Background: Investigations into identifying novel immunohistochemical indicators are on the rise, aiming to elucidate tumor characteristics better. Therefore, this study examined the expression and distribution characteristics of nestin within the pleomorphic adenoma (PA) stromal component, and explored the potential mechanism underlying its formation, providing a comprehensive understanding of nestin's role as an immunohistochemical indicator.

Methods: This retrospective study included 51 patients diagnosed with PA of the parotid glands. The expression levels in the stromal component were assessed in patients' tissue specimens using immunostaining. Combined with computed tomography (CT) image analysis, the chi-square test, paired-sample t-test, Pearson's correlation coefficient, and principal component analysis (PCA) were used to explore the potential relationship between nestin expression characteristics and tumor parameters.

Results: Nestin was diffusely expressed in PA sections. The cellular arrangement in stromal components resulting from pseudopod-like protrusions appeared in “loose” and “reticular” patterns. These patterns were visualized through immunohistochemical staining, and the images were semi-quantitatively analyzed using ImageJ software. The two cellular arrangements showed statistical differences in the histomorphology of the stromal component, particularly between the “mucus-rich” and “cell-rich” (p < 0.05). CT imaging combined with Principal Component Analysis (PCA) revealed that the key factors affecting these two histomorphology types of stromal components were tumor area and CT value.

Conclusions: The formation of stromal components in PA is associated with changes in pseudopod-like cellular protrusions under different microenvironments. The tumor microenvironment and adjacent host tissue likely play a role in the development of PA. Furthermore, the “mucus-rich” structure tends to be more frequent in larger tumors. Nestin expression may contribute to maintaining the benign nature of PA, despite the stromal component being invasive. These results highlight the potential of nestin as an immunohistochemical marker, strongly associated with the formation and structural organization of stromal components.