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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 14  |  Issue : 4  |  Page : 208-215

Evaluation of carcinoma-associated fibroblasts in oral squamous cell carcinoma: a potential role for cathepsin K


Department of Oral Pathology, Faculty of Dentistry, Tanta University, Tanta, Egypt

Date of Submission14-May-2017
Date of Acceptance03-Aug-2017
Date of Web Publication21-Dec-2017

Correspondence Address:
Basant H Abou Zaid
Department of Oral Pathology, Faculty of Dentistry, Tanta University, Medical Campus, Algeysh Street, Tanta
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tdj.tdj_31_17

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  Abstract 

Aim
The aim of this work was to identify carcinoma-associated fibroblasts (CAFs) in the stroma of oral squamous cell carcinoma (OSCC), through examining α-smooth muscle actin (α-SMA) expression immunohistochemically. Also, to investigate the immunohistochemical expression pattern of cathepsin K (CTSK) in CAFs as well as to assess the expression level of CTSK mRNA in CAFs, by real-time reverse transcription PCR (RT-PCR) technique. Furthermore, to correlate CTSK expression in different histopathological grades of OSCC, in order to identify its potential role in tumor progression and aggressiveness.
Materials and methods
Paraffin-embedded OSCC specimens were stained immunohistochemically using antibodies against α-SMA and CTSK. At the same time, level of CTSK mRNA expression was evaluated in fibroblasts indirectly cocultured with OSCC cells by means of real-time RT-PCR technique.
Results
In addition to CTSK coimmunolocalization with α-SMA in stromal CAFs, its expression by OSCC cells was significantly correlated with increased malignant criteria. Stromal CAFs showed upregulated CTSK expression in poorly differentiated OSCC. Real-time RT-PCR results revealed enhanced CTSK mRNA expression by fibroblasts in indirect coculture.
Conclusion
Collectively, the results propose an evolving role for CTSK in OSCC progression.

Keywords: carcinoma-associated fibroblasts, cathepsin K, oral cancer, oral squamous cell carcinoma


How to cite this article:
Abou Zaid BH, Abo-Azma NE, Megahed EM, Deraz EM. Evaluation of carcinoma-associated fibroblasts in oral squamous cell carcinoma: a potential role for cathepsin K. Tanta Dent J 2017;14:208-15

How to cite this URL:
Abou Zaid BH, Abo-Azma NE, Megahed EM, Deraz EM. Evaluation of carcinoma-associated fibroblasts in oral squamous cell carcinoma: a potential role for cathepsin K. Tanta Dent J [serial online] 2017 [cited 2018 May 24];14:208-15. Available from: http://www.tmj.eg.net/text.asp?2017/14/4/208/221380


  Introduction Top


Worldwide, more than 500 000 new cases of oral cancer are detected annually making it the sixth most common cancer [1],[2]. Among these, more than 90% are diagnosed as oral squamous cell carcinoma (OSCC) [3]. OSCC incidence is generally more prevalent in developing countries [4].

A report of the Middle-East Cancer Consortium of the National Cancer Institute in Bethesda, USA, depicted that Egypt had one of the highest overall incidence rates of cancer of oral cavity and pharynx among the Middle-East Cancer Consortium countries. Furthermore, overall urban incidence of head and neck squamous cell carcinoma was twice or more that of rural incidence in some parts of Egypt [5]. Such variation in incidence signals for differences in lifestyle exposure to specific risk factors amongst them, tobacco carries the greatest burden [6],[7].

Microscopically, OSCC incorporates large number of nontransformed cells as well as a specialized extracellular matrix (ECM) collectively designated as the stroma [8]. Ancillary stromal cells include fibroblasts, myofibroblasts, endothelial cells, leukocytes and bone marrow-derived cells which contribute to tumor microenvironment (TME) complexity [8].

