Patient-Derived Xenograft vs. Organoids: A Preliminary Analysis of Cancer Research Output, Funding and Human Health Impact in 2014-2019.

Cancer remains a major threat to mortality and morbidity globally, despite intense research and generous funding. Patient-derived xenograft (PDX) models-where tumor biopsies are injected into an animal-were developed to improve the predictive capacity of preclinical animal models. However, recent observations have called into question the clinical relevance, and therefore the translational accuracy, of these. Patient-derived organoids (PDO) use patient tumor samples to create in vitro models that maintain aspects of tumor structure and heterogeneity. We undertook a preliminary analysis of the number of breast, colorectal, and lung cancer research studies using PDX or PDO published worldwide between 2014-2019. We looked for evidence of impacts of this research on human health. The number of publications that focused on PDO is gradually increasing over time, but is still very low compared to publications using PDX models. Support for new research projects using PDO is gradually increasing, a promising indicator of a shift towards more human-relevant approaches to understanding human disease. Overall, increases in total funding for these three major cancer types does not appear to be translating to any consequential increase in outputs, defined for this purpose as publications associated with clinical trials. With increasing public discomfort in research using animals and demands for 'alternative' methods, it is timely to consider how to implement non-animal methods more effectively.

Anaerobiosis influences virulence properties of Pseudomonas aeruginosa cystic fibrosis isolates and the interaction with Staphylococcus aureus.

The airways of individuals with cystic fibrosis (CF) are abundantly colonised by Staphylococcus aureus and Pseudomonas aeruginosa. Co-infecting hypoxic regions of static mucus within CF airways, together with decreases in pulmonary function, mucus plugging and oxygen consumption by host neutrophils gives rise to regions of anoxia. This study determined the impact of anaerobiosis upon S. aureus-P. aeruginosa interactions in planktonic co-culture and mixed species biofilms in vitro. Whilst anoxia reduced the ability for P. aeruginosa CF isolates to dominate over S. aureus, this occurred in an isolate dependent manner. Investigations into the underlying mechanisms suggest that the anti-staphylococcal compound facilitating P. aeruginosa dominance under normoxia and anoxia is greater than 3 kDa in size and is heat-stable. Not all interspecies interactions studied were antagonistic, as S. aureus exoproducts were shown to restore and enhance P. aeruginosa motility under normoxia and anoxia in an isolate dependent manner. Collectively, this study suggests changes in oxygen availability within regions of the CF lung is likely to influence interspecies interactions and in turn, potentially influence disease progression.

An investigation into E-cigarette cytotoxicity in-vitro using a novel 3D differentiated co-culture model of human airways.

Currently there is a lack of consensus on the possible adverse health effects of E-cigarettes (ECs). Important factors including cell model employed and exposure method determine the physiological relevance of EC studies. The present study aimed to evaluate EC cytotoxicity using a physiologically relevant in-vitro multicellular model of human airways. Human bronchial epithelial cells (CALU-3) and pulmonary fibroblasts (MRC-5) were co-cultured at air-liquid-interface for 11-14 days post which they were exposed to whole cigarette smoke (WCS) or EC vapour (ECV) at standard ISO-3308 regime for 7 m using a bespoke aerosol delivery system. ECV effects were further investigated at higher exposure times (1 h-6 h). Results showed that while WCS significantly reduced cell viability after 7 m, ECV decreased cell viability only at exposure times higher than 3 h. Furthermore, ECV caused elevated IL-6 and IL-8 production despite reduced cell viability. ECV exposure also produced a marked increase in oxidative stress. Finally, WCS but not ECV exposure induced caspase 3/7 activation, suggesting a caspase independent death of ECV exposed cells. Overall, our results indicate that prolonged ECV exposure (≥3 h) has a significant impact on pro-inflammatory mediators' production, oxidative stress and cell viability but not caspase 3/7 activity.

A comparative study of electronic cigarette vapor extracts on airway-related cell lines in vitro.

