Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Prognostic value of indoleamine 2,3 dioxygenase in patients with higher-risk myelodysplastic syndromes treated with azacytidine.

Hypomethylating agents (HMAs) are widely used in patients with higher-risk myelodysplastic syndromes (MDS) not eligible for stem cell transplantation; however, the response rate is <50%. Reliable predictors of response are still missing, and it is a major challenge to develop new treatment strategies. One current approach is the combination of azacytidine (AZA) with checkpoint inhibitors; however, the potential benefit of targeting the immunomodulator indoleamine-2,3-dioxygenase (IDO-1) has not yet been evaluated. We observed moderate to strong IDO-1 expression in 37% of patients with high-risk MDS. IDO-1 positivity was predictive of treatment failure and shorter overall survival. Moreover, IDO-1 positivity correlated inversely with the number of infiltrating CD8+ T cells, and IDO-1+ patients failed to show an increase in CD8+ T cells under AZA treatment. In vitro experiments confirmed tryptophan catabolism and depletion of CD8+ T cells in IDO-1+ MDS, suggesting that IDO-1 expression induces an immunosuppressive microenvironment in MDS, thereby leading to treatment failure under AZA treatment. In conclusion, IDO-1 is expressed in more than one-third of patients with higher-risk MDS, and is predictive of treatment failure and shorter overall survival. Therefore, IDO-1 is emerging as a promising predictor and therapeutic target, especially for combination therapies with HMAs or checkpoint inhibitors.

Improved overall survival in head and neck cancer patients after specific therapy of distant metastases.

PURPOSE: While metastases directed therapy for oligometastatic disease is recommended in different cancer entities, the treatment of solitary metastases in head and neck squamous cell carcinoma (HNSCC) patients is not clearly defined. METHODS: A retrospective analysis was performed on data from 143 HNSCC patients treated between 2001 and 2016 in a tertiary university hospital. Clinical factors and outcome were measured using the median survival of patients receiving metastases specific therapy in comparison with matched control patients. RESULTS: In 37 patients, distant metastases were treated specifically with either surgery and/or stereotactic ablative radiotherapy and had with 23.97 months a more than three times higher median survival than 10 untreated matched controls with potentially treatable distant metastases (7.07 months). CONCLUSIONS: Our retrospective analysis demonstrates a significant survival benefit for HNSCC patients who received a specific therapy regarding distant metastasis irrespective of localization as compared to a matched control cohort.

High NOTCH1 mRNA Expression Is Associated with Better Survival in HNSCC.

The clinical impact of the expression of NOTCH1 signaling components in squamous cell carcinoma of the pharynx and larynx has only been evaluated in subgroups. The aim of this study was therefore to evaluate NOTCH1 expression in head and neck squamous cell cancer (HNSCC) patient tissue and cell lines. We analyzed tissue from 195 HNSCCs and tissue from 30 normal patients for mRNA expression of NOTCH1, NOTCH3, HES1, HEY1, and JAG1 using quantitative real-time PCR. Association of expression results and clinical orpathological factors was examined with multivariate Cox regression. NOTCH1 expression was determined in three Human Papilloma Virus (HPV)-positive and nine HPV-negative HNSCC cell lines. High expression of NOTCH1 was associated with better overall survival (p = 0.013) and disease-free survival (p = 0.040). Multivariate Cox regression confirmed the significant influence of NOTCH1 expression on overall survival (p = 0.033) and disease-free survival (p = 0.029). A significant correlation was found between p16 staining and NOTCH1 mRNA expression (correlation coefficient 0.28; p = 0.01). NOTCH1 was expressed at higher levels in HPV-positive HNSCC cell lines compared with HPV-negative cell lines, which was not statistically significant (p = 0.068). We conclude that NOTCH1 expression is associated with overall survival, and that inhibition of NOTCH1 therefore seems less promising.

Post-therapeutic microRNA-146a in liquid biopsies may determine prognosis in metastatic gastrointestinal cancer patients receiving 90Y-radioembolization.

