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ORIGINAL RESEARCH
Year : 2014  |  Volume : 1  |  Issue : 1  |  Page : 14-17

Assessment of collagen fiber nature, spatial distribution, hue and its correlation with invasion and metastasis in oral squamous cell carcinoma and surgical margins using Picro Sirius red and polarized microscope


Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Dental College and Hospital, Pimpri, Pune, Maharashtra, India

Date of Web Publication31-Jan-2014

Correspondence Address:
Ketki K Kalele
Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Dental College and Hospital, Pimpri, Pune, Maharashtra
India
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Source of Support: Department of Oral Pathology and Microbiology. DPU Pune, Conflict of Interest: None


DOI: 10.4103/2348-3172.126159

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  Abstract 

Introduction: Oral squamous cell carcinoma (OSCC) comprises a bulk of all the oral malignancies and is posing a major health problem among the population. It is an established fact that tumor stroma plays a vital role in tumor progression. Therefore, methods to detect, quantify, and analyze collagen are of immense value in this regard. Picro Sirius red, which has the capability to detect thin fibers, although frequently used, is seldom exploited to the fullest extent. Aim: Our goal is not only to identify nature of fibers, but also to assess the fiber hue and the spatial distribution of different colors in various grades of OSCC and correlate it with the metastasis of the tumor. The study has also analyzed the nature of stromal elements along the clear, close and involved surgical margins of OSCC. Materials and Methods: Ten cases each of well, moderately and poorly differentiated OSCC as well as clear, close and involved margins were stained with haematoxylin eosin and picrosirius red staining for evaluation under polarized microscope. Results: In this study we found that the birefringence of the collagen fiber changed from orange red to yellowish green from well to poorly differentiated OSCC. The collagen fibers in well-differentiated carcinoma revealed polarizing colors of reddish orange around the tumor islands in the majority of the fields. To the best of our knowledge is not being studied so far in the English literature. Conclusion: In the present study, it has been observed that stromal changes at the invading front of the tumor islands and with increasing grade of the tumor can be evaluated more efficiently with the use of Picro Sirius red stain.

Keywords: Metastasis, Picro Sirius red, polarized microscope


How to cite this article:
Kalele KK, Managoli NA, Roopa N M, Kulkarni M, Bagul N, Kheur S. Assessment of collagen fiber nature, spatial distribution, hue and its correlation with invasion and metastasis in oral squamous cell carcinoma and surgical margins using Picro Sirius red and polarized microscope. J Dent Res Rev 2014;1:14-7

How to cite this URL:
Kalele KK, Managoli NA, Roopa N M, Kulkarni M, Bagul N, Kheur S. Assessment of collagen fiber nature, spatial distribution, hue and its correlation with invasion and metastasis in oral squamous cell carcinoma and surgical margins using Picro Sirius red and polarized microscope. J Dent Res Rev [serial online] 2014 [cited 2019 Dec 14];1:14-7. Available from: http://www.jdrr.org/text.asp?2014/1/1/14/126159


  Introduction Top


Collagen which is a chief component of the extra cellular matrix (ECM) plays a vital role in maintenance of structural integrity and tissue function of the body. Stromal components are the key factors for provision of nutrition and growth to any tumor as also they act as a barrier for spread of the tumor. It is a known fact that migration of normal cells is controlled by proteolysis of the ECM. Proteolytic remodeling of the ECM facilitates invasion of tumor cells in cancer, which further leads to an abundant change in the collagenous stroma promoting tumor progression. [1]

Therefore, methods to detect, quantify, and evaluate the nature of collagen in the stroma carries significance in this regard. Routine stains employed to stain collagen have disadvantages, which primarily includes poor specificity for thin fibers. In contrast, Picro Sirius red has the capability to detect thin fibers and also to differentiate mature and immature fibers. Although this special stain is frequently used, it is seldom utilized to the fullest extent. [2]

Determination of the cause(s) and effect(s) of the stromal response will further add on to our understanding of tumor cell interactions with stroma, and will help us to recognize prognostic indicators for patients with oral squamous cell carcinoma (OSCC). Thorough understanding of the stromal remodeling/change in nature of collagen fibers and their association with grade or aggressiveness of tumor can help us to target our existing treatments more effectively. The examination of collagen fibers by Picro Sirius red serves as a procedure to differentiate mature and immature fibers. [3]

Aim and objectives

Assessment of collagen fiber nature, spatial distribution, hue and its correlation with the grade in OSCC using Picro Sirius red and polarized microscope. To observe the changes in collagen fiber in various grades of OSCC, and in clear, close and involved surgical margins.