The use of electronic cigarettes (ECs) is rapidly increasing worldwide; however, scientific evidence regarding EC cytotoxicity is limited. The aim of this study was to evaluate the acute cytotoxicity of EC vapor extract (ECE) on airway-related cells in vitro. Cigarette smoke extract (CSE), vapor extract of fifteen brands/flavors of ECs and the extract from the E-vehicle (propylene glycol and glycerin) was collected. Extracts, in concentrations of 100-12.5%, were added to human bronchial epithelial (BEAS-2B, IB3-1 and C38), fibroblast (Wi-38) and macrophage (J774 and THP-1) cell lines. Viability was assessed after 24 h using a standard XTT assay. Viability of <70% of control (no extract) was considered cytotoxic according to UNI EN ISO 10993-5 standards. CSE displayed a concentration-dependent influence on cell viability across all four cell lines with 100% producing the most toxic effect, therefore validating the model and indicating higher cytotoxicity than in ECEs. ECEs did reduce viability although this was not correlated with nicotine content or the E-vehicle. However, several flavors proved cytotoxic, with variation between different brands and cell lines. These data indicate that not all ECs are the same and that use of a particular flavor or brand may have differing effects. The cell line used is also an important factor. More research is crucial to ascertain the health effects of different ECs before they can be accepted as a safe alternative to tobacco cigarettes.

Towards a 21st-century roadmap for biomedical research and drug discovery: consensus report and recommendations.

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and non-governmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathways-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this.

Investigation of the enhanced antimicrobial activity of combination dry powder inhaler formulations of lactoferrin.

The airways of most people with cystic fibrosis are colonized with biofilms of the Gram-negative, opportunistic pathogen Pseudomonas aeruginosa. Delivery of antibiotics directly to the lung in the form of dry powder aerosols offers the potential to achieve high local concentrations directly to the biofilms. Unfortunately, current aerosolised antibiotic regimes are unable to efficiently eradicate these biofilms from the airways. We investigated the ability of the innate antimicrobial, lactoferrin, to enhance the activity of two aminoglycoside antibiotics (tobramycin and gentamicin) against biofilms of P. aeruginosa strain PAO1. Biofilms were prepared in 96 well polystyrene plates. Combinations of the antibiotics and various lactoferrin preparations were spray dried. The bacterial cell viability of the various spray dried combinations was determined. Iron-free lactoferrin (apo lactoferrin) induced a 3-log reduction in the killing of planktonic cell by the aminoglycoside antibiotics (p<0.01) and also reduced both the formation and persistence of P. aeruginosa biofilms (p<0.01). Combinations of lactoferrin and an aminoglycoside displays potential as an effective new therapeutic strategy in the treatment of P. aeruginosa biofilms infections such as those typical of the CF lungs.

The N-terminus of CD14 acts to bind apoptotic cells and confers rapid-tethering capabilities on non-myeloid cells.

Cell death and removal of cell corpses in a timely manner is a key event in both physiological and pathological situations including tissue homeostasis and the resolution of inflammation. Phagocytic clearance of cells dying by apoptosis is a complex sequential process comprising attraction, recognition, tethering, signalling and ultimately phagocytosis and degradation of cell corpses. A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within this process. The role of myeloid cell CD14 in mediating apoptotic cell interactions with macrophages has long been known though key molecules and residues involved have not been defined. Here we sought to further dissect the function of CD14 in apoptotic cell clearance. A novel panel of THP-1 cell-derived phagocytes was employed to demonstrate that CD14 mediates effective apoptotic cell interactions with macrophages in the absence of detectable TLR4 whilst binding and responsiveness to LPS requires TLR4. Using a targeted series of CD14 point mutants expressed in non-myeloid cells we reveal CD14 residue 11 as key in the binding of apoptotic cells whilst other residues are reported as key for LPS binding. Importantly we note that expression of CD14 in non-myeloid cells confers the ability to bind rapidly to apoptotic cells. Analysis of a panel of epithelial cells reveals that a number naturally express CD14 and that this is competent to mediate apoptotic cell clearance. Taken together these data suggest that CD14 relies on residue 11 for apoptotic cell tethering and it may be an important tethering molecule on so called 'non-professional' phagocytes thus contributing to apoptotic cell clearance in a non-myeloid setting. Furthermore these data establish CD14 as a rapid-acting tethering molecule, expressed in monocytes, which may thus confer responsiveness of circulating monocytes to apoptotic cell derived material.

