A quantitative and qualitative comparative analysis of collagen fibers to determine the role of connective tissue stroma in oral squamous cell carcinoma using special stains and polarized microscopy

Abstract

Background: Solid tumors such as oral squamous cell carcinoma (OSCC) are composed of malignant epithelial cells and the stroma in which these cells are dispersed. As the tumor progresses, the extracellular matrix undergoes dramatic morphological and architectural changes. Special stains make analysis easy and less erroneous by highlighting the area of interest and can be used to study these changes.

Aim: The aim of the study was to analyze morphological changes in collagen fibers in various histological grades of OSCC using Masson's trichrome (MT) and Picrosirius red (PSR).

Study design: The study comprised 74 tissue samples, divided into two groups: Group I consisted of 63 cases of histologically proven OSCC (39 cases of well-differentiated squamous cell carcinoma [WDSCC], 17 moderately differentiated squamous cell carcinoma [MDSCC] and 7 poorly differentiated squamous cell carcinoma [PDSCC]) and Group II consisted of 11 cases of normal mucosa as controls.

Materials and methods: Sections were stained with hematoxylin and eosin, MT and PSR and observed under light and polarizing microscope, respectively.

Statistical analysis: ANOVA, Tukey's honestly significant difference post hoc multiple comparison test, Chi-square test and paired t-test were used for the statistical analysis.

Results: As the grade of OSCC progressed, collagen fibers became thin, loosely packed and haphazard. The mean area fraction also decreased. They exhibited orange-red hue and strong birefringence in WDSCC, yellowish-orange hue and strong birefringence in MDSCC and greenish-yellow hue and weak birefringence in PDSCC.

Conclusion: Initially, there is a reorganization of the collagen fibers in an attempt to prevent the invasion of tumor cells, but as cancer progresses, the stromal change enhances movement of the tumor cells within it, leading to metastasis.

Keywords: Collagen; masson's trichrome; oral squamous cell carcinoma; picrosirius red; polarizing microscopy; tumor microenvironment.

Figure 1

Histopathological image of Masson's trichrome-stained section of well-differentiated oral squamous cell carcinoma showing densely packed collagen fibers (arrow) exhibiting parallel arrangement (×400)

Figure 2

Histopathological image of Masson's trichrome-stained section of moderately differentiated oral squamous cell carcinoma showing haphazardly arranged loosely packed collagen fibers (arrow) at ×400

Figure 3

Histopathological image of Masson's trichrome-stained section of poorly differentiated oral squamous cell carcinoma showing haphazardly arranged loosely packed collagen fibers (arrow) at ×400

Figure 4

Histopathological image of Picrosirius red-stained section of well-differentiated oral squamous cell carcinoma showing densely packed collagen fibers (arrow) exhibiting parallel arrangement and orange–red birefringence (×400)

Figure 5

Histopathological image of Picrosirius red-stained section of moderately differentiated oral squamous cell carcinoma showing haphazardly arranged loosely packed collagen fibers (arrow) exhibiting yellowish-orange birefringence (×400)

Figure 6

Histopathological image of Picrosirius red-stained section of poorly differentiated oral squamous cell carcinoma showing haphazardly arranged loosely packed collagen fibers (arrow) exhibiting greenish-yellow birefringence (×400)