Background: Head and neck squamous cell carcinomas (HNSCC) are phenotypically and
molecularly heterogeneous and frequently develop therapy resistance. Reliable patient-derived 3D
tumor models are urgently needed to further study the complex pathogenesis of these tumors and
to overcome treatment failure. Methods: We developed a three-dimensional organotypic co-culture
(3D-OTC) model for HNSCC that maintains the architecture and cell composition of the individual
tumor. A dermal equivalent (DE), composed of healthy human-derived fibroblasts and viscose fibers,
served as a scaffold for the patient sample. DEs were co-cultivated with 13 vital HNSCC explants
(non-human papillomavirus (HPV) driven, n = 7; HPV-driven, n = 6). Fractionated irradiation was
applied to 5 samples (non-HPV-driven, n = 2; HPV-driven n = 3). To evaluate expression of ki-67,
cleaved caspase-3, pan-cytokeratin, p16INK4a, CD45, ∝smooth muscle actin and vimentin over time,
immunohistochemistry and immunofluorescence staining were performed Patient checkup data
were collected for up to 32 months after first diagnosis. Results: All non-HPV-driven 3D-OTCs
encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of
primaries and 3D-OTCs were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth patterns (i.e., invasive, expansive, silent). Cancer-associated fibroblasts and
leukocytes could be detected for up to 21 days. HPV DNA was detectable in both primary and
3D-OTCs (day 14) of HPV-driven tumors. However, p16INK4a expression levels were varying.
Morphological alterations and radioresistant tumor cells were detected in 3D-OTC after fractionated
irradiation in HPV-driven and non-driven samples. Conclusions: Our 3D-OTC model for HNSCC
supports cancer cell survival and proliferation in their original microenvironment. The model enables
investigation of invasive cancer growth and might, in the future, serve as a platform to perform
sensitivity testing upon treatment to predict therapy response