  Materials and Methods Top


Ten cases of histologically diagnosed well, moderate and poorly differentiated OSCC as well as surgical margins (clear, close and involved) were taken from the histopathological records. Two sections of normal buccal mucosa as the control were also obtained from the archives of the Department of Oral Pathology, Dr. D.Y. Patil Dental College, Pune. The sections were stained with hematoxylin and eosin [Figure 1], [Figure 2], [Figure 3] and also Picro Sirius red (Sirius red F3B (C.I.35780) in a saturated picric acid solution. [4]
Figure 1: Photomicrograph of well-differentiated OSCC (×40)

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Figure 2: Photomicrograph of moderately differentiated OSCC (×40)

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Figure 3: Photomicrograph of moderately differentiated OSCC (×40)

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Analysis of collagen

Picro Sirius red stained sections are observed under a polarizing microscope. A total of 10 high power fields from each section are visualized and their color was noted by three blind observers for interpretation and comparison of collagen fiber hue along the various grades of OSCC and surgical margins [Table 1].
Table 1: Assessment criteria based on the hue

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  Results Top


Assessment criteria

In this study we found that the birefringence of the collagen fiber changed from orange red to yellowish green from well [Figure 4] to poorly differentiated OSCC [Figure 5] and [Figure 6]. The collagen fibers in well-differentiated carcinoma revealed polarizing colors of reddish orange around the tumor islands in the majority of the fields.
Figure 4: Photomicrograph of well-differentiated squamous cell carcinoma showing reddish birefringence in Picro Sirius red stain (×4 magnification)

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Figure 5: Photomicrograph of moderately differentiated squamous cell carcinoma showing yellowish red birefringence in Picro Sirius red stain (×4 magnification)

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Figure 6: Photomicrograph of poorly differentiated squamous cell carcinoma showing greenish birefringence in Picro Sirius red stain (×4 magnification)

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A gradual change in the polarizing colors from yellowish orange to greenish yellow was seen in moderately differentiated carcinoma. However, in poorly differentiated squamous cell carcinoma predominantly showed greenish yellow polarizing color and weak birefringence of this color was seen surrounding the tumor islands.

A similar color variance was noted in clear, close and involved surgical margins, respectively. One striking feature we observed in the section of close margin stroma surrounding the tumor islands was, it showed yellowish green and the rest of the normal tissue around tumor islands showed weak yellowish red color.


  Discussion Top


Stromal elements surrounding the tumor (Tumor stroma), which includes inflammatory cells, endothelial cells and fibroblasts, play a very important role in tumor progression as stated throughout the literature. Stroma consisting of myofibroblast-driven reaction, predominantly contains fibrillar collagens (types I and III), are stated to be a poor prognostic indicator. [5] ECM provides the vascular supply, nourishment, oxygen supply, and also limits the influx of inflammatory cells, thereby acting as a barrier for immunological rejection. [3]

Collagen is a triple helical structure and its molecules are rich in basic amino acids, thus they have strong affinity toward acidic dyes giving strong reaction with the acidic dyes. Sirius red is a strongly acidic azo dye which contains six sulfonic groups, with a molecular weight of 1372; hence, it is effectively used to stain collagen in tissue sections. [6]

Biochemical structure of Sirius red which is an elongated dye molecule is such that it reacts with collagen and amplifies its normal birefringence. This is because many dye molecules are parallely aligned with the long axis of each collagen molecule. [6] Also, it has been stated that this dye binds to collagen through a strong interaction of its acid sulfonic groups to collagen molecules.

This study examines spectrum of collagen fiber birefringence in various grades of OSCC along with surgical margins. Our results are consistent with the study done by Venigella and Charu, the results of which showed that there was a gradual change in the polarizing color along with advancing grade of the tumor from yellowish orange to greenish yellow. The color change was boldly noticed immediately adjacent to the invading islands of tumor. [3] One of the causes for this can be linked to the deposition of thick bands collagen fibers which are composed of densely packed fibrils. Type I collagen fibers (thick fibers) showed a strong birefringence of red, orange and yellow color and a weak birefringence of green when the fibers were Type III collagen (thin fibrillar) by polarizing microscopy. [7] In this study, birefringence of the collagen fiber changed from orange red to yellowish green from well to poorly differentiated OSCC. A similar color variance was noted in clear, close and involved margins, respectively. In the section of close margin stroma surrounding the tumor islands showed yellowish green and the rest of the tissue showed weak greenish red color.