PURPOSE: The role of microRNA-146a (miR-146a) in defining the tumor immune microenvironment (TIME) is well established. The aim of this study was to evaluate circulating miR-146a as an early prognostic marker of 90Y-radioembolization (90Y-RE) in metastatic liver cancer and to assess the correlation between circulating miR-146a and TIME cellular composition in distant, yet untreated metastases. METHODS: Twenty-one patients with bilobar liver lesions from gastro-intestinal cancer underwent lobar 90Y-RE. Biopsy of contralateral lobe abscopal tumors was acquired at the onset of a second treatment session at a median of 21 days after initial RE, immediately prior to ablation therapy of the contralateral lobe tumor. miR-146a was measured by RT-qPCR in plasma collected 24 h before (T1) and 48 h after (T2) initial unilobar 90Y-RE. The level of miR-146a was correlated with the infiltration of CD4 + , CD8 + , FoxP3 T cells, CD163 + M2 macrophages and immune-exhausted T cells in the abscopal tumor tissue acquired before the second treatment session. RESULTS: Plasma samples collected at T2 showed a higher concentration of miR-146a with respect to T1 in 43% of the patients (p = 0.002). In these patients, tumors revealed a pro-tumorigenic immune composition with enrichment of Tim3 + immune exhausted cells (p = 0.021), in combination with a higher infiltration of CD163 + M2 macrophages and a lower infiltration of CD8 + T cells. Patients with a higher level of miR-146a after 90Y-RE showed a trend to shorter OS (p = 0.055). CONCLUSION: miR-146a may represent a novel prognostic biomarker for 90Y-radioembolization in metastatic liver cancer.

Development of MDS in Pediatric Patients with GATA2 Deficiency: Increased Histone Trimethylation and Deregulated Apoptosis as Potential Drivers of Transformation.

GATA2 deficiency is a heterogeneous, multisystem disorder associated with a high risk of developing myelodysplastic syndrome (MDS) and the progression to acute myeloid leukemia. The mechanisms underlying malignant transformation in GATA2 deficiency remain poorly understood, necessitating predictive markers to assess an individual's risk of progression and guide therapeutic decisions. In this study, we performed a systematic analysis of bone marrow biopsies from 57 pediatric MDS patients. Focusing on hematopoiesis and the hematopoietic niche, including its microenvironment, we used multiplex immunofluorescence combined with multispectral imaging, gene expression profiling, and multiplex RNA in situ hybridization. Patients with a GATA2 deficiency exhibited a dysregulated GATA2 transcriptional network. Disease progression (GATA2-EB, n = 6) was associated with increased GATA2 mRNA levels, restored expression of the GATA2 target EZH2, and increased H3K27me3. GATA2-EB was further characterized by the high expression of the anti-apoptotic protein BCL2, a feature absent in children with a GATA2 deficiency and refractory cytopenia of childhood (GATA2-RCC, n = 24) or other pediatric MDS subgroups (RCC, n = 17; MDS-EB, n = 10). The multispectral imaging analysis of additional BCL2 family members revealed significantly elevated Mediators of Apoptosis Combinatorial (MAC) scores in GATA2-EB patients. Taken together, our findings highlight the potential drivers of disease progression in GATA2 deficiency, particularly increased histone trimethylation and dysregulated apoptosis. Furthermore, upregulated BCL2 and EZH2 and increased MAC scores provide a strong rationale for the use of venetoclax and azacitidine in therapeutic regimens for GATA2-EB.

Adrenal tropism of SARS-CoV-2 and adrenal findings in a post-mortem case series of patients with severe fatal COVID-19.

