy. CD10 expression in gastrointestinal cancer is thought to be one of a small intestine-related phenotype. Recently, in spite of still unclearness of CD10
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induction, CD10 might associated with cancer stemness through the enzymatic activity affecting stem niche and the intracellular signaling associated with AKT (30). CD10 increment in metastatic CRC might be newly translated as an increase of stemness in cancer cells. We have earlier reported that CD10 knockdown inhibits liver metastasis of CRC cells by using a mouse model (31). Racecadotril, an anti-relaxant drug, Thiorphan and AHU377 used as anti-hypertensives are anti-CD10 drugs (32-34). Application of these drugs to CRC treatment in large clinical trials might be needed to identify effective anti-metastatic strategies in CRC. 10
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Acknowledgment This work was supported in part by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, Japan
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. Conflict of Interest Statement
We declare that there is not any Financial Support or Relationships which may pose a conflict
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of interest in the contents of the submitted manuscript. All authors have approved the
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comments.
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Table 1. Serum CD10 cutoff values for detecting liver metastasis of CRC ___________________________________________________ Serum CD10 cutoff ________________________ >1000 pg/mL
>750 pg/mL
26
0
0
Dialysis
19
2
6
Stage II
29
0
3
Stage III
26
1
Liver met*
20
14
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4
17
___________________________________________________ Sensitivity
Specificity
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Cutoff
___________________________________________________ >1000 pg/mL
93%
85%
76%
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>750 pg/mL
70%
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*Patients in stage IV with liver metastasis and patients with metachronous liver metastasis
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Figure 1. Relationship between serum CD10 levels and CT26 tumor weights (a) CD10 concentration determined by performing ELISA. (b) CD10 concentration and tumor
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weight.
Figure 2. Serum CD10 levels in patients with CRC
Serum CD10 concentration was examined by performing ELISA. Data is shown as Mean ±
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Figure 3. Serum CD10 levels in patients with CRC and patients undergoing hemodialysis Serum CD10 concentration was examined by performing ELISA. Green circles in stage III
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indicate metachronous liver metastasis.
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