Keywords: Organoid, Tonsil, HPV

Background: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide.1 The oropharynx, specifically palatine tonsil, is one of the most common sites of HNSCC, with varying presentations induced by human papillomavirus (HPV+) and those caused otherwise (HPV-). Clinically, these subsets of malignancy provide a unique environment for tumorigenesis and differential response to therapy. Previous studies by our research team demonstrated successful development of a 3D tonsillar organoid protocol that responds to treatment with chemotherapy and radiation.2-4

Objective: Currently, the literature is absent of a tonsillar organoid model that recapitulates B and T cell populations in a human tonsil. Our study seeks to investigate our tonsillar organoid model for existing immune cell populations, with plans on initiating immunotherapy treatments in future studies.

Methods: Previous description of our organoid development can be found in the literature.3 For the current study, pediatric tonsils with tonsillar hypertrophy were used for investigating media effects on organoid growth and immune populations. Pneumacult, Pneumacult and serum, RPMI, and RPMI and serum were used for selection of optimal organoid growth and immune cell presence. For experimental analysis of immune populations, HPV+ tumor and contralateral normal tonsil were collected from the same patient. All cells were collected at day zero and at the end of the protocol and subsequently stained with B and T cell panels for flow cytometry analysis.5

Results: Pediatric tonsil yielded 42.2%, 41.4%, 38%, and 38.6% B-cell’s in selected media, and 46.7%, 48.9%, 53.8%, 51.6% T-cell’s, respectively. Organoid growth was comparable between groups.

Conclusions: Pneumacult media with pediatric tonsil revealed abundant B and T-cell populations with successful organoid growth at day 5. The experimental protocol utilizing patient derived HPV+ tonsil tumor and contralateral normal tonsil for organoid growth in Pneumacult is currently underway.

1. Barsouk, A., Aluru, J. S., Rawla, P., Saginala, K., & Barsouk, A. (2023). Epidemiology, Risk Factors, and Prevention of Head and Neck Squamous Cell Carcinoma. Medical sciences (Basel, Switzerland), 11(2), 42. https://doi.org/10.3390/medsci11020042
2. Zhao, Z., Chen, X., Dowbaj, A.M. et al. Organoids. Nat Rev Methods Primers 2, 94 (2022). https://doi.org/10.1038/s43586-022-00174-y
3. Nguyen, H. T. L., & Soragni, A. (2020). Patient-Derived Tumor Organoid Rings for Histologic Characterization and High-Throughput Screening. STAR protocols, 1(2), 100056. https://doi.org/10.1016/j.xpro.2020.100056
4. Phan, N., Hong, J.J., Tofig, B. et al. A simple high-throughput approach identifies actionable drug sensitivities in patient-derived tumor organoids. Commun Biol 2, 78 (2019). https://doi.org/10.1038/s42003-019-0305-x
5. Sanz, I., Wei, C., Jenks, S. A., Cashman, K. S., Tipton, C., Woodruff, M. C., Hom, J., & Lee, F. E. (2019). Challenges and Opportunities for Consistent Classification of Human B Cell and Plasma Cell Populations. Frontiers in immunology, 10, 2458. https://doi.org/10.3389/fimmu.2019.02458