Fibroblasts, the primary stromal component, undergo activation becoming myofibroblasts in wound healing, fibrosis [9] as well as in cancer where they and referred to as cancer-associated fibroblasts (CAFs) [10]. CAFs are spindle-shaped cells that share features of smooth muscle cells and fibroblasts [11]. They can be identified immunohistochemically by different myoid markers such as α-smooth muscle actin (α-SMA) [12],[13].

CAFs are able to secrete a plethora of growth factors, cytokines, chemokines, inflammatory mediators, adhesion proteins and most abundantly ECM proteins [14] which all favor cancer cell proliferation and thereby, tumor progression [15]. Cathepsins are lysosomal peptidases that encompass cysteine, serine and aspartic protease classes [16] and are characterized by outstanding broad spectrum of functions in almost all tissues and cell types [17]. Cathepsin K (CTSK) is a lysosomal cysteine protease, first described in osteoclasts as a mediator of bone resorption [18]. Despite its known powerful collagenolytic activity [19], importance of CTSK expression in OSCC has not been sufficiently clarified in the current literature. Therefore, this study aims to shed light on the potential role of CTSK in OSCC progression.


  Materials and Methods Top


Surgical material

This study was approved by the Ethics Committee of Faculty of Dentistry, Tanta University. Forty-two paraffin-embedded OSCC specimens were obtained from archives of Oral Pathology Department, Faculty of Dentistry, Tanta University. Serial sections were cut at thickness of 4 μm. One set of the specimens was routinely stained with hematoxylin and eosin, and then graded histopathologically according to WHO criteria [20] into well differentiated, moderately differentiated and poorly differentiated OSCC. The other sets were utilized for immunohistochemical staining of α-SMA and CTSK. Normal-appearing oral mucosa adjacent to OSCC tissue utilized as internal control. In addition, blood vessels within connective tissue of immunohistochemically stained sections served as positive internal control for α-SMA.

Immunohistochemistry

Mouse monoclonal antibody against α-SMA (clone 1A4, diluted at 1: 50) was purchased from Dako (Glostrup, Denmark) and a human monoclonal antibody against CTSK (clone 3F9, diluted at 1: 100) was purchased from Abcam (UK). Immunohistochemical procedures were performed using Streptavidin Biotin Complex Universal Kit (Neomarkers, Fremont, California, USA) according to manufacturer instructions.

Cell culture

Cell lines growth and maintenance

One OSCC cell line (SCC-9) and one normal fibroblast cell line (BJ) were purchased from the holding company for biological products and vaccines (VACSERA, Giza, Egypt). SCC-9 and BJ cells were subcultured individually in T25 flask equipped by 3 ml of Dulbecco's modified Eagle's medium (DMEM) (low glucose) at a concentration of 5 × 104 cells/ml. Cultures were maintained in incubator at 37°C in 5% CO2 in air until confluency [Figure 1].
Figure 1: Phase contrast microscope images of confluent cell cultures: (a) normal human fibroblast cell line (BJ) shows elongated morphology with bipolar or multipolar outline. (b) oral squamous cell carcinoma cell line (SCC-9) exhibits polygonal shapes with more regular dimensions. (a, b, original magnification ×200).

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Indirect coculture

Upon confluency, BJ and SCC-9 cells were cocultured in a 12-well plate without physical contact using ThinCert 12-well cell culture inserts with 1.0 μm pore size polyethylene terephthalate membrane (Greiner Bio-one, GmbH). BJ cells were seeded in the lower well at concentration of 5 × 103 in 2 ml of DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 μg/ml fungizone. Shortly after seeding of BJ cells, the insert chambers were positioned on their conforming wells and then SCC-9 cells were seeded in the insert at concentration of 5 × 103 in 1 ml of DMEM. In another well, BJ cells were seeded at the same concentration in 2 ml of DMEM and the above insert chamber was placed containing only 1 ml of DMEM. This well served as a control. Both indirect coculture and control were maintained for 7 days, with medium being changed every 3 days. After 7 days, cells in the wells and inserts were collected for RNA isolation and subsequent PCR test.

