Bio Protoc. 2026 Nov 5;16(21):e5861. doi: 10.21769/BioProtoc.5861. eCollection 2026 Nov 5.

ABSTRACT

Syngeneic and orthotopic injection of cancer cells in mice is a gold standard model in pre-clinical cancer research. Herein, we detail a protocol of orthotopic intratongue injection as a model for upper respiratory tract cancer studies. This model recapitulates tumor growth and invasion in an orthotopic site, the oral cavity mucosa. It can be experimentally designed in a syngeneic context or used to study patient-derived cells in immunocompromised animals. Procedurally, mice are anesthetized and injected with a small volume of cancer cell suspension near the lateral border of the tongue, right under the mucous membrane. Post-injection, animal welfare is carefully attended to by appropriate analgesia, while providing soft food for the duration of the experiment to ensure the ability of the mice to eat ad libitum. Tongues and potential metastatic organs are dissected and can be sampled and analyzed using relevant state-of-the-art methods, including -omics and imaging approaches. We describe a broad range of distinct tumor profiling and analysis methods applicable to this model. These include physical measurements of elasticity with atomic force microscopy on fresh tissue, hematoxylin-eosin staining and standard immunohistochemistry, and confocal microscopy applied to paraffin-embedded tongue sections. This model is orthotopic, accessible without surgery, easy to monitor, and rapid to perform. Key features • Orthotopic and clinically relevant head and neck squamous cell carcinoma (HNSCC) model. • Rapid, reproducible, and minimally invasive procedure. • Compatible with multimodal downstream analyses. • Versatile platform for preclinical studies.

PMID:43022610 | PMC:PMC13757187 | DOI:10.21769/BioProtoc.5861