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Keywords:

  • oral cancer;
  • mucin;
  • MUC4;
  • immunohistochemistry;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

MUC4 mucin is now known to be expressed in various normal and cancer tissues. We have previously reported that MUC4 expression is a novel prognostic factor in several malignant tumors; however, it has not been investigated in oral squamous cell carcinoma (OSCC). The aim of our study is to evaluate the prognostic significance of MUC4 expression in OSCC. We examined the expression profile of MUC4 in OSCC tissues from 150 patients using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed. MUC4 was expressed in 61 of the 150 patients with OSCC. MUC4 expression was significantly correlated with higher T classification (p = 0.0004), positive nodal metastasis (p = 0.049), advanced tumor stage (p = 0.002), diffuse invasion of cancer cells (p = 0.004) and patient's death (p = 0.004) in OSCC. Multivariate analysis showed that MUC4 expression (p = 0.011), tumor location (p = 0.032) and diffuse invasion (p = 0.009) were statistically significant risk factors. Backward stepwise multivariate analysis demonstrated MUC4 expression (p = 0.0015) and diffuse invasion (p = 0.018) to be statistically significant independent risk factors of poor survival in OSCC. The disease-free and overall survival of patients with MUC4 expression was significantly worse than those without MUC4 expression (p < 0.0001 and p = 0.0001). In addition, the MUC4 expression was a significant risk factor for local recurrence and subsequent nodal metastasis in OSCC (p = 0.017 and p = 0.0001). We first report MUC4 overexpression is an independent factor for poor prognosis of patients with OSCC; therefore, patients with OSCC showing positive MUC4 expression should be followed up carefully.

Oral cancer is a serious problem in many parts of the world. The annual incidence of oral cancer is estimated at around 275,000, with oral squamous cell carcinomas (OSCCs) representing >90% of these cases.1 Generally, OSCC invades normal adjacent tissue at an early stage of growth: 25–50% of patients with OSCC have lymph node metastases.2 Unfortunately, while lymph node metastasis is one of the main prognosticators of survival in OSCC, it is quite often subclinical or occult at the time of diagnosis and treatment.3 Additionally, although complete surgical resection of tumor is one of the effective ways to improve the survival rate of patients with OSCC, patients treated by curative resection often have a poor outcome due to unexpected local recurrence and/or subsequent nodal metastasis. Thus, investigation of the prognostic factors that predict poor outcome is very valuable in treatment of patients with OSCC.

Mucins are membrane-bound or membrane-secreted glycoproteins expressed in epithelial cells.4, 5 They contain a polymorphic central domain that comprises a variable number of tandem repeats, a hallmark of the mucin family.4, 5 To date, 21 different mucins have been found in humans, and these are functionally classified into two major categories: secreted (MUC2, MUC5AC, MUC5B, MUC6-MUC8 and MUC19) and membrane-bound (MUC1, MUC3, MUC4, MUC12, MUC13, MUC15-MUC17, MUC20 and MUC21).6, 7 These mucins serve to protect normal epithelial tissue throughout the body.4, 5 In addition, mucins are involved in the differentiation and renewal of the epithelium and modulation of cell adhesion, immune response and cell signaling.4–6

Among the group of mucins, MUC4 is a membrane-bound mucin encoded by a gene on chromosome 3q29.6 The largest and most distinct feature of MUC4 is its extracellular tandem repeat domain, which comprises a stretch of 16 amino acids repeated in tandem up to 500 times.6 A monoclonal antibody, clone 8G7, which was generated by Batra,8 recognizes the tandem repeat domain of MUC4. Using this antibody, aberrant overexpression of MUC4 has been discovered in many human neoplasms.9–15 In addition, our immunohistochemical studies using this antibody have shown that aberrant MUC4 expression is related to aggressive tumor behavior and a poor outcome in patients with intrahepatic cholangiocarcinoma, invasive ductal carcinoma of the pancreas, extrahepatic bile duct carcinoma and lung adenocarcinoma.7, 10–12, 14, 16 However, profile of MUC4 expression in normal oral squamous epithelium and OSCC still remains unknown.