Real-time reverse transcription polymerase chain reaction

Total RNA was extracted from harvested cells using RNA-spin Total RNA Extraction Kit (INTRON Biotechnology Inc., Korea) according to manufacturer's protocol. cDNA synthesis and RT-PCR were performed using TIANScript RT Kit (TIANGEN Biotech, Beijing, China). For CTSK, we used forward (5′-ccgcagtaatgacacccttt-3′) and reverse (5′-gcacccacagagctaaaagc-3′) primer sequences whereas β-actin (forward: 5′-aactgggacgacatggagaaaa-3′ and reverse: 5′-agaggcgtacagggatagcaca-3′) was utilized as an internal housekeeping gene. In brief, PCR amplification was done using 1.5 μl aliquot of cDNA samples in the 7500 real-time PCR thermal cycler (Applied Biosystems Inc., USA). A total of 40 PCR cycles at 95°C for denaturation then at 60°C for annealing was done using Taq DNA polymerase enzyme as specified by manufacturer instructions. All experiments were done in duplicates.

Assessment of immunohistochemical results

Identification of carcinoma-associated fibroblasts through evaluating α-smooth muscle actin immunostaining

α-SMA stained sections were examined under light microscope at ×100 followed by ×400 magnification. Noninflammatory and nonendothelial spindle cells exhibited brown intracytoplasmic α-SMA staining were considered as CAFs, regardless the staining intensity. Representative fields from each slide were identified and matched with their corresponding serial sections with positive CTSK staining.

Evaluation of cathepsin K expression

Three representative CTSK-positive sections, at the same locations according to paired serial sections with positive α-SMA were evaluated at ×400 magnification. CTSK immunostaining was assessed in overall tumor epithelial cells and overall stromal CAFs as described elsewhere [21]. Cells were assigned to one of four-point numerical scale (0, no staining is observed; 1, weak staining; 2, moderate staining and 3, strong staining).

Statistical analysis

Data obtained were then statistically analyzed using SPSS statistics for Windows, version 17.0 (SPSS Inc., Chicago, Illinois, USA). Correlations among groups were done using one-way analysis of variance test. Subsequent comparisons between groups were assessed using Mann–Whitney U test. Significance of changes in CTSK expression versus histopathological grade was tested by Pearson's correlation. The level of statistical significance was at P value less than 0.05.


  Results Top


Immunohistochemistry

Immunoreactivity in normal internal control tissue

Within normal mucosa adjacent to OSCC, there was no evidence for α-SMA-immunoreactive CAFs in the subepithelial connective tissue. Only the smooth muscles in blood vessel walls in all specimens were positive to α-SMA, hence they served as internal positive control. CTSK was not expressed neither by normal oral keratinocytes nor by connective tissue fibroblasts. Yet, positive CTSK expression was evident in inflammatory cells found in some specimens.

Cathepsin K immunohistochemical expression in oral squamous cell carcinoma specimens

Expression pattern of α-SMA and CTSK immunostaining

α-SMA expression was observed in all of the 42 (100%) specimens as brown staining in the cytoplasm of stromal CAFs [Figure 2]a, [Figure 2]c, [Figure 2]e, [Figure 3]a, [Figure 3]c, [Figure 3]e, [Figure 4]a, [Figure 4]c and [Figure 5]a and in blood vessel walls [Figure 3]c and [Figure 5]a. CTSK expression was detected in 41 out of total 42 (97.6%) specimens in both tumor epithelial cells as well as peritumoral stromal CAFs in the form of diffuse, granular, brown, cytoplasmic staining with mean intensity of 1.8 ± 0.77 for tumor cells and 2.3 ± 0.75 for stromal CAFs [Table 1].
Table 1: Cathepsin K expression in different oral squamous cell carcinoma histopathological grades

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Figure 2: Immunohistochemical staining for α-smooth muscle actin (a, c, e) and cathepsin K (CTSK) (b, d, f) in well differentiated oral squamous cell carcinoma (×400). (b) Weak CTSK expression in the cytoplasm of invading tumor islands as well as within stromal carcinoma-associated fibroblasts (CAFs). Moderate CTSK expression is evident in (d) and (f) in tumor cells. (f) Strong CTSK expression by scattered peritumoral CAFs.

