Stroma-derived extracellular vesicle mRNA signatures inform histological nature of prostate cancer.

Histological assessment of prostate cancer is the key diagnostic test and can predict disease outcome. This is however an invasive procedure that carries associated risks, hence non-invasive assays to support the diagnostic pathway are much needed. A key feature of disease progression, and subsequent poor prognosis, is the presence of an altered stroma. Here we explored the utility of prostate stromal cell-derived vesicles as indicators of an altered tumour environment. We compared vesicles from six donor-matched pairs of adjacent-normal versus disease-associated primary stromal cultures. We identified 19 differentially expressed transcripts that discriminate disease from normal stromal extracellular vesicles (EVs). EVs isolated from patient serum were investigated for these putative disease-discriminating mRNA. A set of transcripts including Caveolin-1 (CAV1), TMP2, THBS1, and CTGF were found to be successful in discriminating clinically insignificant (Gleason = 6) disease from clinically significant (Gleason > 8) prostate cancer. Furthermore, correlation between transcript expression and progression-free survival suggests that levels of these mRNA may predict disease outcome. Informed by a machine learning approach, combining measures of the five most informative EV-associated mRNAs with PSA was shown to significantly improve assay sensitivity and specificity. An in-silico model was produced, showcasing the superiority of this multi-modal liquid biopsy compared to needle biopsy for predicting disease progression. This proof of concept highlights the utility of serum EV analytics as a companion diagnostic test with prognostic utility, which may obviate the need for biopsy.

Aquaglyceroporin-3's Expression and Cellular Localization Is Differentially Modulated by Hypoxia in Prostate Cancer Cell Lines.

Aquaporins are required by cells to enable fast adaptation to volume and osmotic changes, as well as microenvironmental metabolic stimuli. Aquaglyceroporins play a crucial role in supplying cancer cells with glycerol for metabolic needs. Here, we show that AQP3 is differentially expressed in cells of a prostate cancer panel. AQP3 is located at the cell membrane and cytoplasm of LNCaP cell while being exclusively expressed in the cytoplasm of Du145 and PC3 cells. LNCaP cells show enhanced hypoxia growth; Du145 and PC3 cells display stress factors, indicating a crucial role for AQP3 at the plasma membrane in adaptation to hypoxia. Hypoxia, both acute and chronic affected AQP3's cellular localization. These outcomes were validated using a machine learning classification approach of the three cell lines and of the six normoxic or hypoxic conditions. Classifiers trained on morphological features derived from cytoskeletal and nuclear labeling alongside corresponding texture features could uniquely identify each individual cell line and the corresponding hypoxia exposure. Cytoskeletal features were 70-90% accurate, while nuclear features allowed for 55-70% accuracy. Cellular texture features (73.9% accuracy) were a stronger predictor of the hypoxic load than the AQP3 distribution (60.3%).

Dog breeds and body conformations with predisposition to osteosarcoma in the UK: a case-control study.

BACKGROUND: Osteosarcoma is an aggressive and painful bone neoplasm in dogs. Previous studies have reported epidemiological associations suggesting that large body mass, long bone length and the genetics of certain breeds including the Rottweiler are associated with elevated osteosarcoma risk. However, these studies were often limited by selection bias and confounding factors, and have rarely offered insights into breed-associated protection for osteosarcoma. The current study includes 1756 appendicular and axial osteosarcoma cases presenting to VPG Histology (Bristol, UK) compared against a control population of 905,211 dogs without osteosarcoma from primary care electronic patient records in the VetCompass™ dataset. METHODS AND STUDY DESIGN: Retrospective, case-control study. Multivariable logistic regression analysis explored associations between demographic risk factors (including breed, chondrodystrophy, age, sex/neuter status, skull-shape, and body mass) and osteosarcoma of all anatomical sites. RESULTS: We identified several breeds with increased and reduced odds of osteosarcoma. At highest risk were the Rottweiler and Great Dane, with > 10 times the odds of osteosarcoma compared with crossbreds, and the Rhodesian Ridgeback, which has not featured in previous lists of at-risk breeds for osteosarcoma, and had an odds ratio of 11.31 (95% confidence interval 7.37-17.35). Breeds at lowest risk of osteosarcoma (protected breeds) included the Bichon Frise, the French Bulldog and the Cavalier King Charles Spaniel, all with odd ratios of less than 0.30 compared with crossbreds. Body mass was strongly associated with osteosarcoma risk; dogs over 40 kg exhibited osteosarcoma odds of 45.44 (95% confidence interval 33.74-61.20) compared with dogs less than 10 kg. Chondrodystrophic breeds had an osteosarcoma odds ratio of 0.13 (95% confidence interval 0.11-0.16) compared with non-chondrodystrophic breeds. CONCLUSIONS: This study provides evidence of strong breed-associated osteosarcoma risk and protection, suggesting a genetic basis for osteosarcoma pathogenesis. It highlights that breeds selected for long legs/large body mass are generally overrepresented amongst at-risk breeds, whilst those selected for short leg length/small body mass are generally protected. These findings could inform genetic studies to identify osteosarcoma risk alleles in canines and humans; as well as increasing awareness amongst veterinarians and owners, resulting in improved breeding practices and clinical management of osteosarcoma in dogs.

Design of experiment (DoE)-driven in vitro and in vivo uptake studies of exosomes for pancreatic cancer delivery enabled by copper-free click chemistry-based labelling.

