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.

Nano Meets Micro-Translational Nanotechnology in Medicine: Nano-Based Applications for Early Tumor Detection and Therapy.

Nanomaterials have great potential for the prevention and treatment of cancer. Circulating tumor cells (CTCs) are cancer cells of solid tumor origin entering the peripheral blood after detachment from a primary tumor. The occurrence and circulation of CTCs are accepted as a prerequisite for the formation of metastases, which is the major cause of cancer-associated deaths. Due to their clinical significance CTCs are intensively discussed to be used as liquid biopsy for early diagnosis and prognosis of cancer. However, there are substantial challenges for the clinical use of CTCs based on their extreme rarity and heterogeneous biology. Therefore, methods for effective isolation and detection of CTCs are urgently needed. With the rapid development of nanotechnology and its wide applications in the biomedical field, researchers have designed various nano-sized systems with the capability of CTCs detection, isolation, and CTCs-targeted cancer therapy. In the present review, we summarize the underlying mechanisms of CTC-associated tumor metastasis, and give detailed information about the unique properties of CTCs that can be harnessed for their effective analytical detection and enrichment. Furthermore, we want to give an overview of representative nano-systems for CTC isolation, and highlight recent achievements in microfluidics and lab-on-a-chip technologies. We also emphasize the recent advances in nano-based CTCs-targeted cancer therapy. We conclude by critically discussing recent CTC-based nano-systems with high therapeutic and diagnostic potential as well as their biocompatibility as a practical example of applied nanotechnology.

Early Alterations of Endothelial Nitric Oxide Synthase Expression Patterns in the Guinea Pig Cochlea After Noise Exposure.

Constitutively expressed endothelial nitric oxide synthase (eNOS) is supposed to play a role in noise-induced nitric oxide (NO)-production. It is commonly known that intense noise exposure results in inducible NOS (iNOS) expression and increased NO-production, but knowledge about a contribution of the eNOS isoform is still lacking. Effects of noise exposure on eNOS immunolabeling were determined in male guinea pigs (n=24). For light microscopic analysis, 11 animals were exposed to 90 dB for 1 hr and 6 animals were used as controls. After exposure, eNOS immunostaining was performed on paraffin sections, and the staining intensities were quantified for 4 cochlear regions. For electron microscopic analysis, 2 animals were exposed for 2 hr to 90 dB and 5 animals were used as controls. The intensity of eNOS immunolabeling was found to be already comprehensively increased 1 hr after noise exposure to 90 dB. At the ultrastructural level, a clear increase in eNOS immunolabeling was found in microtubules-rich areas of cochlear cuticular structures. Hence, our findings indicate that the reticular lamina forming the endolymph-perilymph barrier at the apical side of the organ of Corti is involved in a fast intrinsic otoprotective mechanism of the cochlea.

Is small smarter? Nanomaterial-based detection and elimination of circulating tumor cells: current knowledge and perspectives.

Circulating tumor cells (CTCs) are disseminated cancer cells. The occurrence and circulation of CTCs seem key for metastasis, still the major cause of cancer-associated deaths. As such, CTCs are investigated as predictive biomarkers. However, due to their rarity and heterogeneous biology, CTCs' practical use has not made it into the clinical routine. Clearly, methods for the effective isolation and reliable detection of CTCs are urgently needed. With the development of nanotechnology, various nanosystems for CTC isolation and enrichment and CTC-targeted cancer therapy have been designed. Here, we summarize the relationship between CTCs and tumor metastasis, and describe CTCs' unique properties hampering their effective enrichment. We comment on nanotechnology-based systems for CTC isolation and recent achievements in microfluidics and lab-on-a-chip technologies. We discuss recent advances in CTC-targeted cancer therapy exploiting the unique properties of nanomaterials. We conclude by introducing developments in CTC-directed nanosystems and other advanced technologies currently in (pre)clinical research.

Staging of oropharyngeal squamous cell carcinoma of the head and neck: Prognostic features and power of the 8th edition of the UICC staging manual.

