Abstract
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.
Highlights
Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance
Thirteen HNSCC samples were cultivated on dermal equivalents for up to 21 days (Figure S1)
A total of 6 out of HNSCC were cultivated for up to days, due to a pilot phase, in which the experimental setting was designed for 14 days of cultivation (HNSCC1, 2 and 3), detachment of dermal equivalent (DE) (HNSCC10), suspicion of contamination (HNSCC11) or small tumor mass and limited material for tissue explants (HNSCC13)
Summary
Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance. 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. DEs were co-cultivated with 13 vital HNSCC explants (non-human papillomavirus (HPV) driven, n = 7; HPV-driven, n = 6). Head and neck squamous cell carcinoma (HNSCC) is characterized by inter- and intra-tumorigenic heterogeneity [1] with a variable response to therapy and a high rate of treatment failure. Besides tobacco and alcohol consumption, infection with high-risk types of human papillomavirus (HPV), in particular HPV 16, is a risk factor for oropharyngeal squamous cell carcinoma (OPSCC) [4]. Reliable molecular biomarkers for early detection of primary and recurrent tumors, as well as prediction of treatment response, in HNSCC are sparse. There is an unmet need for predictive assays for ex ante analysis of the individual patient’s response to standard therapies, and to investigate the therapeutic efficacy of novel drugs
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