Organotypic Co-Cultures as a Novel 3D Model for Head and Neck Squamous Cell Carcinoma

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.

Ex Vivo Culture Models to Indicate Therapy Response in Head and Neck Squamous Cell Carcinoma

“…Contamination with fibroblasts could also be a practical challenge, since fibroblasts are able to overgrow the culture because of their high proliferation rate [86,87]. For primary cultures of HNSCC specifically, it remains a challenge to successfully culture and maintain HPV-positive cells and tissues in vitro [49,88,89]. The exact explanation is still unknown, but it is thought that tumor cells must have acquired traits or mutations compatible with survival and immortality to be able to survive in the unnatural in vitro environment.…”

“…In contrast, the often relatively quick occurrence of tissue deterioration during culturing might influence the outcome of drug sensitivity assays [36]. Improving tissue viability over time, for example by the use of a dermal equivalent (consisting of viscose fibers and human-derived fibroblasts) as tissue support, could increase reliability of the histoculture model and allow for prolonged ex vivo drug exposure [49]. In addition, microdevices might offer a chance to increase tissue viability by providing a controlled culture environment and continuous perfusion and nutrient supply to the tumor tissue.…”

“…This makes them not suitable for culturing for extended periods of time at this moment. Tissue viability might be prolonged by the use of a supportive matrix [49]. Nevertheless, the histoculture model is hampered by a relatively short viable period of 3-6 days and other disadvantages such as low throughput and low reproducibility.…”

“…The success percentage of the histoculture model varies from 59% to 100%, with an average of 90% and a median of 98%, as reported by 21 out of 29 articles. Studies describe a culture duration 1 up to 21 days [32,49,82]. In general, the average culture duration of histocultures was 5 days, with a median of 5 days.…”

“…The most recent study on HNSCC histocultures by Engelmann et al described the longest culture duration so far [49]. With the use of a dermal equivalent (DE), they were able to maintain tumor explants of all their non-HPV-driven HNSCCs up to 21 days in vitro.…”