INTRODUCTION: Recognizing the prognostic power differentiating HPV-associated oropharyngeal squamous cell cancer (OPSCC) from OPSCC with other causes, the UICC Cancer Staging Manual 8th edition realizes significant changes from the 7th edition. Purpose of this study was to evaluate the differences of prognostic impact between the 7th and the latest edition of TNM Classification as well as to examine risk factors like extranodal extension (ENE) and lymph node ratio (LNR) for HPV-mediated OPSCC. MATERIAL AND METHODS: The study includes 255 patients with OPSCC and initial diagnosis between 2008 and 2015. HPV status was determined according to p16 immunohistochemistry (IHC) and all patients were classified as defined by 7th and 8th edition of UICC. Prognostic influence of ENE and LNR was analyzed for patients with HPV-mediated OPSCC. RESULTS: 41.2% of the OPSCC were p16-positive. Implementation of the 8th edition of the UICC lead to a better differentiation between the respective stages. Regarding HPV-positive OPSCC, Kaplan-Meier survival curves showed a significantly better overall survival (OS) for patients with a LNR ≤10% as well as for patients with negative ENE status (p = 0.004, p = 0.008). CONCLUSION: 8th edition of UICC achieves to differentiate properly between the UICC stages. However, the staging rule of ignoring ENE in HPV-mediated OPSCC should be further analyzed. Moreover LNR might be a possible additional prognostic factor - especially regarding HPV-positive tumors.

Nanomedical detection and downstream analysis of circulating tumor cells in head and neck patients.

The establishment of novel biomarkers in liquid biopsies of cancer patients has come more into focus in prognostic and diagnostic research efforts. Due to their prognostic relevance disseminated tumor cells or circulating tumor cells are the subject of intensive research and are discussed as early diagnostic indicators for treatment failure and the formation of micrometastases. A potential association of this early-systemic tumor component with poor prognosis of cancer patients could be already demonstrated for various entities including breast, colon, lung, melanoma, ovarian and prostate cancers. Thus, the detection of circulating tumor cells seems to be also applicable for minimal-invasive monitoring of therapy progress in head and neck cancer patients. A major problem of the use in clinical routine is that circulating tumor cells could not be detected by modern imaging techniques. To overcome these limitations highly sensitive detection methods and techniques for their molecular characterization are urgently needed allowing mechanistic understanding and targeting of circulating tumor cells. Especially the medical application of nanotechnology (nanomedical methods) has made valuable contributions to the field. Here, we want to provide a comprehensive overview on (nanomedical) detection methods for circulating tumor cells and discuss their merits, pitfalls and future perspectives especially for head and neck solid squamous cell carcinoma (HNSCC) patients.

Disease-relevant signalling-pathways in head and neck cancer: Taspase1's proteolytic activity fine-tunes TFIIA function.

Head and neck cancer (HNC) is the seventh most common malignancy in the world and its prevailing form, the head and neck squamous cell carcinoma (HNSCC), is characterized as aggressive and invasive cancer type. The transcription factor II A (TFIIA), initially described as general regulator of RNA polymerase II-dependent transcription, is part of complex transcriptional networks also controlling mammalian head morphogenesis. Posttranslational cleavage of the TFIIA precursor by the oncologically relevant protease Taspase1 is crucial in this process. In contrast, the relevance of Taspase1-mediated TFIIA cleavage during oncogenesis of HNSCC is not characterized yet. Here, we performed genome-wide expression profiling of HNSCC which revealed significant downregulation of the TFIIA downstream target CDKN2A. To identify potential regulatory mechanisms of TFIIA on cellular level, we characterized nuclear-cytoplasmic transport and Taspase1-mediated cleavage of TFIIA variants. Unexpectedly, we identified an evolutionary conserved nuclear export signal (NES) counteracting nuclear localization and thus, transcriptional activity of TFIIA. Notably, proteolytic processing of TFIIA by Taspase1 was found to mask the NES, thereby promoting nuclear localization and transcriptional activation of TFIIA target genes, such as CDKN2A. Collectively, we here describe a hitherto unknown mechanism how cellular localization and Taspase1 cleavage fine-tunes transcriptional activity of TFIIA in HNSCC.