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Figure 3: Immunohistochemical staining for α-smooth muscle actin (a, c, e) and cathepsin K (CTSK) (b, d, f) in moderately differentiated oral squamous cell carcinoma (×400). Tumor epithelial cells show weak (b), moderate (d) and strong (f) cytoplasmic CTSK expression. Peritumoral carcinoma-associated fibroblasts exhibit moderate (b, d) and strong (f) CTSK immunostaining. Positive staining is noted in chronic inflammatory cells (black arrow) (d).

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Figure 4: Immunohistochemical staining for α-smooth muscle actin (a and c) and cathepsin K. (CTSK) (b and d) in poorly differentiated oral squamous cell carcinoma (×400). Moderate CTSK expression is observed in the cytoplasm of tumor cells as well as in stromal carcinoma-associated fibroblasts (CAFs) (b). Strong cytoplasmic CTSK immunostaining is evident in both tumor cells and scattered peritumoral CAFs (d).

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Figure 5: (a) Invading front of well differentiated oral squamous cell carcinoma with α-smooth muscle actin immunostaining highlighting bands of carcinoma-associated fibroblasts (CAFs) as well as internal control blood vessels (black arrowheads) (×400). (b) The same previous fields shows moderate cathepsin K expression in tumor cell cytoplasm and strong expression in stromal CAFs (×400).

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Semiquantitative analysis of CTSK expression

Well differentiated OSCC: Within tumor epithelial cells, most of specimens exhibited weak [Figure 2]b to moderate staining (66.7 and 33.3%, respectively) [Figure 2]d and [Figure 2]f, while none of them showed strong CTSK expression. On the other hand, stromal CAFs exhibited moderate CTSK expression in 60% of specimens [Figure 2]d, 26.7% showed strong expression [Figure 2]f and 13.3% displayed weak CTSK expression [Figure 2]b and [Table 1].

Moderately differentiated OSCC: More than half of specimens (56.25%) exhibited moderate CTSK expression in tumor cells [Figure 3]d, 25% showed weak expression [Figure 3]b and only two specimens (12.5%) exhibited strong CTSK expression [Figure 3]f. Regarding the stromal CAFs, the majority of specimens demonstrated strong [Figure 3]f to moderate [Figure 3]b and [Figure 3]d CTSK expression (43.75 and 37.5%, respectively). Two (12.5%) specimens showed weak CTSK immunostaining.

Poorly differentiated OSCC: In the invading tumor cells, all specimens exhibited strong [Figure 4]d to moderate [Figure 4]b CTSK immunostaining (54.6 and 45.4%, respectively). Most of the specimens (72.7%) exhibited strong CTSK staining within stromal CAFs [Figure 4]d, whereas the remainder (27.3%) showed moderate CTSK expression [Figure 4]b.

CTSK expression in invading tumor front

Out of the total 42 OSCC specimens, only 26 (62%) included deep invasive tumor fronts; where malignant tumor cells appear to invade the surrounding tissues in a less cohesive pattern with lesser degrees of cellular differentiation compared to other parts of the tumor. All these specimens exhibited strong to moderate CTSK expression in the cytoplasm of tumor cells (50% for each). In addition, more than half of specimens (65.4%) displayed strong CTSK expression within surrounding stromal CAFs [Figure 5]b and the remainder (35.6%) showed moderate CTSK immunostaining. It is noteworthy that CTSK expression was also evident in chronic inflammatory cells infiltrating the tumor stroma, in various tumor areas including the deep invasive front [Figure 3]d and [Figure 5]b.

