Optimization of a Three-Dimensional Culturing Method for Assessing the Impact of Cisplatin on Notch Signaling in Head and Neck Squamous Cell Carcinoma (HNSCC)

SIMPLE SUMMARY: Most experimental research on head and neck squamous cell carcinoma (HNSCC) relies on two- or three-dimensional (2D/3D) cell- and tissue-culture model systems. Commonly used methods like 2D monolayer cultures or 3D organoid models typically lack critical components of the tumor microenvironment (TME), such as cancer-associated fibroblast (CAFs), that likely influence chemosensitivity versus drug resistance of the tumor cells. In addition, based on the small experimental scale, it is often difficult to isolate a sufficient number of cells, proteins, or RNA from miniaturized 3D cultures for subsequent molecular analyses. In this manuscript, we describe a novel, more robust, and simultaneously larger-scale model system in which tumor cells and CAFs spontaneously generate tumor microtissues that mimic the architecture and histology of HNSCC biopsies. We have used this “3D sheet model” to investigate the assumed functional connection between NOTCH signaling and sensitivity to the chemotherapeutic drug cisplatin in HNSCC. ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type, with cisplatin being a primary treatment approach. However, drug resistance and therapy failure pose a significant challenge, affecting nearly 50% of patients over time. This research had two aims: (1) to optimize a 3D cell-culture method for assessing the interplay between tumor cells and cancer-associated fibroblasts (CAFs) in vitro; and (2) to study how cisplatin impacts the Notch pathway, particularly considering the role of CAFs. Using our optimized “3D sheet model” approach, we tested two HNSCC cell lines with different cisplatin sensitivities and moderate, non-mutated NOTCH1 and -3 expressions. Combining cisplatin with a γ-secretase inhibitor (crenigacestat) increased sensitivity and induced cell death in the less sensitive cell line, while cisplatin alone was more effective in the moderately sensitive line and sensitivity decreased with the Notch inhibitor. Cisplatin boosted the expression of core Notch signaling proteins in 3D monocultures of both lines, which was counteracted by crenigacestat. In contrast, the presence of patient-derived CAFs mitigated effects and protected both cell lines from cisplatin toxicity. Elevated NOTCH1 and NOTCH3 protein levels were consistently correlated with reduced cisplatin sensitivity and increased cell survival. Additionally, the Notch ligand JAG2 had additional, protective effects reducing cell death from cisplatin exposure. In summary, we observed an inverse relationship between NOTCH1 and NOTCH3 levels and cisplatin responsiveness, overall protective effects by CAFs, and a potential link between JAG2 expression with tumor cell survival.