A Review for Uncovering the "Protein-Nanoparticle Alliance": Implications of the Protein Corona for Biomedical Applications.

Understanding both the physicochemical and biological interactions of nanoparticles is mandatory for the biomedical application of nanomaterials. By binding proteins, nanoparticles acquire new surface identities in biological fluids, the protein corona. Various studies have revealed the dynamic structure and nano-bio interactions of the protein corona. The binding of proteins not only imparts new surface identities to nanoparticles in biological fluids but also significantly influences their bioactivity, stability, and targeting specificity. Interestingly, recent endeavors have been undertaken to harness the potential of the protein corona instead of evading its presence. Exploitation of this 'protein-nanoparticle alliance' has significant potential to change the field of nanomedicine. Here, we present a thorough examination of the latest research on protein corona, encompassing its formation, dynamics, recent developments, and diverse bioapplications. Furthermore, we also aim to explore the interactions at the nano-bio interface, paving the way for innovative strategies to advance the application potential of the protein corona. By addressing challenges and promises in controlling protein corona formation, this review provides insights into the evolving landscape of the 'protein-nanoparticle alliance' and highlights emerging.

Revolution in Cancer Treatment: How Are Intelligently Designed Nanostructures Changing the Game?

Nanoparticles (NPs) are extremely important tools to overcome the limitations imposed by therapeutic agents and effectively overcome biological barriers. Smart designed/tuned nanostructures can be extremely effective for cancer treatment. The selection and design of nanostructures and the adjustment of size and surface properties are extremely important, especially for some precision treatments and drug delivery (DD). By designing specific methods, an important era can be opened in the biomedical field for personalized and precise treatment. Here, we focus on advances in the selection and design of nanostructures, as well as on how the structure and shape, size, charge, and surface properties of nanostructures in biological fluids (BFs) can be affected. We discussed the applications of specialized nanostructures in the therapy of head and neck cancer (HNC), which is a difficult and aggressive type of cancer to treat, to give an impetus for novel treatment approaches in this field. We also comprehensively touched on the shortcomings, current trends, and future perspectives when using nanostructures in the treatment of cancer.

The Magic Triangle in Oral Potentially Malignant Disorders: Vitamin D, Vitamin D Receptor, and Malignancy.

OPMDs (oral potentially malignant disorders) are a group of disorders affecting the oral mucosa that are characterized by aberrant cell proliferation and a higher risk of malignant transformation. Vitamin D (VitD) and its receptor (VDR) have been extensively studied for their potential contributions to the prevention and therapeutic management of various diseases and neoplastic conditions, including oral cancer. Observational studies suggest correlations between VitD deficiency and higher cancer risk, worse prognosis, and increased mortality rates. Interestingly, emerging data also suggest a link between VitD insufficiency and the onset or progression of OPMDs. Understanding the role of the VitD-VDR axis not only in established oral tumors but also in OPMDs might thus enable early detection and prevention of malignant transformation. With this article, we want to provide an overview of current knowledge about OPMDs and VitD and investigate their potential association and ramifications for clinical management of OPMDs.

Exosomes in Cancer Progression and Therapy Resistance: Molecular Insights and Therapeutic Opportunities.

The development of therapy resistance still represents a major hurdle in treating cancers, leading to impaired treatment success and increased patient morbidity. The establishment of minimally invasive liquid biopsies is a promising approach to improving the early diagnosis, as well as therapy monitoring, of solid tumors. Because of their manifold functions in the tumor microenvironment, tumor-associated small extracellular vesicles, referred to as exosomes, have become a subject of intense research. Besides their important roles in cancer progression, metastasis, and the immune response, it has been proposed that exosomes also contribute to the acquisition and transfer of therapy resistance, mainly by delivering functional proteins and RNAs, as well as facilitating the export of active drugs or functioning as extracellular decoys. Extensive research has focused on understanding the molecular mechanisms underlying the occurrence of resistance and translating these into strategies for early detection. With this review, we want to provide an overview of the current knowledge about the (patho-)biology of exosomes, as well as state-of-the-art methods of isolation and analysis. Furthermore, we highlight the role of exosomes in tumorigenesis and cancer treatment, where they can function as therapeutic agents, biomarkers, and/or targets. By focusing on their roles in therapy resistance, we will reveal new paths of exploiting exosomes for cancer diagnosis and treatment.