To date, there has been no extensive research on the relationship between MUC4 expression and patient prognosis in OSCC. Here, we examined the expression profile of MUC4 in 150 patients with OSCC and found MUC4 expression to be an independent prognostic factor for poor outcome in patients with OSCC.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patients and tissue samples

Surgically resected OSCCs from 150 patients (97 males, 53 females) were studied. All tissue specimens were registered from 1992 to 2006 and filed in the Department of Oral Surgery, Kagoshima University Hospital. The mean age of the patients was 64.5 years (range 25–88 years). Anatomical sites, differentiation, TNM classification and stage grouping of tumor in patients with OSCC were classified according to TNM classification of malignant tumors.17 Using biopsy specimens, aggressiveness of invasion in invasive front was assessed by classification of mode of invasion.18 The study was approved by the Human Ethics Committee of Kagoshima University Graduate School of Medical and Dental Sciences. Informed consent was obtained to use surgical specimens for our study from all the patients. All specimens were fixed in formalin, embedded in paraffin and cut into 4-μm-thick sections for immunohistochemistry, in addition to the usual hematoxylin and eosin staining.

Immunohistochemistry

Antibodies

Immunohistochemistry for MUC4 was performed using mouse monoclonal antibody, clone 8G7, which recognizes a tandem repeat sequence of human MUC4.8 It has been confirmed that this monoclonal antibody was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry and confocal analysis.8 Biotinylated affinity-purified horse anti-mouse IgG and avidin-biotinylated horseradish peroxidase complex (ABC) were purchased from Vector Laboratories (Burlingame, CA) as the Vectastain Elite ABC Kit.

Staining procedure

Immunohistochemical stainings were done using an immunoperoxidase method with the ABC complex as described previously.10–12, 14 Each section was deparaffinized with xylene. Endogenous peroxidase was blocked by incubating the sections in 0.3% hydrogen peroxidase in absolute methanol at room temperature for 30 min. After hydration in decreasing concentrations of ethanol in water, the sections were washed in 0.01 M phosphate-buffered saline (PBS, pH 7.4). Epitope retrieval was achieved by water-bath pretreatment at 80°C for 20 min in 0.01 M citrate buffer (pH 6.0).

The sections were washed twice with PBS, and then 2% horse serum in PBS was applied for 30 min at room temperature to prevent nonspecific staining. For staining using antibody, the sections were incubated with dilutions of the primary antibody (1:3,000) in PBS with 1% bovine serum albumin for 16 h at 4°C. The sections were washed three times with PBS and incubated with the biotinylated secondary antibodies for 30 min at room temperature, then washed a further three times with PBS. All sections were then treated with ABC for 30 min. After being washed with PBS three more times, the sections were reacted with diaminobenzidine substrate for 10 min for visualization, rinsed with tap water, counterstained with haematoxylin and mounted. Reaction products were not present when hybridoma culture medium, normal mouse serum or PBS was used instead of the primary antibodies. Positive controls for antibody staining were run using tissues of pulmonary bronchus for MUC4.

Evaluation of the results by scoring

Three blinded investigators (W. T., T. H. and M. N.) independently evaluated the immunostaining. When their evaluations differed, a consensus decision was reached. The immunohistochemical staining was evaluated by the percentage of positively stained carcinoma cells. According to preliminary statistical analysis (Supporting Information p1), specimens that showed more than 5% positively stained carcinoma cells were graded as MUC4-positive.