Progressive respiratory failure and hyperinflammatory response is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. Despite mounting evidence of disruption of the hypothalamus-pituitary-adrenal axis in COVID-19, relatively little is known about the tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to adrenal glands and associated changes. Here we demonstrate adrenal viral tropism and replication in COVID-19 patients. Adrenal glands showed inflammation accompanied by inflammatory cell death. Histopathologic analysis revealed widespread microthrombosis and severe adrenal injury. In addition, activation of the glycerophospholipid metabolism and reduction of cortisone intensities were characteristic for COVID-19 specimens. In conclusion, our autopsy series suggests that SARS-CoV-2 facilitates the induction of adrenalitis. Given the central role of adrenal glands in immunoregulation and taking into account the significant adrenal injury observed, monitoring of developing adrenal insufficiency might be essential in acute SARS-CoV-2 infection and during recovery.

Depicting adoptive immunotherapy for prostate cancer in an animal model with magnetic resonance imaging.

Genetically modified natural killer (NK) cells that recognize tumor-associated surface antigens have recently shown promise as a novel approach for cancer immunotherapy. To determine NK cell therapy response early, a real-time, noninvasive method to quantify NK cell homing to the tumor is desirable. The purpose of this study was to evaluate if MR imaging could provide a noninvasive, in vivo diagnosis of NK cell accumulation in epithelial cell adhesion molecule (EpCAM)-positive prostate cancers in a rat xenograft model. Genetically engineered NK-92-scFv(MOC31)-ζ cells, which express a chimeric antigen receptor specific to the tumor-associated EpCAM antigen, and nontargeted NK-92 cells were labeled with superparamagnetic particles of iron-oxides (SPIO) ferumoxides. Twelve athymic rats with implanted EpCAM positive DU145 prostate cancers received intravenous injections of 1.5×10(7) SPIO labeled NK-92 and NK-92-scFv(MOC31)-ζ cells. EpCAM-positive prostate cancers demonstrated a progressive and a significant decline in contrast-to-noise-ratio data at 1 and 24 h after injection of SPIO-labeled NK-92-scFv(MOC31)-ζ cells. Conversely, tumor contrast-to-noise-ratio data did not change significantly after injection of SPIO-labeled parental NK-92 cells. Histopathology confirmed an accumulation of the genetically engineered NK-92-scFv(MOC31)-ζ cells in prostate cancers. Thus, the presence or absence of a tumor accumulation of therapeutic NK cells can be monitored with cellular MR imaging. EpCAM-directed, SPIO labeled NK-92-scFv(MOC31)-ζ cells accumulate in EpCAM-positive prostate cancers.

Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways.

BACKGROUND: Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro. METHODS: Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis. RESULTS: In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis. CONCLUSION: Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy.

Indocyanine green-enhanced imaging of antigen-induced arthritis with an integrated optical imaging/radiography system.

OBJECTIVE: To evaluate a combined indocyanine green-enhanced optical imaging/radiography system for the detection of arthritic joints in a rat model of antigen-induced arthritis. METHODS: Arthritis of the knee and ankle joints was induced in 6 Harlan rats, using peptidoglycan-polysaccharide polymers. Three rats served as untreated controls. Optical imaging of the knee and ankle joints was done with an integrated optical imaging/radiography system before and up to 24 hours following intravenous injection of 10 mg/kg indocyanine green. The fluorescence signal intensities of arthritic and normal joints were compared for significant differences, using generalized estimating equation models. Specimens of knee and ankle joints were further processed and evaluated by histology. RESULTS: Immediately after administration, indocyanine green provided a significant increase in the fluorescence signal of arthritic joints compared with baseline values (P < 0.05). The fluorescence signal of arthritic joints was significantly higher compared with that of nonarthritic control joints at 1-720 minutes after intravenous injection (P < 0.05). Fusion of indocyanine green-enhanced optical imaging and radiography allowed for anatomic coregistration of the inflamed tissue with the associated joint. Hematoxylin and eosin staining confirmed marked synovial inflammation of arthritic joints and the absence of inflammation in control joints. CONCLUSION: Indocyanine green-enhanced optical imaging is a clinically applicable tool for detection of arthritic tissue. Using relatively high doses of indocyanine green, long-term enhanced fluorescence of arthritic joints can be achieved. This may facilitate simultaneous evaluations of multiple joints in a clinical setting. Fusion of indocyanine green-enhanced optical imaging scans with radiography increases anatomic resolution.

