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, p16^INK4a, 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, p16^INK4a 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.

Background: Head and neck squamous cell carcinoma (HNSCC) patients frequently develop treatment resistance to cetuximab, a monoclonal antibody against EGFR, as well as radiotherapy. Here we addressed extracellular signal-regulated kinase 1/2 (ERK1/2) regulation by cetuximab or fractionated irradiation (IR) and conducted in silico prognostic evaluation of the EGFR-MAPK axis in HNSCC. Methods: Expression of ERK1/2 phosphorylation (pERK1/2) was determined in HNSCC cell lines, which were treated with cetuximab or fractionated-IR. Furthermore, the effect of fractionated IR on pERK1/2 was confirmed in an ex vivo HNSCC tissue culture model. Expression and prognostic significance of EGFR-ERK axis was evaluated in a cohort of radiotherapy plus cetuximab-treated HNSCC. Correlations among EGFR-MAPK signalling components and association between transcript and protein expression profiles and patient survival in HNSCC were analysed using publicly available databases. Results: ERK1/2 phosphorylation was rebounded by prolonged cetuximab administration and was induced by fractionated IR, which could be suppressed by a MEK inhibitor as a radiosensitiser. In silico assessments suggested that EGFR-MAPK cascade genes and proteins could predict HNSCC patients’ survival as a prognostic signature. Conclusions: Activation of ERK1/2 signalling contributes to the cellular defence of HNSCC against cetuximab and fractionated IR treatment. EGFR-MAPK axis has a prognostic significance in HNSCC.

Despite the current progress in the development of new concepts of precision medicine for head and neck squamous cell carcinoma (HNSCC), in particular targeted therapies and immune checkpoint inhibition (CPI), overall survival rates have not improved during the last decades. This is, on the one hand, caused by the fact that a significant number of patients presents with late stage disease at the time of diagnosis, on the other hand HNSCC frequently develop therapeutic resistance. Distinct intratumoral and intertumoral heterogeneity is one of the strongest features in HNSCC and has hindered both the identification of specific biomarkers and the establishment of targeted therapies for this disease so far. To date, there is a paucity of reliable preclinical models, particularly those that can predict responses to immune CPI, as these models require an intact tumor microenvironment (TME). The “ideal” preclinical cancer model is supposed to take both the TME as well as tumor heterogeneity into account. Although HNSCC patients are frequently studied in clinical trials, there is a lack of reliable prognostic biomarkers allowing a better stratification of individuals who might benefit from new concepts of targeted or immunotherapeutic strategies. Emerging evidence indicates that cancer stem cells (CSCs) are highly tumorigenic. Through the process of stemness, epithelial cells acquire an invasive phenotype contributing to metastasis and recurrence. Specific markers for CSC such as CD133 and CD44 expression and ALDH activity help to identify CSC in HNSCC. For the majority of patients, allocation of treatment regimens is simply based on histological diagnosis and on tumor location and disease staging (clinical risk assessments) rather than on specific or individual tumor biology. Hence there is an urgent need for tools to stratify HNSCC patients and pave the way for personalized therapeutic options. This work reviews the current literature on novel approaches in implementing three-dimensional (3D) HNSCCin vitroandin vivotumor models in the clinical daily routine. Stem-cell based assays will be particularly discussed. Those models are highly anticipated to serve as a preclinical prediction platform for the evaluation of stable biomarkers and for therapeutic efficacy testing.

Immunotherapy has evolved into a powerful tool in the fight against a number of types of cancer, including head and neck squamous cell carcinomas (HNSCC). Although checkpoint inhibition (CPI) has definitely enriched the treatment options for advanced stage HNSCC during the past decade, the percentage of patients responding to treatment is widely varying between 14‑32% in second‑line setting in recurrent or metastatic HNSCC with a sporadic durability. Clinical response and, consecutively, treatment success remain unpredictable in most of the cases. One potential factor is the expression of target molecules of the tumor allowing cancer cells to acquire therapy resistance mechanisms. Accordingly, analyzing and modeling the complexity of the tumor microenvironment (TME) is key to i) stratify subgroups of patients most likely to respond to CPI and ii) to define new combinatorial treatment regimens. Particularly in a heterogeneous disease such as HNSCC, thoroughly studying the interactions and crosstalking between tumor and TME cells is one of the biggest challenges. Sophisticated 3D models are therefore urgently needed to be able to validate such basic science hypotheses and to test novel immuno‑oncologic treatment regimens in consideration of the individual biology of each tumor. The present review will first summarize recent findings on immunotherapy, predictive biomarkers, the role of the TME and signaling cascades eliciting during CPI. Second, it will highlight the significance of current promising approaches to establish HNSCC 3D models for new immunotherapies. The results are encouraging and indicate that data obtained from patient‑specific tumors in a dish might be finally translated into personalized immuno‑oncology.

