Patient-derived 3D organotypic co-cultures as a long-term ex vivo model for pleomorphic adenoma of the parotid gland

Pleomorphic adenoma is the most common benign salivary gland tumor and most frequently arises in the parotid gland. Although benign, it can recur and may undergo malignant transformation, underscoring the need for experimental models that preserve native tissue features over time. In this study, fresh tumor tissue from six patients with primary histologically benign pleomorphic adenoma was divided into fragments of approximately 3 × 3 mm in surface dimensions and approximately 3 mm in thickness and maintained in a patient-derived three-dimensional organotypic co-culture for up to 21 days. The primary longitudinal analysis compared baseline tissue with matched fragments harvested at days 14 and 21. Histology and immunohistochemistry were assessed in three randomly selected high-power fields within morphologically viable, tumor-containing regions; necrotic or degenerative areas were excluded. Ki-67-positive cell nuclei remained within the predefined low category at all retained time points. PLAG1 positivity was retained in all cases but was more variable by day 21, while epithelial and myoepithelial-associated markers showed marker- and case-dependent patterns. CD45-positive cells remained detectable in sampled viable compartments, whereas SSTR2 was commonly reduced after baseline. These findings support the use of the 3D-OTC platform for longitudinal histomorphologic and selected biomarker assessment within surviving viable tissue compartments. Because whole-fragment necrosis and diffusion of oxygen, nutrients, or test compounds were not quantified, the present data do not establish uniform full-thickness viability or validate the platform for therapeutic-response testing.