Radiotherapy is the standard treatment for head and neck squamous cell carcinoma (HNSCC), however, radioresistance remains a major clinical problem despite significant improvements in treatment protocols. Therapeutic outcome could potentially be improved if a patient's tumour response to irradiation could be predicted ex vivo before clinical application. The present study employed a bespoke microfluidic device to maintain HNSCC tissue whilst subjecting it to external beam irradiation and measured the responses using a panel of cell death and proliferation markers. HNSCC biopsies from five newly-presenting patients [2 lymph node (LN); 3 primary tumour (PT)] were divided into parallel microfluidic devices and replicates of each tumour were subjected to single-dose irradiation (0, 5, 10, 15 and 20 Gy). Lactate dehydrogenase (LDH) release was measured and tissue sections were stained for cytokeratin (CK), cleaved-CK18 (cCK18), phosphorylated-H2AX (γH2AX) and Ki‑67 by immunohistochemistry. In addition, fragmented DNA was detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Compared with non‑irradiated controls, higher irradiation doses resulted in elevated CK18-labelling index in two lymph nodes [15 Gy; 34.8% on LN1 and 31.7% on LN2 (p=0.006)] and a single laryngeal primary tumour (20 Gy; 31.5%; p=0.014). Significantly higher levels of DNA fragmentation were also detected in both lymph node samples and one primary tumour but at varying doses of irradiation, i.e., LN1 (20 Gy; 27.6%; p=0.047), LN2 (15 Gy; 15.3%; p=0.038) and PT3 (10 Gy; 35.2%; p=0.01). The γH2AX expression was raised but not significantly in the majority of samples. The percentage of Ki‑67 positive nuclei reduced dose-dependently following irradiation. In contrast no significant difference in LDH release was observed between irradiated groups and controls. There is clear inter- and intra-patient variability in response to irradiation when measuring a variety of parameters, which offers the potential for the approach to provide clinically valuable information.

BackgroundThough the management of malignancies has improved vastly in recent years, many treatment options lack the desired efficacy and fail to adequately augment patient morbidity and mortality. It is increasingly clear that patient response to therapy is unique to each individual, necessitating personalised, or ‘precision’ medical care. This demand extends to thyroid cancer; ~ 10% patients fail to respond to radioiodine treatment due to loss of phenotypic differentiation, exposing the patient to unnecessary ionising radiation, as well as delaying treatment with alternative therapies.MethodsHuman thyroid tissue (n = 23, malignant and benign) was live-sliced (5 mm diameter × 350-500 μm thickness) then analysed or incorporated into a microfluidic culture device for 96 h (37 °C). Successful maintenance of tissue was verified by histological (H&E), flow cytometric propidium iodide or trypan blue uptake, immunohistochemical (Ki67 detection/ BrdU incorporation) and functional analysis (thyroxine [T4] output) in addition to analysis of culture effluent for the cell death markers lactate dehydrogenase (LDH) and dead-cell protease (DCP). Apoptosis was investigated by Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Differentiation was assessed by evaluation of thyroid transcription factor (TTF1) and sodium iodide symporter (NIS) expression (western blotting).ResultsMaintenance of gross tissue architecture was observed. Analysis of dissociated primary thyroid cells using flow cytometry both prior to and post culture demonstrated no significant change in the proportion of viable cells. LDH and DCP release from on-chip thyroid tissue indicated that after an initial raised level of release, signifying cellular damage, detectable levels dropped markedly. A significant increase in apoptosis (p < 0.01) was observed after tissue was perfused with etoposide and JNK inhibitor, but not in control tissue incubated for the same time period. No significant difference in Ki-67 positivity or TTF1/NIS expression was detected between fresh and post-culture thyroid tissue samples, moreover BrdU positive nuclei indicated on-chip cellular proliferation. Cultured thyroid explants were functionally viable as determined by production of T4 throughout the culture period.ConclusionsThe described microfluidic platform can maintain the viability of thyroid tissue slices ex vivo for a minimum of four days, providing a platform for the assessment of thyroid tissue radioiodine sensitivity/adjuvant therapies in real time.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P &lt; .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P &lt; .05) and 149.3 ± 9.7 (P &lt; .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA–plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

Introduction: Gastroesophageal reflux plays a significant role in idiopathic pulmonary fibrosis (IPF).Given the morbidity and mortality associated with IPF, understanding the mechanisms responsible for reflux is essential if patients are to receive optimal treatment and management, especially given the lack of clear benefit of anti-reflux therapies. Our aim was to understand the inter-relationships between esophageal motility, lung mechanics and reflux (particularly proximal reflux -a prerequisite of aspiration), and pulmonary function in IPF patients. Methods:We prospectively recruited 35 IPF patients (aged 53-75yrs; 27 male) who underwent highresolution impedance manometry and 24-hr pH-impedance, together with pulmonary function assessment.Results: Twenty-two (63%) patients exhibited dysmotility, 16(73%) ineffective esophageal motility (IEM) and 6(27%) esophagogastric junction outflow obstruction. Patients with IEM had more severe pulmonary disease (%FVC:p=0.032) and more proximal reflux (p=0.074) than patients with normal motility. In patients with IEM, intra-thoracic pressure inversely correlated with the number of proximal events (r=-0.429;p=0.098). Surprisingly, inspiratory lower esophageal sphincter pressure (LESP) positively correlated with the percentage of reflux events reaching the proximal esophagus (r=0.583;p=0.018), whilst in patients with normal motility it inversely correlated with the bolus exposure time (r=-0.478;p=0.098) and number of proximal events (r=-0.542;p=0.056). %FVC in IEM patients inversely correlated with the percentage of reflux events reaching the proximal esophagus (r=-0.520;p=0.039) and inspiratory LESP (r=-0.477;p=0.062), and positively correlated with intrathoracic pressure (r=0.633;p=0.008). Conclusions:We have shown that pulmonary function is worse in patients with IEM which is associated with more proximal reflux events, the latter correlating with lower intra-thoracic pressures and higher LESPs.