MEK1/2 regulates APOBEC3B and polymerase iota-induced mutagenesis in head and neck cancer cells.

Resistance to chemotherapy provides a major challenge in treatment of metastatic cancer. Prolonged exposure to almost any drug regimen leads to the formation of resistant subclones in almost all advanced solid tumors. Tumor heterogeneity because of intrinsic genetic instability is seen as one of the major contributing factors. In this work, we present evidence that genetic instability measured by mutation frequency is induced by treatment with the EGFR inhibitor afatinib or cisplatin in head and neck squamous cancer cells. We find that APOBEC3B and polymerase iota are upregulated, and inhibition of MEK1/2 by U0126 leads to downregulation on the protein level. Costimulation of afatnib and cisplatin with U0126 leads to a significantly lower mutation frequency. These findings may represent a molecular mechanism for dynamically controlling genetic instability during chemotherapy in head and neck squamous cell carcinoma (HNSCC) cancer cells.

Breast cancers: MR imaging of folate-receptor expression with the folate-specific nanoparticle P1133.

PURPOSE: To assess the capability of the folate receptor (FR)-targeted ultrasmall superparamagnetic iron oxide (USPIO) P1133 to provide FR-specific enhancement of breast cancers on magnetic resonance (MR) images. MATERIALS AND METHODS: This study was approved by the institutional Animal Care and Use Committee. The FR-targeted contrast agent P1133 was incubated with various FR-positive human breast cancer cell lines, with and without free folic acid (FFA) as a competitor. Labeling efficiencies were evaluated with MR imaging and inductively coupled plasma mass spectrometry. Subsequently, six athymic rats with implanted FR-positive MDA-MB-231 breast cancers underwent MR imaging at 3 T before and up to 1 hour and 24 hours after injection of P1133. Six athymic rats with implanted FR-positive MDA-MB-231 cancers injected with the non-FR-targeted USPIO P904 and nine athymic rats with implanted FR-negative A549 lung cancers injected with P1133 (n = 6) or P904 (n = 3) served as controls. Data of the in vitro studies were compared for significant differences with the Wilcoxon test for two independent samples. Tumor signal-to-noise-ratios (SNRs) were compared between different experimental groups by using the Kruskal-Wallis test and were correlated with histopathologic findings. Differences with P < .05 were considered significant. RESULTS: FR-positive breast cancer cells showed a significant P1133 uptake which was inhibited by FFA. MDA-MB-231 cells showed the highest level of P1133 uptake and the strongest T2 effect on MR images. In vivo, all tumors showed an initial perfusion effect. At 24 hours after injection, only MDA-MB-231 tumors injected with P1133 showed significantly decreased SNR data compared with baseline data (P < .05). MR findings were confirmed by using histopathologic findings. CONCLUSION: The FR-targeted USPIO P1133 demonstrates a specific retention in FR-positive breast cancers. Because FR expression correlates with tumor aggressiveness and prognosis, persistent P1133 tumor enhancement may be used as a noninvasive indicator for tumors with poor outcome.

Non-invasive tracking of human haemopoietic CD34(+) stem cells in vivo in immunodeficient mice by using magnetic resonance imaging.