The Apoptosis Inhibitor Protein Survivin Is a Critical Cytoprotective Resistor against Silica-Based Nanotoxicity.

Exposure to nanoparticles is inevitable as they become widely used in industry, cosmetics, and foods. However, knowledge of their (patho)physiological effects on biological entry routes of the human body and their underlying molecular mechanisms is still fragmented. Here, we examined the molecular effects of amorphous silica nanoparticles (aSiNPs) on cell lines mimicking the alveolar-capillary barrier of the lung. After state-of-the-art characterization of the used aSiNPs and the cell model, we performed cell viability-based assays and a protein analysis to determine the aSiNP-induced cell toxicity and underlying signaling mechanisms. We revealed that aSiNPs induce apoptosis in a dose-, time-, and size-dependent manner. aSiNP-induced toxicity involves the inhibition of pro-survival pathways, such as PI3K/AKT and ERK signaling, correlating with reduced expression of the anti-apoptotic protein Survivin on the protein and transcriptional levels. Furthermore, induced Survivin overexpression mediated resistance against aSiNP-toxicity. Thus, we present the first experimental evidence suggesting Survivin as a critical cytoprotective resistor against silica-based nanotoxicity, which may also play a role in responses to other NPs. Although Survivin's relevance as a biomarker for nanotoxicity needs to be demonstrated in vivo, our data give general impetus to investigate the pharmacological modulation of Survivin`s functions to attenuate the harmful effects of acute or chronic inhalative NP exposure.

Exploiting Vitamin D Receptor and Its Ligands to Target Squamous Cell Carcinomas of the Head and Neck.

Vitamin D (VitD) and its receptor (VDR) have been intensively investigated in many cancers. As knowledge for head and neck cancer (HNC) is limited, we investigated the (pre)clinical and therapeutic relevance of the VDR/VitD-axis. We found that VDR was differentially expressed in HNC tumors, correlating to the patients' clinical parameters. Poorly differentiated tumors showed high VDR and Ki67 expression, whereas the VDR and Ki67 levels decreased from moderate to well-differentiated tumors. The VitD serum levels were lowest in patients with poorly differentiated cancers (4.1 ± 0.5 ng/mL), increasing from moderate (7.3 ± 4.3 ng/mL) to well-differentiated (13.2 ± 3.4 ng/mL) tumors. Notably, females showed higher VitD insufficiency compared to males, correlating with poor differentiation of the tumor. To mechanistically uncover VDR/VitD's pathophysiological relevance, we demonstrated that VitD induced VDR nuclear-translocation (VitD < 100 nM) in HNC cells. RNA sequencing and heat map analysis showed that various nuclear receptors were differentially expressed in cisplatin-resistant versus sensitive HNC cells including VDR and the VDR interaction partner retinoic acid receptor (RXR). However, RXR expression was not significantly correlated with the clinical parameters, and cotreatment with its ligand, retinoic acid, did not enhance the killing by cisplatin. Moreover, the Chou-Talalay algorithm uncovered that VitD/cisplatin combinations synergistically killed tumor cells (VitD < 100 nM) and also inhibited the PI3K/Akt/mTOR pathway. Importantly, these findings were confirmed in 3D-tumor-spheroid models mimicking the patients' tumor microarchitecture. Here, VitD already affected the 3D-tumor-spheroid formation, which was not seen in the 2D-cultures. We conclude that novel VDR/VitD-targeted drug combinations and nuclear receptors should also be intensely explored for HNC. Gender-specific VDR/VitD-effects may be correlated to socioeconomic differences and need to be considered during VitD (supplementation)-therapies.

Cloning Strategy for HDAC1/HDAC2 Hybrid Protein Expression in Mammalian Cells.

