Organoids in Head and Neck Cancer

Abstract

Head and neck cancer (HNC) is the seventh most common malignancy worldwide, with approximately one million new cases and 465,000 deaths in 2020. More than 95% of these tumors are head and neck squamous cell carcinomas (HNSCC), arising from the mucosal epithelium of the upper aerodigestive tract. Despite therapeutic advances, including targeted agents, 5-year survival has improved only modestly to around 66%. One major limitation is the absence of robust biomarkers capable of predicting treatment outcome and guiding therapy selection. Traditional in vitro models inadequately reflect tumor heterogeneity and 3D architecture, emphasizing the need for physiologically relevant systems such as organoids. In Chapter 2, many biomarkers in the EGFR–PI3K–AKT–mTOR pathway are reviewed. Although several candidates have shown promise, only a few have been confirmed in clinical trials. Progress is hindered by small sample sizes, patient heterogeneity, and inconsistent treatment protocols. A more systematic and patient-centered approach—integrating biomarker research within clinical trials—is urgently needed. Chapter 3 describes expanding the biobank of long-term HNSCC organoids established by Driehuis et al. from 31 to 110 models, including additional HNC subtypes. In an adjuvant radiotherapy cohort, radioresistant organoids corresponded to earlier patient relapse (n=15), whereas cisplatin sensitivity did not correlate with clinical outcome. Drug screening showed that cisplatin and carboplatin generally increased radiosensitivity, consistent with clinical practice, while cetuximab reduced radiosensitivity, reflecting clinical findings of poorer outcomes with cetuximab-based chemoradiation. Nevertheless, most organoids still demonstrated higher toxicity with cetuximab plus radiotherapy compared to radiotherapy alone. Chapter 3 also reports organoid responses to targeted therapies. Although PIK3CA mutations are proposed biomarkers for alpelisib, PIK3CA-mutant organoids did not respond significantly better than wild-type models. Using CRISPR base editing, we generated isogenic pairs carrying the common PIK3CA E545K mutation. Variation between patients exceeded the mutation-specific effect, highlighting the importance of patient heterogeneity when interpreting biomarker performance. In Chapter 4, high p-mTOR expression in HPV-negative HNSCC patients treated with primary chemoradiotherapy was associated with significantly worse overall survival. In parallel, p-mTOR levels in organoids correlated with sensitivity to the mTOR inhibitor everolimus, underscoring the translational potential of organoids for predicting targeted therapy responses. Chapter 5 examines the mechanism of jawbone invasion in oral squamous cell carcinoma (OSCC). The findings suggest that OSCC promotes osteoclast activation through regulation of RANKL and RANK. HNSCC organoids expressed RANKL, OPG, and RANK, demonstrating their utility for studying bone-invasion biology and identifying potential therapeutic targets such as RANKL inhibition. However, the clinical relevance of denosumab in HNC remains uncertain. Finally, Chapter 6 investigates factors influencing organoid establishment. Organoids derived from older patients grew less effectively, while no other clinical parameters correlated with outgrowth. Pre-selecting samples with adequate epithelial cell content increased organoid establishment to 75.2%, indicating that improved tissue collection strategies can substantially enhance success rates.