Cathepsin K expression correlated with different oral squamous cell carcinoma grades

Within tumor cells, CTSK expression revealed to be upregulated with increased malignancy grade – that is, poorly differentiated OSCC exhibited significantly higher CTSK expression (P < 0.05) as compared to moderately and well differentiated [Figure 6]. The difference in CTSK expression between moderately and poorly differentiated OSCC was significantly increased (P < 0.05) in favor of poorly differentiated OSCC [Table 2]. Stromal CAFs showed significantly higher CTSK expression (P < 0.05) in poorly differentiated OSCC than in well differentiated [Table 2] and [Figure 6]. CTSK expression by tumor cells was highly significantly (P<0.01) correlated to its expression by stromal CAFs [Figure 7].
Figure 6: Statistical analysis of cathepsin K (CTSK) expression by tumor epithelial cells and stromal carcinoma-associated fibroblasts in different oral squamous cell carcinoma histopathological grades.

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Table 2: Cathepsin K expression by tumor cells and stromal carcinoma-associated fibroblasts in different oral squamous cell carcinoma grades assessed by Mann-Whitney U test

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Figure 7: Cathepsin K (CTSK) expression by tumor cells is highly significantly correlated with its expression by stromal carcinoma-associated fibroblasts in oral squamous cell carcinoma histopathological grades.

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Real-time reverse transcription polymerase chain reaction

CTSK mRNA expression was evaluated in control fibroblasts grown individually and in those maintained in the transwell coculture system with OSCC cells. Normal control fibroblasts found to express CTSK mRNA constitutively. After 7 days growth in indirect coculture with OSCC cells, expression of CTSK mRNA by fibroblasts showed two folds increase as compared to normal control cells [Figure 8].
Figure 8: Amplification curves of mRNA for the control gene β-actin (yellow color) and CTSK gene (red color).