Dynamic deacetylation of non-histone proteins by histone deacetylases (HDACs) is a key regulator of protein functions, interactions, and turnover. Among class I HDACs, human HDAC1 and HDAC2 share more than 80% global homology at the amino acid level. However, despite the high redundancy, there are examples for differential substrate specificities of HDAC1 and HDAC2. Until now it remains quite unclear how specific and overlapping functions of HDAC1/HDAC2 are regulated in different contexts. Here, we describe molecular cloning techniques for the generation of HDAC1/HDAC2 hybrid proteins, HDAC1/HDAC2 mutants lacking known interaction domains, and HDAC1/HDAC2 hybrid proteins with interchanged N-terminal domains. These proteins are tools for the analysis of specific protein interactions and functions in mammalian cells.

The Vitamin D Receptor-BIM Axis Overcomes Cisplatin Resistance in Head and Neck Cancer.

Treatment success of head and neck squamous cell carcinoma (HNSCC) is often hindered by cisplatin resistance. As inherent and acquired therapy resistance counteracts improvement in long-term survival, novel multi-targeting strategies triggering cancer cell apoptosis are urgently required. Here, we identify the vitamin D receptor (VDR) as being significantly overexpressed in tumors of HNSCC patients (n = 604; p = 0.0059), correlating with tumor differentiation (p = 0.0002), HPV status (p = 0.00026), and perineural invasion (p = 0.0087). The VDR, a member of the nuclear receptor superfamily, is activated by its ligand vitamin D (VitD) and analogs, triggering multiple cellular responses. As we found that the VDR was also upregulated in our cisplatin-resistant HNSCC models, we investigated its effect on overcoming cisplatin resistance. We discovered that VitD/cisplatin combinations synergistically killed even cisplatin-resistant cells at clinically achievable levels. Similar results were obtained for the clinically used VitD analog Maxacalcitol. Moreover, VitD/cisplatin combinations inhibited tumor cell migration by E-cadherin upregulation. Signaling pathway analyses revealed that VitD co-treatments triggered cancer cell death by increasing the expression of the pro-apoptotic BCL-2 family protein BIM. BIM's pro-apoptotic activity in HNSCC cells was confirmed by ectopic overexpression studies. Importantly, BIM expression is positively associated with HNSCC patients' (n = 539) prognosis, as high expression correlated with improved survival (p = 0.0111), improved therapy response (p = 0.0026), and remission (p = 0.004). Collectively, by identifying, for the first time, the VDR/BIM axis, we here provide a molecular rationale for the reported anti-cancer activity of VitD/analogs in combination therapies. Our data also suggest its exploitation as a potential strategy to overcome cisplatin resistance in HNSCC and other malignancies by inducing additional pro-apoptotic pathways.

Characterization of a novel microfluidic platform for the isolation of rare single cells to enable CTC analysis from head and neck squamous cell carcinoma patients.

Detailed examination of tumor components is leading-edge to establish personalized cancer therapy. Accompanying research on cell-free DNA, the cell count of circulating tumor cells (CTCs) in patient blood is seen as a crucial prognostic factor. The potential of CTC analysis is further not limited to the determination of the overall survival rate but sheds light on understanding inter- and intratumoral heterogeneity. In this regard, commercial CTC isolation devices combining an efficient enrichment of rare cells with a droplet deposition of single cells for downstream analysis are highly appreciated. The Liquid biopsy platform CTCelect was developed to realize a fully-automated enrichment and single cell dispensing of CTCs from whole blood without pre-processing. We characterized each process step with two different carcinoma cell lines demonstrating up to 87 % enrichment (n = 10) with EpCAM coupled immunomagnetic beads, 73 % optical detection and dispensing efficiency (n = 5). 40 to 56.7 % of cells were recovered after complete isolation from 7.5 ml untreated whole blood (n = 6). In this study, CTCelect enabled automated dispensing of single circulating tumor cells from HNSCC patient samples, qPCR-based confirmation of tumor-related biomarkers and immunostaining. Finally, the platform was compared to commercial CTC isolation technologies to highlight advantages and limitations of CTCelect. This system offers new possibilities for single cell screening in cancer diagnostics, individual therapy approaches and real-time monitoring.