OBJECTIVE: To assess migration of CD34(+) human stem cells to the bone marrow of athymic mice by using magnetic resonance (MR) imaging and Resovist, a contrast agent containing superparamagnetic iron oxide (SPIO) particles. METHODS: All animal and human procedures were approved by our institution's ethics committee, and women had given consent to donate umbilical cord blood (UCB). Balb/c-AnN Foxn1(nu)/Crl mice received intravenous injection of 1 x 10(6) (n=3), 5 x 10(6) (n=3) or 1 x 10(7) (n=3) human Resovist-labelled CD34(+) cells; control mice received Resovist (n=3). MR imaging was performed before, 2 and 24 h after transplantation. Signal intensities of liver, muscle and bone marrow were measured and analysed by ANOVA and post hoc Student's t tests. MR imaging data were verified by histological and immunological detection of both human cell surface markers and carboxydextrancoating of the contrast agent. RESULTS: CD34(+) cells were efficiently labelled by Resovist without impairment of functionality. Twenty-four hours after administration of labelled cells, MR imaging revealed a significant signal decline in the bone marrow, and histological and immunological analyses confirmed the presence of transplanted human CD34(+) cells. CONCLUSION: Intravenously administered Resovist-labelled CD34(+) cells home to bone marrow of mice. Homing can be tracked in vivo by using clinical 1.5-T MR imaging technology.

Bradykinin Receptor B1 and C-Reactive Protein as Prognostic Factors for Pharyngocutaneous Fistula Development After Laryngectomy.

Pharyngocutaneous fistulae (PCF) are one of the most common complications after laryngectomy. Predisposing risk factors have been studied, yet knowledge to determine which patients are prone to developing a fistula remains scarce. This study aims to establish prognostic parameters to identify individual patients at risk for PCF development. As PCF and inflammation seem to be interwoven, this work focuses on markers able to detect an inflammatory response. We retrospectively analyzed all patients who had undergone a laryngectomy at our clinic in the years 2007 to 2017 (n = 182). Immunohistochemical expression of bradykinin type 1 and 2 receptor and vascular endothelial growth factor receptor 2 was studied in all available tumor samples. Additionally, the clinical inflammation parameters 'body temperature', 'pain', 'c-reactive protein (CRP)', and 'leucocytes' were postoperatively tracked in all patients. The times between fistula diagnosis, therapeutic approach, and hospital discharge were recorded. We found a strong correlation between inflammation and the formation of a fistula. High bradykinin 1 receptor expression in the tumor samples correlated with postoperative PCF development. Persistently elevated CRP and leukocyte levels beyond the 6th postoperative day were also risk factors. A decreased time lapse between PCF diagnosis and surgical revision clearly correlated with a shorter hospital stay. In this study, we identified a bradykinin 1 receptor positive patient group at high risk for development of PCF. We recommend close monitoring for fistula formation in these patients to ensure timely intervention.

c-Rel gain in B cells drives germinal center reactions and autoantibody production.

Single-nucleotide polymorphisms and locus amplification link the NF-κB transcription factor c-Rel to human autoimmune diseases and B cell lymphomas, respectively. However, the functional consequences of enhanced c-Rel levels remain enigmatic. Here, we overexpressed c-Rel specifically in mouse B cells from BAC-transgenic gene loci and demonstrate that c-Rel protein levels linearly dictated expansion of germinal center B (GCB) cells and isotype-switched plasma cells. c-Rel expression in B cells of otherwise c-Rel-deficient mice fully rescued terminal B cell differentiation, underscoring its critical B cell-intrinsic roles. Unexpectedly, in GCB cells transcription-independent regulation produced the highest c-Rel protein levels among B cell subsets. In c-Rel-overexpressing GCB cells this caused enhanced nuclear translocation, a profoundly altered transcriptional program, and increased proliferation. Finally, we provide a link between c-Rel gain and autoimmunity by showing that c-Rel overexpression in B cells caused autoantibody production and renal immune complex deposition.

Axonal guidance by surface microstructuring for intracellular transport investigations.