According to Junqueira et al. and Montes et al. [8],[9] the color change can be attributed to the carcinogenic events, action of MMP's, pathological breakdown of the matrix by the tumor cells, abortive stroma, thereby promoting tumor progression. Thus, collagen profiling can be effectively used to correlate the qualitative nature of collagen to the progression and clinical behavior of OSCC. Thus, the color changes seen in the present study are clear indicators of some stromal tissue alterations taking place around the tumor cells, which can be related to the carcinogenic events taking place during tumorigenesis.

Such changes in the collagen of the stromal tissue are being studied by different authors on various other malignancies. Breast cancer studies [10] have shown that as there is increase in the collagen content of the extracellular matrix the more is the mechanical stiffness of the stromal tissue, which also impart transport resistance to the tumors. As, also a study on myocardial infarction concluded that the collagen degradation and loss after myocardial infarction is associated with infarct expansion and followed by a functional decline. [11] One of the studies conducted on human osteosarcomas by Junqueira et al.[12] inferred that, both type I and III collagens were present in the fibroblastic areas of the tumor: In the anaplastic areas Type III and in the chondroblastic areas Type II collagen dominated in osteosarcomas.


  Conclusion Top


In the present study, it has been observed that stromal changes at the invading front of the tumor islands and with increasing grade of the tumor can be evaluated more efficiently with the use of Picro Sirius red stain. This finding further can prove to be an important factor in predicting tumor behavior. Determination of collagen fiber nature in different grades of oral squamous cell can help for targeting the stroma for various treatment strategies. Further research with larger sample size is required in this direction.

 
  References Top

1.Rich L, Whittaker PC, Picrosirius RS. A polarized light assessment of fibrillar hue and spatial distribution. Braz J Morphol Sci 2005;22:97-104.  Back to cited text no. 1
    
2.Junqueira LC, Cossermelli W, Brentani R. Differential staining of collagens type i, ii and iii by sirius red and polarization microscopy. Arch Histol Jpn 1978;41:267-74.  Back to cited text no. 2
[PUBMED]    
3.Venigella A, Charu S. Evaluation of collagen in different grades of oral squamous cell carcinoma by using the picrosirius red stain: A histochemical study. J Clin Diagn Res 2010;4:3444-9.  Back to cited text no. 3
    
4.Montes GS, Junqueira LC. The use of Picrosirius-Polarization method for the study of the biopathology of collagen. Mem Inst Oswaldo Cruz 1991;86: Suppl 3:S1-11.  Back to cited text no. 4
    
5.De Wever O, Demetter P, Mareel M, Bracke M. Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer 2008;123:2229-38.  Back to cited text no. 5
[PUBMED]    
6.Dayan D, Hiss Y, Hirshberg A, Bubis JJ, Wolman M. Are the polarization colours of Picrosirius red-stained collagen determined only by the diameter of the fibres. Histochemistry 1989;93:27-9.  Back to cited text no. 6
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7.Allon I, Vered M, Buchner A, Dayan D. Stromal differences in salivary gland tumours of a common histopathogenesis but with different biological behaviour: A study with picrosirius red and polarizing microscopy. Acta Histochem 2006;108:259-64.  Back to cited text no. 7
[PUBMED]    
8.Montes GS, Krisztan RM, Shigihara KM, Tokoro R, Mourão PA, Junqueira LC. Histochemical and morphological characterization of reticular fibres. Histochemistry 1980;65:131-41.  Back to cited text no. 8
    
9.van den Hooff A. Stromal involvement in malignant growth. Adv Cancer Res 1988;50:159-96.  Back to cited text no. 9
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10.Monsky WL, Mouta Carreira C, Tsuzuki Y, Gohongi T, Fukumura D, Jain RK. Role of host microenvironment in angiogenesis and microvasular functions in human breast cancer xenografts: Mammary fat pad versus cranial tumours. Clin cancer Res 2002;8:1008-13.  Back to cited text no. 10
[PUBMED]    
11.Whittaker P, Boughner DR, Kloner RA. Role of collagen in acute myocardial infarct expansion. Circulation 1991;84:2123-34.  Back to cited text no. 11
[PUBMED]    
12.Jonquiere LC, Assis Figueiredo MT, Torloni H, Montes GS. Differential histologic diagnosis of osteoid: A study on human osteosarcoma collagen by the histochemical picrosirius-polarization method. J Pathol 1986;148:189-96.  Back to cited text no. 12
    


    Figures

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

  [Table 1]


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