Impact of Secretion-Active Osteoblast-Specific Factor 2 in Promoting Progression and Metastasis of Head and Neck Cancer.

Treatment success of head and neck cancer (HNC) is still hampered by tumor relapse due to metastases. Our study aimed to identify biomarkers by exploiting transcriptomics profiles of patient-matched metastases, primary tumors, and normal tissue mucosa as well as the TCGA HNC cohort data sets. Analyses identified osteoblast-specific factor 2 (OSF-2) as significantly overexpressed in lymph node metastases and primary tumors compared to normal tissue. High OSF-2 levels correlate with metastatic disease and reduced overall survival of predominantly HPV-negative HNC patients. No significant correlation was observed with tumor localization or therapy response. These findings were supported by the fact that OSF-2 expression was not elevated in cisplatin-resistant HNC cell lines. OSF-2 was strongly expressed in tumor-associated fibroblasts, suggesting a tumor microenvironment-promoting function. Molecular cloning and expression studies of OSF-2 variants from patients identified an evolutionary conserved bona fide protein secretion signal (1MIPFLPMFSLLLLLIVNPINA21). OSF-2 enhanced cell migration and cellular survival under stress conditions, which could be mimicked by the extracellular administration of recombinant protein. Here, OSF-2 executes its functions via ß1 integrin, resulting in the phosphorylation of PI3K and activation of the Akt/PKB signaling pathway. Collectively, we suggest OSF-2 as a potential prognostic biomarker and drug target, promoting metastases by supporting the tumor microenvironment and lymph node metastases survival rather than by enhancing primary tumor proliferation or therapy resistance.

Identification of cytokeratin24 as a tumor suppressor for the management of head and neck cancer.

To improve management of head and neck squamous cell carcinoma patients, we need to increase our understanding of carcinogenesis, to identify biomarkers, and drug targets. This study aimed to identify novel biomarkers by providing transcriptomics profiles of matched primary tumors, lymph node metastasis, and non-malignant tissue of 20 HNSCC patients as well as by bioinformatic analyses of a TCGA HNSCC cohort, comprising 554 patients. We provide cancer cell signaling networks differentially expressed in tumors versus metastases, such as mesenchymal-epithelial transition, and structural integrity networks. As a proof of principle study, we exploited the data sets and performed functional analyses of a novel cytokeratin, cytokeratin24 (cKRT24), which had not been described as biomarker for tumors before. Survival analysis revealed that low cKRT24 expression correlated with poor overall survival in HNSCC. Experimentally, downregulation of cKRT24 in primary tumors, metastases, and HNSCC cell lines was verified on mRNA and protein level. Cloning and ectopic overexpression of cKRT24 not only affected viability and growth of HNSSC cell lines, but also inhibited tumor growth in murine xenograft studies. We conclude that cKRT24 functions as a tumor suppressor in HNSCC, and may serve as an additional prognostic biomarker and novel target to support current HNSCC treatments.

IsoMAG-An Automated System for the Immunomagnetic Isolation of Squamous Cell Carcinoma-Derived Circulating Tumor Cells.