Intracellular transport, a complex interplay of diverse processes, is fundamental for the development, function and survival of cells. Passive diffusion and active transport phases alternate in living cells, with active phases arising from molecular motors, such as kinesin or dynein, pulling cargoes along microtubules. A better understanding of stochasic mechanisms involved in motor-microtubule interactions and in diffusion processes, which enable efficient active transport over long distances in motor neurons, requires a better link between theoretical models and live-cell experiments. Herein, we establish one-dimensional (1D) intracellular transport geometries, suitable for comparing experimental findings with recent theoretical 1D model predictions, by guiding axonal outgrowth of pheochromocytoma (PC12) cells along predefined chemical surface structures with a strip width of 2 microm, fabricated by means of microscale plasma-initiated patterning (microPIP method). Quantification of the intracellular transport of quantum dots (QDs) in straight axons, which exhibit almost parallel microtubules, is obtained by our recently developed algorithm based on a time-resolved mean-square displacement (MSD) analysis. Such a thorough dissection of experimental data will be useful for validation and clarification of current theoretical transport models.

Tracking of [18F]FDG-labeled natural killer cells to HER2/neu-positive tumors.

INTRODUCTION: The objective of this study was to label the human natural killer (NK) cell line NK-92 with [(18)F]fluoro-deoxy-glucose (FDG) for subsequent in vivo tracking to HER2/neu-positive tumors. METHODS: NK-92 cells were genetically modified to NK-92-scFv(FRP5)-zeta cells, which express a chimeric antigen receptor that is specific to the tumor-associated ErbB2 (HER2/neu) antigen. NK-92 and NK-92-scFv(FRP5)-zeta cells were labeled with [(18)F]FDG by simple incubation at different settings. Labeling efficiency was evaluated by a gamma counter. Subsequently, [(18)F]FDG-labeled parental NK-92 or NK-92-scFv(FRP5)-zeta cells were intravenously injected into mice with implanted HER2/neu-positive NIH/3T3 tumors. Radioactivity in tumors was quantified by digital autoradiography and correlated with histopathology. RESULTS: The NK-92 and NK-92-scFv(FRP5)-zeta cells could be efficiently labeled with [(18)F]FDG by simple incubation. Optimal labeling efficiencies (80%) were achieved using an incubation period of 60 min and additional insulin (10 IU/ml). After injection of 5x10(6) [(18)F]FDG-labeled NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, digital autoradiography showed an increased uptake of radioactivity in HER2/neu-positive tumors at 60 min postinjection. Conversely, injection of 5x10(6) NK-92 cells not directed against HER2/neu receptors did not result in increased uptake of radioactivity in the tumors. Histopathology confirmed an accumulation of the NK-92-scFv(FRP5)-zeta cells, but not the parental NK cells, in tumor tissues. CONCLUSION: The human NK cell line NK-92 can be directed against HER2/neu antigens by genetic modification. The genetically modified NK cells can be efficiently labeled with [(18)F]FDG, and the accumulation of these labeled NK cells in HER2/neu-positive tumors can be monitored with autoradiography.

The Aurora-Kinase A Phe31-Ile polymorphism as possible predictor of response to treatment in head and neck squamous cell carcinoma.

Recently the Aurora-Kinases (Aurk) moved into the focus as novel disease related biomarkers and therapeutic targets. Elevated Aurora-Kinase expression has been found in a number of malignancies, amongst them HNSCC. For esophageal cancer, the AurkA Phe31-Ile polymorphism has previously been associated with tumor progression. Here we evaluated the treatment efficiency of HNSCC cell radiation as a function of Aurora-Kinases in HNSCC cell lines. Moreover, we investigated a potential sensitization to radiation by a cell treatment with the inhibitors Alisertib, Barasertib, Docetaxel and VX-680. In parallel the radiation dependent expression and regulation of AurkA/B, p-Akt Ser 473 and Survivin and the AurkA polymorphism were investigated in primary tumor samples. We identified a high-risk collective with elevated AurkA and Survivin or AurkA and p-Akt Ser 473 expression. High AurkA, AurkB, and p-Akt Ser 473 expression was exclusively found in the heterozygous cell line. We found a polymorphism dependent sensitivity to treatments with different Aurk inhibitors: The homozygous cell line UD-SCC-5 could be sensitized to radiation with Docetaxel in combination with any of the Aurora-Kinase inhibitors. In contrast, treatment with Docetaxel or radiation did not enhance the inhibitory effect of Barasertib or VX-680 in the heterozygous SAS cell line. These findings indicate that the Aurora-Kinase A Phe31-Ile-polymorphism is a possibly predictive factor for response to radiation in combination with Docetaxel and Aurora-Kinase inhibitor treatments.