Statistical analysis

Statistical analysis was performed using JMP (version 5.0; SAS Institute, Cary, NC). Association between MUC4 expression and clinicopathological characteristics was evaluated using the χ2 test. Overall and disease-free survival of the patients was compared between the MUC4-positive and MUC4-negative groups according to the Kaplan–Meier method. Overall survival was measured from the date of surgery to the date of the last follow-up examination or death, whereas disease-free survival was measured from the date of surgery to the date when recurrence and/or subsequent nodal metastasis were first detected. Differences between the survival curves were tested using the log-rank test. Univariate and multivariate survival analyses were performed using the Cox proportional hazards regression model. For the multivariate model, we used 0.05 as the cutoff P value to select the analyzed factors from the univariate analysis data. Backward stepwise multivariate analysis was also used to find independent prognostic factors. A probability of p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patient characteristics

Clinicopathologic features of patients with OSCC are listed in Supporting Information p2. At the last follow-up, 107 of 150 patients (71%) remained alive, 34 of 150 patients (23%) had died of OSCC and 9 of 150 patients (6%) had died of other causes. The 5-year overall survival rate of all patients was 77.8%, and the 5-year disease-free survival rate was 64.5%. The mean observation time was 70.4 months (range 1–206 months).

MUC4 expression in normal oral squamous epithelium, cancer-adjacent dysplastic epithelium and OSCC

MUC4 was not expressed in almost all normal squamous epithelium of the oral cavity (upper area of Fig. 1a). Only six patients (4%) showed MUC4 positivity (more than 5% of cells stained) mainly in the keratinized cells and the prickle-cell layer. In other eight patients, a slight staining was observed only in the surface of keratinized layer. Aberrant expression of MUC4 was often observed in cancer-adjacent dysplastic epithelium in MUC4-positive patients (Fig. 1b, upper area of C, left side of D). Interestingly, some patients showed no MUC4 expression in the cancer tissue even though MUC4 was expressed in the cancer-adjacent dysplastic epithelium (Fig. 1d).

thumbnail image

Figure 1. Immunohistochemical staining of MUC4 mucin in oral normal squamous epithelium and oral squamous cell carcinoma. (a) MUC4 showed no expression in normal oral squamous epithelium (upper area) whereas strong positive staining was seen in OSCC cells (lower area, original magnification ×40). (b) In cancer-adjacent dysplastic epithelium, MUC4 was expressed throughout the entire thickness of the epidermis with absence of stain in basal layer of epithelium (original magnification ×200). (c) Moderate MUC4 expression was often observed in cancer-adjacent dysplastic epithelium (upper area) of MUC4-positive patients. Strong MUC4 staining was observed in cancer cells beneath epithelium (lower area, original magnification ×40). (d) Some patients showed no MUC4 expression in cancer tissue (right side) although MUC4 expression was noted in cancer-adjacent dysplastic epithelium (left side, original magnification ×100). (e) Strong cytoplasmic MUC4 expression was noted in the nests of well-differentiated OSCC (original magnification ×200). (f) Positive staining of MUC4 was noted in the diffuse invasive front of OSCC (original magnification ×200).

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Representative expression patterns of MUC4 in OSCC are illustrated in Figures 1e and 1f. Positive staining (more than 5% of cells stained) of MUC4 was seen in the carcinoma cells in 61 (41%) OSCC cases although most normal squamous epithelium showed no expression. The mean and median of the amount of MUC4 expression in all 150 patients was 8.9 and 3%, respectively. The range of staining was 0–80%. The details of distribution of MUC4 immunohistochemistry are described in Supporting Information p3. MUC4 was expressed predominantly in the cytoplasm of tumor cells, with occasional positivity in cell membrane. Stromal tissues of carcinoma except precious few cells were negative for MUC4.

The relationship between MUC4 expression and clinicopathologic features

Association between MUC4 expression and clinicopathologic characteristics including age, gender, tumor location, T classification, nodal metastasis, tumor stage, tumor differentiation, mode of invasion and vital status are summarized in Table 1. The percentages of higher T classification, positive nodal metastasis, advanced tumor stage and diffuse invasion of cancer cells were significantly higher in the MUC4-positive group than the MUC4-negative group (p = 0.0004, 0.049, 0.002, 0.004, respectively; Table 1). Death from OSCC was significantly correlated with MUC4 expression (p = 0.004; Table 1). When comparing clinicopathological factors between MUC4 high expression group (>50% cells stained) and low expression group (5–49% cells stained), there was no significant difference in two groups (listed in Supporting Information p4).