Free secretory component from cystic fibrosis sputa displays the cystic fibrosis glycosylation phenotype.

Secretory IgA contributes to humoral defense mechanisms against pathogens targeting mucosal surfaces, and secretory component (SC) fulfills multiple roles in this defense. The aims of this study were to quantify total SC and to analyze the form of free SC in sputa from normal subjects, subjects with asthma, and subjects with cystic fibrosis (CF). Significantly higher levels of SC were detected in CF compared with both other groups. Gel filtration chromatography revealed that SC in CF was relatively degraded. Free SC normally binds interleukin (IL)-8 and inhibits its function. However, in CF sputa, IL-8 binding to intact SC was reduced. Analysis of the total carbohydrate content of free SC signified overglycosylation in CF compared with normal subjects and subjects with asthma. Monosaccharide composition analysis of free SC from CF subjects revealed overfucosylation and undersialylation, in agreement with the reported CF glycosylation phenotype. SC binding to IL-8 did not interfere with the binding of IL-8 to heparin, indicating distinct binding sites on IL-8 for negative regulation of function by SC and heparin. We suggest that defective structure and function of SC contribute to the characteristic sustained inflammatory response in the CF airways.

Safety and use of sputum induction in children with cystic fibrosis.

We assessed the safety and use of induced sputum (IS) in children with cystic fibrosis (CF). Forty-eight children (19 males) with CF, mean age 12.6 (range, 7.3-17.0) years and median forced expired volume in 1 sec (FEV(1)) 48% (range, 14-77%) predicted were recruited. Patients spontaneously expectorated sputum and then performed sputum induction by inhalation of nebulized 7% hypertonic saline. Samples were sent for bacteriological culture, and for measurement of the following inflammatory mediators: interleukin-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase activity. FEV(1) was performed before and after inhalation of hypertonic saline. There was no increase in mediator levels in IS compared to expectorated sputum (ES) samples. Only 3 patients demonstrated significant bronchoconstriction following inhalation of hypertonic saline, by the method used. From the ES samples, Pseudomonas aeruginosa was isolated in 13 patients, Staphylococcus aureus in 7 patients, Stenotrophomonas maltophilia in 1 patient, and both Pseudomonas aeruginosa and Staphylococcus aureus in 5 patients. All these organisms were found in the IS samples. However, in 2 patients whose ES grew no organisms, one patient's IS grew Pseudomonas aeruginosa, and the other patient's IS grew Staphylococcus aureus. In our study, sputum induction was safe, with no proinflammatory effect.

Effects of recombinant human DNase and hypertonic saline on airway inflammation in children with cystic fibrosis.

Recombinant human DNase (rhDNase) is an established treatment in cystic fibrosis (CF), but it may liberate cationic mediators bound to DNA in the airways. An alternative mucolytic therapy is hypertonic saline (HS); however, HS may potentiate neutrophilic inflammation. We compared the effect of rhDNase and HS on cationic proinflammatory mediators in CF sputum. In a randomized, crossover trial, 48 children with CF were allocated consecutively to 12 weeks of once-daily 2.5 mg rhDNase, alternate-day 2.5 mg rhDNase, and twice-daily 7% HS. Sputum levels of total interleukin-8 (IL-8), free IL-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase (NE) activity were measured before and after each treatment. The change in mediator levels from baseline with daily rhDNase and HS was not significant; however, with alternate-day rhDNase, there was an increase in free IL-8. When changes in mediator levels with daily rhDNase were compared with alternate-day rhDNase and HS, no significant differences were detected. Only changes in NE activity were associated with changes in lung function. In summary, we were unable to show that rhDNase or HS promote airway inflammation in CF.