Optimizing Preclinical Models for Oral Cancer: The Influence of 4NQO Administration Routes on Tumor Development

Abstract

Citation: Van den Bosch, J.; Caz, N.; Martens, S.; Erens, C.; Rasking, L.; Gervois, P.; Nijsten, K.; Himmelreich, U.; Van Cauter, S.; Hillen, L.M.; et al. Optimizing Preclinical Models for Oral Cancer: The Influence of 4NQO Administration Routes on Tumor Development. Cancers 2025, 17, 2108. These authors contributed equally to this work. Simple Summary Understanding how oral cancer develops and progresses is essential to improve treatment strategies. This study focuses on a commonly used animal model for oral cancer that closely mimics the stages of human disease, from early tissue changes to invasive tumors. By tracking tumor growth over time, researchers can identify key moments when treatment may be most effective. However, without careful observation, important aspects of tumor evolution may be overlooked. By combining MRI and microscopic analysis at different time points, tumor development can be tracked. This approach enables assessment of disease progression, helping refine the use of this model in preclinical research. Abstract Background/Objectives: Oral squamous cell carcinoma (OSCC) is the most common oral cancer, progressing from hyperplasia to dysplasia, carcinoma in situ (CIS), and finally invasive squamous cell carcinoma (ISCC). Developing an animal model that mimics both early and advanced OSCC stages has been challenging. The 4-Nitroquinoline 1-oxide (4NQO) model is considered one of the most suitable, as it represents all stages of OSCC. Nevertheless, thoroughly understanding the properties of the 4NQO model is essential for preclinical testing of novel therapeutics. Methods: We aimed to characterize the 4NQO rat model using two application methods-drinking water and topical application-over eight months. Monthly sacrifices allowed histopathological analysis and ex vivo magnetic resonance imaging (MRI) to track tumor progression. Results: CIS was observed at three months in the drinking water group, evolving into ISCC by six months, while topical application induced CIS at eight months without ISCC formation. The tongue was divided into three regions and histological properties, lesion size, and invasion depth Cancers 2025, 17, 2108 https://doi.org/10.3390/cancers17132108 Cancers 2025, 17, 2108 2 of 20 were analyzed. In the drinking water group, particularly in the body of the tongue, we saw earlier CIS development, larger lesions, and deeper invasion. Additionally, assessment of proliferative properties showed an increased cell division in dysplastic lesions that reduced upon invasion. MRI was able to show macroscopic tumoral lesions, in concordance with histology. Conclusions: Overall, the drinking water method closely mimics human OSCC, validating the 4NQO model for translational OSCC research.