Table 1. Association between MUC4 expression and clinicopathologic characteristics in patients with OSCC (N = 150)
inline image

Univariate analysis of prognostic factors in OSCC

The univariate analysis of prognostic factors of OSCC is summarized in Table 2. For statistical analysis of prognostic factors, tumor locations were classified into two groups; better survival group (lip, cheek, tongue and mandible, n = 109) and poor survival group (maxilla and floor of mouth, n = 41) according to preliminary survival analyses (Supporting Information p5). T classification, tumor stage, tumor differentiation and mode of invasion were also divided by two groups as well (Table 2). Tumor location (maxilla and floor of mouth vs. lip, cheek, tongue and mandible; p = 0.017), positive nodal metastasis (p = 0.012), advanced tumor stage (stages III and IV vs. stages I and II; p = 0.026), diffuse invasion (mode of invasion grades 4c and 4d vs. grades 1 to 3; p = 0.003) and positive MUC4 expression (p = 0.0002) were statistically significant risk factors affecting the survival of patients with OSCC.

Table 2. Univariate analysis of prognostic factors in OSCC (N = 150)
inline image

Multivariate analysis of prognostic factors

The multivariate analysis of prognostic factors of OSCC is summarized in Table 3. Tumor location (p = 0.032), diffuse invasion (p = 0.009) and positive MUC4 expression (p = 0.011) were found to be statistically significant independent factors for poor prognosis. By backward stepwise multivariate analysis of the five prognostic factors listed in Table 3, diffuse invasion (mode of invasion grade 1–3 vs. grade 4; p = 0.0176) and positive MUC4 expression (p = 0.0015) were found to be statistically significant independent factors for poor prognosis in OSCC (Table 3).

Table 3. Multivariate analysis of prognostic factors in OSCC (N = 150)
inline image

The relationship between MUC4 expression and survival rate

Of the 150 patients examined, 34 died during the follow-up period. Median and mean lengths of survival for patients with OSCC were 62.6 and 70.4 months, respectively. The disease-free survival of patients with MUC4 expression (61 patients) was significantly worse than those with no MUC4 expression (89 patients; p < 0.0001, Fig. 2a). Also, the overall survival of patients with high MUC4 expression was significantly worse than that of those with no MUC4 expression (p = 0.0001, Fig. 2b). The 1-, 3- and 5-year overall survival rates of MUC4-positive patients were 89.4, 74.0 and 65.5%, respectively, whereas those of MUC4-negative patients were 97.7, 90.3 and 86.0%, respectively (Table 4).

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Figure 2. Correlation between MUC4 expression and survival rate in 150 patients with oral squamous cell carcinoma, as determined by the Kaplan–Meier method. (a) The disease-free survival of patients with expression of MUC4 was statistically worse than that of those with low MUC4 expression (log-rank; p < 0.0001). (b) The overall survival of patients with expression of MUC4 was statistically worse than that of those with low MUC4 expression (log-rank; p = 0.0001).

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Table 4. Prognostic Significance of MUC4 Expression in OSCC (N = 150)
inline image

Prognostic significance of MUC4 expression in OSCC

The prognostic significance of aberrant MUC4 expression in OSCC is summarized in Table 4. In the patients with MUC4 expression, 13 of 61 (21%) patients showed local recurrence after surgery and 21 of 61 (34%) patients showed subsequent nodal metastasis. MUC4 expression showed statistically significant correlation with local recurrence and subsequent nodal metastasis (χ2, p = 0.017, p = 0.0001). Both the 5 years disease-free and overall survival rates of patients with MUC4 expression were significantly worse than those of MUC4-negative patients (log-rank test, p < 0.0001, p = 0.0001, Fig. 2 and Table 4). Taken together, unfavorable events and poor survival after surgery were quite well reflected by aberrant MUC4 expression in OSCC.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In our study, our results indicate that MUC4 expression in cancer cells correlates with nodal metastasis, diffuse invasion and tumor progression of OSCC, and that disease-free and overall survival is significantly worse in patients with high MUC4 expression compared to those with no MUC4 expression. In addition, MUC4 expression showed statistically significant correlation with local recurrence and subsequent nodal metastasis after first therapy. Taken together, unfavorable events and poor survival after surgery were quite well reflected by aberrant MUC4 expression in OSCC. Thus, understanding of a patient's MUC4 expression status might impact decisions regarding therapeutic method, operation method, range of resection safety margin and indication for elective neck dissection. Moreover, OSCC patients with positive MUC4 expression should be followed up carefully.

