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.

Numerous studies have established a proof of concept that abnormal expression and function of estrogen receptors (ER) are crucial processes in initiation and development of hormone-related cancers and also affect the efficacy of anti-cancer therapy. Radiotherapy has been applied as one of the most common and potent therapeutic strategies, which is synergistic with surgical excision, chemotherapy and targeted therapy for treating malignant tumors. However, the impact of ionizing radiation on ER expression and ER-related signaling in cancer tissue, as well as the interaction between endocrine and irradiation therapy remains largely elusive. This review will discuss recent findings on ER and ER-related signaling, which are relevant for cancer radiotherapy. In addition, we will summarize pre-clinical and clinical studies that evaluate the consequences of anti-estrogen and irradiation therapy in cancer, including emerging studies on head and neck cancer, which might improve the understanding and development of novel therapeutic strategies for estrogen-related cancers.

Molecular mechanisms of intrinsic or acquired radioresistance serve as critical barrier for curative therapy of head and neck squamous cell carcinoma (HNSCC) and remain a major obstacle for progression-free and disease-specific survival. HNSCC cell lines were treated with a protocol of fractionated irradiation (IR, 4× 2Gy) alone or in combination with antagonists of estrogen receptor signaling and viability was determined by a colony-forming assay (CFA). Expression of submaxillary gland androgen-regulated protein 3A (SMR3A) and estrogen receptor 2 (ESR2) were assessed in tumor cells in vitro by RQ-PCR, Western blot analysis and immunofluorescence staining, and by immunohistochemical staining of tissue microarrays containing tumor sections from patients with oropharyngeal squamous cell carcinoma (OPSCC), which were treated by definitive or adjuvant radiotherapy. Subgroups with distinct SMR3A and ESR2 expression patterns were correlated with clinical parameters and survival outcome including multivariable analysis. Fractionated irradiation (IR) revealed an accumulation of tumor cells with prominent SMR3A expression, which was accompanied by an up-regulation of the estrogen receptor 2 (ESR2). ESR2-dependent regulation of SMR3A was supported by induced expression after stimulation with estradiol (E2), which was impaired by co-treatment with 4-Hydroxytamoxifen (TAM) or Fulvestrant, respectively. Both drugs significantly sensitized FaDu cells to fractionated IR as determined by a CFA and accelerated apoptosis. These data suggest a critical role of ESR2 in radioresistance and that SMR3A might serve as a surrogate marker for active ESR2 signaling. In line with this assumption, ESR2-positive oropharyngeal squamous cell carcinoma (OPSCC) with high SMR3A expression had an unfavorable progression-free and disease-specific survival as compared to those tumors with low SMR3A expression. In summary, our findings provide compelling experimental evidence that HNSCC with SMR3A and ESR2 co-expression have a higher risk for treatment failure and these patients might benefit from clinically well-established drugs targeting estrogen receptor signaling.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) forms a distinct tumor entity with better survival clinical outcome. Numerous underlying molecular mechanisms have been postulated for differences in treatment response, but the impact of MEK/ERK signaling, a main driver of carcinogenesis in various cancers including OPSCC and key player mediating therapy resistance remains elusive. In a retrospective experimental cohort study, primary tumor samples from OPSCC patients (n = 124) were available on tissue microarrays (TMAs) and expression levels of phosphorylated ERK1/2 (pERK1/2) were detected by immunohistochemical staining. Correlations of pERK1/2 expression patterns with clinicopathological features and clinical outcome were evaluated by statistical analysis. A low pERK1/2 expression was strongly associated with HPV-related OPSCC, while primary tumors with high pERK1/2 staining showed a distinctly worse survival outcome and were associated with higher cellular differentiation. Co-activation of both ERK1/2 and AKT was a common event and was associated with unfavorable prognosis in our cohort. However, the combinatorial analysis of pAKT (Ser473) and pERK1/2 did not strengthen the predictive power of pERK1/2, suggesting that pERK1/2 plays a more significant function in OPSCC. In summary, our data provide a compelling experimental and statistical evidence that low levels of tumor cell intrinsic ERK1/2 activation contribute at least in part to the favorable outcome of HPV-related OPSCC. On the other hand, presented findings indicate that non-HPV-related OPSCC with elevated ERK phosphorylation are at high risk for treatment failure and might benefit from targeted therapy of MEK/ERK signaling.

Soft tissue sarcomas (STS), including rhabdomyosarcoma (RMS), exhibit significant heterogeneity and limited responsiveness to immune checkpoint blockade (ICB). Unsupervised tumor immune phenotype based on multi-omics expression profiling of STS has been less studied. To reveal the tumor immune phenotype of STS and identify promising therapeutic targets, multi-omics expression profiling across various subtypes of STS was investigated. Here, we established a novel molecular classifier based on immune cell subsets related to TGFβ1 and IFNγ to identify distinct immune phenotypes with higher or lower cytotoxic contents. Immune-high clusters demonstrated enriched immune cell infiltration, elevated IFNγ-related signatures, and favorable clinical outcomes. In contrast, immune-low clusters were enriched for immunosuppressive cell types and exhibited poor survival. CHEK1 emerged as a key node associated with immunosuppressive phenotypes and was significantly overexpressed in immune-low tumors. In situ analysis of independent validation cohorts revealed the significant correlation between CHEK1 and tumor-infiltrating immune cells. Collectively, our findings establish a novel risk assessment strategy for RMS and STS patients, and highlight the potential of CHEK1 as a promising therapeutic target in combination with immune checkpoint inhibitor therapy.

Persistent high-risk HPV infection drives tumorigenesis in various human malignancies, including cervical, oropharyngeal, anal, and vulvar carcinomas. Although HPV-related tumors arise in several different sites, they share many common genetic and epigenetic events. Complex and heterogeneous genomic aberrations and mutations induced by high-risk HPV contribute to the initiation and progression of cervical cancer (CC). However, the associations between high-risk HPV infection and DNA methylation have not been clearly investigated. In the present study, HPV-related gene promoter methylation signature was comprehensively analyzed using multiple interactive platforms. CC patients were successfully classified into high-risk and low-risk groups with significant differences in clinical outcomes based on the HPV-related gene promoter methylation signature. Moreover, the protein levels of ALDH1A2 and clinical prognostic value were confirmed in the CC patients cohort. In summary, our study provides compelling evidence that HPV-related gene promoter methylation signature serves as a strong prognostic signature for CC patients. Clinical investigations in large CC patient cohorts are greatly needed to pave the way to implement epigenetic biomarkers into better clinical management.