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

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.

Snail and E47 repressors of E-cadherin induce distinct invasive and angiogenic properties in vivo.

The transcription factors Snail and E47 are direct repressors of E-cadherin, with both inducing a full epithelial-mesenchymal transition and invasive behaviour in vitro when expressed in the prototypic epithelial MDCK cell line. The role of these repressors in the invasive process and in other tumorigenic properties is, nevertheless, still poorly understood. However, organotypic cultures and in vivo transplantation assays indicate that cells expressing MDCK-Snail and MDCK-E47 exhibit significant differences. MDCK-Snail cells have a higher infiltrative potential than MDCK-E47 cells. Interestingly, both cell types induce angiogenesis of the host stromal tissue in transplantation assays, but this property is greatly enhanced in transplants of MDCK-E47 cells. Xenografted tumours induced in nude mice also show signs of strong angiogenic potential, again markedly increased in tumours induced by MDCK-E47 which exhibit a higher vessel density and proliferation rate than those induced by MDCK-Snail cells. These results suggest differential roles for Snail and E47 E-cadherin repressors in tumour progression where Snail is implicated in promoting the initial invasion and E47 plays an active role in tumour cell growth by promoting angiogenesis.

Discriminating expression of differentiation markers evolves in transplants of benign and malignant human skin keratinocytes through stromal interactions.

Accumulating evidence indicates a decisive role for the adjacent stroma in tumour growth and dissemination. However, it is not clear how far altered differentiation such as expression of aberrant keratins and vimentin, common in invasive human carcinomas, may reflect intrinsic cell properties or a response to the tumour environment. We have addressed this by transplanting benign and malignant human HaCaT-ras keratinocytes, seeded on collagen matrix, onto nude mice. Initially, epithelia derived from benign and malignant cells, being separated from host stroma by collagen, were poorly organized and exhibited the same differentiation markers, as identified by immunofluorescence and in situ hybridization. Epidermal basal and suprabasal keratins were expressed persistently even upon contact with newly formed stroma and malignant cell invasion. In contrast, non-epidermal keratins (K4/K13, K8/18, K19), which were similarly synthesized by benign and malignant cells in culture and in early transplants, were differentially regulated with increasing stromal vicinity. While both proteins and mRNAs were downregulated in benign epithelia, the malignant, invasive tumour cells continuously expressed these non-epidermal keratins throughout (K19), suprabasally (K4/13) or at invasive sites (K8/18). Furthermore, the mesenchymal protein vimentin was expressed de novo in invasive areas confronting tumour stroma. Thus, atypical tissue markers, similarly synthesized in isolated cells in vitro, are downregulated in benign but maintained and upregulated in malignant epithelia. This is presumably caused by the neighbouring stroma being permanently activated by malignant epithelia.