Our series of immunohistochemical studies of MUC4 has been performed in intrahepatic cholangiocarcinoma,10 pancreatic adenocarcinoma,12 extrahepatic bile duct carcinoma11 and lung adenocarcinoma.14 Multivariate analyses have confirmed that high MUC4 expression is an independent poor prognostic factor in these malignant tumors. In addition, other investigators have shown the prognostic significance of MUC4 in many human cancers including pancreatic,19, 20 periampullary,21 colorectal22 and cervical cancers.9 We have now shown MUC4 expression to be an independent poor prognostic factor of OSCC. Together, these results support the theory that MUC4 (over)expression causes cancer cells to behave in a more aggressive fashion.

In our study, MUC4 expression was significantly correlated with higher T classification, positive nodal metastasis, advanced tumor stage, diffuse invasion of cancer cells and patient's death in OSCC. These clinical results were consistent with in vitro studies previously reported. MUC4, by virtue of its large sized extracellular domain, serves as a barrier for some cell–cell and cell–extracellular matrix interactions via steric hindrance.6 In addition, MUC4 has many unique domains that suggest possible functions associated with tumor cell interaction with the extracellular matrix (via NIDO and AMOP domains present the C-terminal region of the MUC4 subunit) and growth factor signaling (via EGF-like domains).6 Moreover, MUC4 promotes tumor progression by repressing apoptosis by multiple mechanisms, both ErbB2 dependent and independent.23 By knockdown and overexpression of MUC4 in cancer cells, the studies have demonstrated the anti-apoptotic function of MUC4.24–26 Furthermore, the previous studies showed the ability of Muc4 to promote tumor metastasis23, 27 and the upregulation of MUC4 in breast tumor metastases but not in primary tumors.28 Taken together, these studies support our data that MUC4 expression correlated with higher T classification, positive nodal metastasis, advanced tumor stage and diffuse invasion of cancer in OSCC. In addition to potential role of MUC4 for tumor progression, invasion and metastasis, it has been shown that Muc4 renders drug resistance to melanoma cell lines.29 Additionally, the presence of MUC4 on the surface of the tumor cells can mask the surface epitopes to the cytotoxic immune cells such as cytotoxic-T lymphocytes or NK cells and, hence, escape from immune response.6, 30 These studies also support our data that MUC4 expression correlated with worse overall and disease-free survival of cancer in OSCC. Thus, all of these features of MUC4 strongly support its significance for a diagnostic tool and a therapeutic target in OSCC.

Previous, early studies into the role of MUC4 expression as a prognostic factor in cancer have mainly examined glandular tissue and adenocarcinoma arising from gland or duct. More recent studies have focused on squamous epithelium and squamous cell carcinomas and have revealed the prognostic value of MUC4 expression.9, 31–33 MUC4 expression has been reported at different levels in cutaneous,33 cervical,9 pulmonary34 and esophageal SCC35 and SCC of the upper aerodigestive tract.31 However, very few reports showed a correlation between MUC4 expression and patient outcome, which includes recurrence, subsequent metastasis and especially patient survival. To our knowledge, by long-term observation focusing patient's survival and unfavorable events, this is the first report to reveal the relationships between MUC4 expression and patient outcome in the area of squamous cell carcinoma. To clarify the universal implication of MUC4 expression in SCC, further extended studies in other organs may be needed.