BACKGROUND: detailed information about circulating tumor cells (CTCs) as an indicator of therapy response and cancer metastasis is crucial not only for basic research but also for diagnostics and therapeutic approaches. Here, we showcase a newly developed IsoMAG IMS system with an optimized protocol for fully automated immunomagnetic enrichment of CTCs, also revealing rare CTC subpopulations. METHODS: using different squamous cell carcinoma cell lines, we developed an isolation protocol exploiting highly efficient EpCAM-targeting magnetic beads for automated CTC enrichment by the IsoMAG IMS system. By FACS analysis, we analyzed white blood contamination usually preventing further downstream analysis of enriched cells. RESULTS: 1 µm magnetic beads with tosyl-activated hydrophobic surface properties were found to be optimal for automated CTC enrichment. More than 86.5% and 95% of spiked cancer cells were recovered from both cell culture media or human blood employing our developed protocol. In addition, contamination with white blood cells was minimized to about 1200 cells starting from 7.5 mL blood. Finally, we showed that the system is applicable for HNSCC patient samples and characterized isolated CTCs by immunostaining using a panel of tumor markers. CONCLUSION: Herein, we demonstrate that the IsoMAG system allows the detection and isolation of CTCs from HNSCC patient blood for disease monitoring in a fully-automated process with a significant leukocyte count reduction. Future developments seek to integrate the IsoMAG IMS system into an automated microfluidic-based isolation workflow to further facilitate single CTC detection also in clinical routine.

Profiling Cisplatin Resistance in Head and Neck Cancer: A Critical Role of the VRAC Ion Channel for Chemoresistance.

Treatment success of head and neck cancers (HNSCC) is often hindered by tumor relapses due to therapy resistances. This study aimed at profiling cisplatin resistance mechanisms and identifying biomarkers potentially suitable as drug targets and for patient stratification. Bioinformatic analyses of suggested resistance factors in a cohort of 565 HNSCC patients identified the VRAC ion channel as a clinically relevant indicator for recurrent diseases following radiochemotherapy (p = 0.042). Other drug import/export transporters, such as CTR1, OCT1, or MRP1, were found to be less relevant. To experimentally verify VRAC's critical role for cisplatin resistance, we used CRISPR/Cas9 knockout resulting in cisplatin-resistant HNSCC cells, which could be resensitized by VRAC expression. Next-generation sequencing further underlined VRAC's importance and identified VRAC-regulated signaling networks, potentially also contributing to cisplatin resistance. CTR1, OCT1, or MRP1 did not contribute to increased cisplatin resistance. In addition to two-dimensional HNSCC models, three-dimensional tumor spheroid cultures confirmed VRAC's unique role for cisplatin sensitivity. Here, resistance correlated with DNA damage and downstream apoptosis. The cisplatin specificity of the identified VRAC pathway was verified by testing paclitaxel and doxorubicin. Our results were independently confirmed in naturally occurring, cisplatin-resistant HNSCC cancer cell models. Collectively, we here demonstrate VRAC's role for cisplatin resistance in HNSCC and its relevance as a potential drug target and/or prognostic biomarker for chemotherapy resistance.

Growth Factor Receptor Expression in Oropharyngeal Squamous Cell Cancer: Her1-4 and c-Met in Conjunction with the Clinical Features and Human Papillomavirus (p16) Status.

This study aimed to assess the distribution of growth factor receptors in oropharyngeal squamous cell cancer (OPSCC) and evaluate their role in the context of human papillomavirus (HPV) status, prognosis and potential relevance for targeted therapy. The protein expression of human epidermal growth factor receptor (Her)1-4 and c-Met were retrospectively assessed using semiquantitative immunohistochemistry on tissue microarrays and analyzed for correlations as well as differences in the clinicopathological criteria. Her1-4 and c-met were overexpressed compared to normal mucosa in 46%, 4%, 17%, 27% and 23%, respectively. Interestingly, most receptors were coexpressed. Her1 and c-Met were inversely correlated with p16 (p = 0.04; p = 0.02). Her2 and c-Met were associated with high tobacco consumption (p = 0.016; p = 0.04). High EGFR, Her3, Her4 and c-Met expression were associated with worse overall and disease-free survival (p ≤ 0.05). Furthermore, EGFR and c-Met expression showed raised hazard ratios of 2.53 (p = 0.02; 95% CI 1.24-5.18) and 2.45 (p = 0.02; 95% CI 1.13-5.35), respectively. Her4 was expressed less in distant metastases than in corresponding primary tumors and was correlated to a higher T category. EGFR and c-Met are relevant negative prognostic factors in OPSCC, independent of known clinicopathological parameters. We suggest dual targeting of EGFR and c-Met as a promising strategy for OPSCC treatment.