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  Discussion Top


As a solid tumor, OSCC has been progressively viewed as a complex of cancer cells and stromal cells that operate in a harmony towards tumor growth and evolution [22]. Out of this tumor milieu, a subpopulation of fibroblasts, known as CAFs, recognized as a prominent effector of cancer progression [23]. CAFs are activated fibroblasts with myofibroblastic features that facilitate its immunohistochemical detection by myoid markers such as α-SMA [12]. CAFs execute their tumor-promoting function through secreting a wide range of chemokines, inflammatory mediators, growth factors and matrix-degrading enzymes [24] including cysteine cathepsins [25]. CTSK is a lysosomal cysteine protease, first recognized as bone resorption mediator in osteoclasts [18]. It is known for its unique, potent collagenolytic activity [19] that can efficiently degrade a variety of collagens in a multitude of physiological and pathological conditions including cancer [25]. In the present study, CTSK expression was absent in normal mucosal keratinocytes as well as normal connective tissue fibroblasts which is in agreement with observations of Bitu et al. [21] and Yan et al.[26]. Furthermore, there was weak, sporadic CTSK immunostaining in chronic inflammatory cells at sites of inflammation in some control tissue specimens. Such observation could be justified by findings of Beklen et al. [27] who reported weak CTSK expression in macrophage-like cells in healthy gingival tissues that thought to contribute to physiologic tissue remodeling. Additionally, ECM homeostasis relies on a dynamic balance between synthesis and degradation of ECM proteins [28]. In this context, despite its lack of expression in normal oral mucosal tissues, CTSK proteolytic activity appears to play a role in re-balancing homeostasis during inflammation as demonstrated by its weak expression in chronic inflammatory cells [28]. In contrast to the healthy tissues, CTSK expression was evident in stromal CAFs in almost all OSCC specimens in the present study, which is consistent with previous observations of Bitu et al. [21] who found CTSK expression in invasive tongue SCC cells in addition to cells of the TME where it promoted cancer cell invasion. Moreover, a number of studies addressed CTSK expression in different tumors including lung adenocarcinoma [29], lung SCC [30] as well as invasive melanoma [31]. Accordingly, the data from all of these studies revealed that CTSK expressed by stromal cells resided in tumor vicinity correlated with tumor cell invasiveness and poor prognosis. In the same context, our indirect coculture study showed two-fold increase in CTSK mRNA expression by fibroblasts in coculture as compared to normal control fibroblasts. This finding would indicate that OSCC cells stimulated fibroblasts to augment CTSK production, which is analogous to findings of Xie et al. [32] who observed that SCC cells enhanced fibroblastic CTSK expression in indirect, noncontact coculture model. Furthermore, they clarified that SCC-derived IL-1α induced CTSK expression by CAFs with subsequent improved SCC invasive capabilities [32]. Taken together, the absence of CTSK in normal fibroblasts and its up-regulation in CAFs would reflect the reciprocal tumor–stromal interaction, which calls for stromal CTSK production by CAFs for the sake of promoting tumor invasion and progression. Not only stromal CAFs demonstrated CTSK expression, but also OSCC cells were CTSK positive in nearly all specimens. This finding is in consensus with previous observations of Bitu et al. [21] who detected CTSK expression in tongue SCC samples, which could suggest an instrumental role for CTSK in OSCC. To the best of our knowledge, there are no available data correlating CTSK expression to different OSCC grades. Our study showed significant correlation between CTSK expression in OSCC cells and increasing grade of malignancy. Such finding would propose a potential role for CTSK in OSCC progression. It was postulated that integral information concerning tumor invasive behavior could be inferred from the tumor invasive front, where perhaps the most aggressive cells reside [33],[34],[35]. This aggressiveness would be exemplified by disturbances in different mechanisms controlling cell differentiation, migration, apoptosis, as well as disordered epithelial–mesenchymal interaction at tumor–host interface, mostly at invasive fronts [35]. Furthermore, ECM proteolytic degradation is predominantly apparent at invasive tumor front reflecting the intimate association with tumor aggressive behavior [36]. In concordance with these concepts, the current study demonstrated enhanced CTSK expression in deep invasive tumor fronts with much prominent expression exhibited by CAFs as compared to OSCC cells. This would underscore the proinvasive role of CAFs by degrading ECM components thus rendering cancer cells with paths for invasion. Another support for these observations came from the work of Kurahara et al.[37], Thomas et al.[38], Hadler-Oslen et al. [39] and who speculated that tumor-linked matrix proteolysis and invasiveness relied more on the TME than being an inherent aspect of cancer cells. Furthermore, CTSK expression was detectable in chronic inflammatory cells within the stroma of all OSCC specimens, a finding that was in line with observations of Bitu et al.[21]. This could emphasize the propensity of CAFs to release proinflammatory cytokines, which attract inflammatory cells to tumor sites and stimulate them further to secrete proteases. This concept could be further confirmed by the notion that upregulation of proteases is prerequisite for activation of many pro-inflammatory cytokines in the TME [25]. Since cancer progression is dependent upon the evolving tumor–stroma cross-talk [40] and due to paucity of studies addressing role of CTSK in OSCC progression, despite its established implication in several other cancers [29],[30],[31],[32],[41],[42], this study tried to clarify whether CTSK released by OSCC cells conform to its expression by nearby CAFs. The present results showed a highly significant correlation (P < 0.01) between OSCC cell-derived CTSK and its expression by stromal CAFs. Comparable to these results, CTSK expression was evident in both tumor cells and tumor-associated fibroblasts in several skin cancers including SCC [37] and basal cell carcinoma [41], where it was found to enhance tumor invasion. In addition, our finding could be verified by the observations of Bitu et al.[21], which highlighted concomitant expression of CTSK by invasive tongue SCC cells and their surrounding stromal cells including CAFs. Consequently, considering the suggestion of Boelens et al. [43] who stated that paracrine communication between stromal cells and cancer cells brings about cancer progression, the present finding would suggest that OSCC cells collaborate with CAFs to promote invasion and progression of oral cancer. It would also indicate that CTSK expression is tightly regulated between OSCC cells and its stroma. Altogether, the results of this study suggest CTSK, a potent cysteine protease, with the potential to be an important effector in OSCC development, invasion and progression.


  Conclusion Top


It can be concluded that upregulation of CTSK expression by CAFs in OSCC, as well as the significant correlation between its expression in OSCC cells and increased malignancy grade propose an evolving role for CTSK in OSCC progression.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

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