Some tumors express mucins that are not normally found in that organ (for example, MUC4 is expressed in premalignant and malignant lesions of the pancreas but is not commonly expressed in normal pancreatic ductal epithelial cells).4, 19 Here, we have observed MUC4 expression in cancer-adjacent dysplastic epithelium in a number of MUC4-positive patients, in whom it was not expressed in the normal squamous epithelium. This finding is supported by previous report that MUC4 was expressed diffusely throughout the dysplastic epithelium while MUC4 expression in the normal squamous epithelium was weak or absent.9, 32, 33 These results, including our data, suggest that MUC4 plays a role in the early stage of carcinogenesis and that it is activated during the process of squamous dysplastic transformation in squamous cell carcinoma. Either way, MUC4 can be a powerful diagnostic marker for early detection of carcinogenesis of OSCC.

Like MUC4, MUC1 is a membrane-bound mucin. These membrane mucins possess specific domains that can make complex associations with various signaling pathways, impacting cell survival through alterations of cell growth, proliferation, death and autophagy.36 Our immunohistochemical studies of mucin expression in various human tumors have shown MUC1 expression to be related to invasive proliferation of tumors and a poor outcome for patients.37–40 We observed MUC1 expression in the luminal surface membrane and/or the cytoplasm of cancer cells. In contrast, MUC4 expression showed a cytoplasmic pattern in the mass-forming type of intrahepatic cholangiocarcinoma,10 pancreatic invasive ductal carcinoma,12 extrahepatic bile duct carcinoma11 and lung adenocarcinoma.14 Both MUC1 and MUC4 are categorized as membrane mucins; however, they are coded in different regions of the genome and act via different mechanisms. For signaling molecules, MUC1 acts as a docking protein, whereas MUC4 acts as a receptor ligand.41 The different expression patterns might point to different mechanisms for MUC1 and MUC4, even though both are membrane mucins with cell signaling function. Thus, the relationship between the expression of MUC4 and MUC1 in the immunohistochemistry of OSCC would be an interesting area of future study.

The MUC4 mucin has been shown to act as an intramembrane ligand and activator for the receptor ErbB2 and thus facilitates its dimerization with other ErbB receptors.25, 42 The MUC4/ErbB2 complex has been observed in various tumors and cancer cell lines,43, 44 suggesting that MUC4 modulates ErbB2 signaling and that overexpression of MUC4 and ErbB2 contributes the promotion of tumor progression.36 Also, studies with rat Muc4 have shown that Muc4 induces limited and specific phosphorylation of ErbB2, which is associated with an upregulation of cell-cycle inhibitor p27kip and a repression of apoptosis.42 This suggests that MUC4 might exert its anti-apoptotic function through ErbB2 downstream signaling.36 On the other hand, recent study has revealed that MUC4 interacts with the HER2 and stabilizes its expression and activity by posttranslational mechanisms.44 Enhanced stabilization of HER2 by MUC4 interaction was associated with enhanced activation of extracellular signal-regulated kinases (Erks) 1 and 2 MAPK.44 Moreover, it has shown that the role of MUC4 in the protection of pancreatic cancer cells from gemcitabine-induced apoptosis through HER2/ERK-dependent phosphorylation and inactivation of the proapoptotic protein Bad.45 Hence, to clarify the biological role of MUC4 in OSCC, colocalization and interaction between MUC4 and these growth factor receptors in OSCC need to be elucidated by future studies.

Recently, epigenetic regulation of human MUC4 has been reported. Both DNA methylation and histone modifications contribute to the regulation of MUC4 in cancer cells.46, 47 Moreover, alternative splicing of the MUC4 transcript has also been observed,48 and some of these MUC4 splice variants are expressed in pancreatic carcinomas but not in the normal pancreas.48–50 Thus, the dysfunction of these regulations of MUC4 expression such as epigenetic alterations and alternative splicing might be a candidate for a molecular marker and a therapeutic target of OSCC in the future.

In conclusion, we first report that the expression of MUC4 is a novel independent poor prognostic factor in OSCC. Therefore, MUC4 is a useful marker for choosing therapy and for predicting the poor outcome of patients with OSCC. Patients with OSCC who show positive MUC4 expression should be followed-up carefully. In addition, MUC4 might be a strong candidate to target of future therapy in OSCC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Mr. Yoshiharu Atsuji and Ms. Yukari Nishimura for their excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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