Although checkpoint inhibitors (CPI) have recently extended the treatment options and improved clinical response of advanced stage head and neck squamous cell carcinoma (HNSCC), treatment success remains unpredictable. Programmed cell death ligand‑1 (PD‑L1) is a key player in immunotherapy. Tumor cells, and exosomes derived therefrom, are carriers of PD‑L1 and efficiently suppress immune responses. The aim of the present study was to analyze the influence of established therapies on PD‑L1 expression of HNSCC cell lines and their exosomes. The HNSCC cell lines, UM‑SCC‑11B, UM‑SCC‑14C and UM‑SCC‑22C were treated with fractionated radiotherapy (RT; 5x2 Gy), cisplatin (CT) and cetuximab (Cetux) as monotherapy, or combined therapy, chemoradiotherapy (CRT; RT and CT) or radioimmunotherapy (RT and Cetux). The expression of PD‑L1 and phosphorylated (p)ERK1/2 as a mediator of radioresistance were assessed using western blotting, immunohistochemistry and an ex vivo vital tissue culture model. Additionally, exosomes were isolated from concentrated supernatants of the (un‑)treated HNSCC cell lines by size exclusion chromatography. Exosomal protein expression levels of PD‑L1 were detected using western blotting and semi‑quantitative levels were calculated. The functional impact of exosomes from the (un‑)treated HNSCC cell lines on the proliferation (MTS assay) and apoptosis (Caspase 3/7 assay) of the untreated HNSCC cell lines were measured and compared. The HNSCC cell lines UM‑SCC‑11B and UM‑SCC‑22B showed strong expression of pERK1/2 and PD‑L1, respectively. RT upregulated the PD‑L1 expression in UM‑SCC‑11B and UM‑SCC‑14C and in exosomes from all three cell lines. CT alone induced PD‑L1 expression in all cell lines. CRT induced the expression of PD‑L1 in all HNSCC cell lines and exosomes from UM‑SCC‑14C and UM‑SCC‑22B. The data indicated a potential co‑regulation of PD‑L1 and activated ERK1/2, most evident in UM‑SCC‑14C. Exosomes from irradiated UM‑SCC‑14C cells protected the unirradiated cells from apoptosis by Caspase 3/7 downregulation. The present study suggested a tumor cell‑mediated regulation of PD‑L1 upon platinum‑based CRT in HNSCC and in exosomes. A co‑regulation of PD‑L1 and MAPK signaling response was hypothesized.

Fractionated irradiation causes premature senescence of tumor cells. Interactions between senescence, the immune system and survival signaling are poorly understood to date. As MAP kinases are implicated in immune resistance, the present study addressed the detection of senescence‑associated modulation of postradiogenic programmed death‑ligand 1 (PD‑L1) and MAP kinase ERK1/2 expression in an in vitro and ex vivo model for head and neck squamous cell carcinoma (HNSCC). Established HNSCC cell lines (UM-SCC-11B, UM-SCC-14C and UM-SCC-22B) were employed to study the expression levels of p21, histone H2AX (γH2AX), PD-L1 and phosphorylated (p)ERK1/2 via immunohistochemistry following application of 4x2 Gy. Using senescence‑associated β‑galactosidase (SA‑ß‑Gal) staining, postradiogenic induction of senescence was additionally assessed. Results were validated in a 3D ex vivo HNSCC model with vital explants. Upon ionizing radiation (IR), senescence‑like subpopulations were observed in all cell lines, showing upregulation of PD‑L1 and pERK1/2 as well as of established senescence markers p21 and γH2AX. SA‑β‑Gal‑positive cells were found in all lines. These results were supported in a 3D tumor model. Fractionated IR can generate a subpopulation of HNSCC cells characterized by senescence‑typical cellular changes and marked expression of PD‑L1 and pERK1/2. Postradiogenic senescence in both 2D and 3D cancer models was possibly related to survival signaling and immune checkpoint regulation, crucial elements in tumor development and progress.