Membrane Hsp70-A Novel Target for the Isolation of Circulating Tumor Cells After Epithelial-to-Mesenchymal Transition.

The presence of circulating tumor cells (CTCs) in the peripheral blood is a pre-requisite for progression, invasion, and metastatic spread of cancer. Consequently, the enumeration and molecular characterization of CTCs from the peripheral blood of patients with solid tumors before, during and after treatment serves as a valuable tool for categorizing disease, evaluating prognosis and for predicting and monitoring therapeutic responsiveness. Many of the techniques for isolating CTCs are based on the expression of epithelial cell surface adhesion molecule (EpCAM, CD326) on tumor cells. However, the transition of adherent epithelial cells to migratory mesenchymal cells (epithelial-to-mesenchymal transition, EMT)-an essential element of the metastatic process-is frequently associated with a loss of expression of epithelial cell markers, including EpCAM. A highly relevant proportion of mesenchymal CTCs cannot therefore be isolated using techniques that are based on the "capture" of cells expressing EpCAM. Herein, we provide evidence that a monoclonal antibody (mAb) directed against a membrane-bound form of Hsp70 (mHsp70)-cmHsp70.1-can be used for the isolation of viable CTCs from peripheral blood of tumor patients of different entities in a more quantitative manner. In contrast to EpCAM, the expression of mHsp70 remains stably upregulated on migratory, mesenchymal CTCs, metastases and cells that have been triggered to undergo EMT. Therefore, we propose that approaches for isolating CTCs based on the capture of cells that express mHsp70 using the cmHsp70.1 mAb are superior to those based on EpCAM expression.

Coexpression of cyclooxygenases (COX-1, COX-2) and vascular endothelial growth factors (VEGF-A, VEGF-C) in esophageal adenocarcinoma.

Cyclooxygenases (COX), especially COX-2, are considered to be involved in carcinogenesis. Our study was initiated to test whether expression of COX isoforms (COX-1 and COX-2) is linked to expression of potent inducers of angiogenesis [vascular endothelial growth factor (VEGF)-A] and lymphangiogenesis (VEGF-C) in esophageal adenocarcinoma. One hundred twenty-three esophageal adenocarcinomas were investigated by means of quantitative reverse transcription-PCR for expression of COX-1, COX-2, VEGF-A, and VEGF-C. Additionally, COX-2 protein expression was determined using immunohistochemistry. Three esophageal cancer cell lines (OE-33, OSC-1, and OSC-2) were treated with COX-inhibiting substances (diclofenac, rofecoxib, and SC-560) and the effect on expression of the four genes was determined. COX-2 protein expression was found in all carcinomas under analysis. RNA expression levels of COX-1 and COX-2 varied markedly in carcinoma tissues and correlated significantly with each other (P < 0.001, r = 0.726). Furthermore, COX expression correlated with expression of VEGF-A (COX-1: P < 0.001, r = 0.753; COX-2: P < 0.001, r = 0.764) and VEGF-C (COX-1: P < 0.001, r = 0.778; COX-2: P < 0.001; r = 0.613). Exposure of esophageal cancer cell lines OE-33, OSC-1, and OSC-2 with three COX-inhibiting substances (diclofenac, rofecoxib, and SC-560) resulted in significantly reduced expression of VEGF-A and VEGF-C. In conclusion, our data suggest that both COX isoforms may be involved in the pathogenesis of esophageal adenocarcinoma, as they are linked to the expression of important modulators of angiogenesis (VEGF-A) and lymphangiogenesis (VEGF-C).