Exosomes (Exo)-based therapy holds promise for treatment of lethal pancreatic cancer (PC). Limited understanding of key factors affecting Exo uptake in PC cells restricts better design of Exo-based therapy. This work aims to study the uptake properties of different Exo by PC cells. Exo from pancreatic carcinoma, melanoma and non-cancer cell lines were isolated and characterised for yield, size, morphology and exosomal marker expression. Isolated Exo were fluorescently labelled using a novel in-house developed method based on copper-free click chemistry to enable intracellular tracking and uptake quantification in cells. Important factors influencing Exo uptake were initially predicted by Design of Experiments (DoE) approach to facilitate subsequent actual experimental investigations. Uptake of all Exo types by PC cells (PANC-1) showed time- and dose-dependence as predicted by the DoE model. PANC-1 cell-derived exosomes (PANC-1 Exo) showed significantly higher uptake in PANC-1 cells than that of other Exo types at the longest incubation time and highest Exo dose. In vivo biodistribution studies in subcutaneous tumour-bearing mice similarly showed favoured accumulation of PANC-1 Exo in self-tissue (i.e. PANC-1 tumour mass) over the more vascularised melanoma (B16-F10) tumours, suggesting intrinsic tropism of PC-derived Exo for their parent cells. This study provides a simple, universal and reliable surface modification approach via click chemistry for in vitro and in vivo exosome uptake studies and can serve as a basis for a rationalised design approach for pre-clinical Exo cancer therapies.

Correction: Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB.

Correction for 'Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB' by Roman Mikhaylov et al., Lab Chip, 2020, 20, 1807-1814, DOI: 10.1039/C9LC01192G.

Novel epoxy-tiglianes stimulate skin keratinocyte wound healing responses and re-epithelialization via protein kinase C activation.

Epoxy-tiglianes are a novel class of diterpene esters. The prototype epoxy-tigliane, EBC-46 (tigilanol tiglate), possesses potent anti-cancer properties and is currently in clinical development as a local treatment for human and veterinary cutaneous tumors. EBC-46 rapidly destroys treated tumors and consistently promotes wound re-epithelialization at sites of tumor destruction. However, the mechanisms underlying these keratinocyte wound healing responses are not completely understood. Here, we investigated the effects of EBC-46 and an analogue (EBC-211) at 1.51 nM-151 µM concentrations, on wound healing responses in immortalized human skin keratinocytes (HaCaTs). Both EBC-46 and EBC-211 (1.51 nM-15.1 µM) accelerated G0/G1-S and S-G2/M cell cycle transitions and HaCaT proliferation. EBC-46 (1.51-151 nM) and EBC-211 (1.51 nM-15.1 µM) further induced significant HaCaT migration and scratch wound repopulation. Stimulated migration/wound repopulation responses were even induced by EBC-46 (1.51 nM) and EBC-211 (1.51-151 nM) with proliferation inhibitor, mitomycin C (1 µM), suggesting that epoxy-tiglianes can promote migration and wound repopulation independently of proliferation. Expression profiling analyses showed that epoxy-tiglianes modulated keratin, DNA synthesis/replication, cell cycle/proliferation, motility/migration, differentiation, matrix metalloproteinase (MMP) and cytokine/chemokine gene expression, to facilitate enhanced responses. Although epoxy-tiglianes down-regulated established cytokine and chemokine agonists of keratinocyte proliferation and migration, enhanced HaCaT responses were demonstrated to be mediated via protein kinase C (PKC) phosphorylation and significantly abrogated by pan-PKC inhibitor, bisindolylmaleimide-1 (BIM-1, 1 µM). By identifying how epoxy-tiglianes stimulate keratinocyte healing responses and re-epithelialization in treated skin, our findings support the further development of this class of small molecules as potential therapeutics for other clinical situations associated with impaired re-epithelialization, such as non-healing skin wounds.

Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB.

Acoustofluidics has been increasingly applied in biology, medicine and chemistry due to its versatility in manipulating fluids, cells and nano-/micro-particles. In this paper, we develop a novel and simple technology to fabricate a surface acoustic wave (SAW)-based acoustofluidic device by clamping electrodes made using a printed circuit board (PCB) with a piezoelectric substrate. The PCB-based SAW (PCB-SAW) device is systematically characterised and benchmarked with a SAW device made using the conventional photolithography process with the same specifications. Microparticle manipulations such as streaming in droplets and patterning in microchannels were demonstrated in the PCB-SAW device. In addition, the PCB-SAW device was applied as an acoustic tweezer to pattern lung cancer cells to form three or four traces inside the microchannel in a controllable manner. Cell viability of ∼97% was achieved after acoustic manipulation using the PCB-SAW device, which proved its ability as a suitable tool for acoustophoretic applications.

Correction: Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir-bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death.

This article was originally published under standard licence, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly.

Energy Stress-Mediated Cytotoxicity in Tuberous Sclerosis Complex 2-Deficient Cells with Nelfinavir and Mefloquine Treatment.

To find new anti-cancer drug therapies, we wanted to exploit homeostatic vulnerabilities within Tuberous Sclerosis Complex 2 (TSC2)-deficient cells with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivity. We show that nelfinavir and mefloquine synergize to selectively evoke a cytotoxic response in TSC2-deficient cell lines with mTORC1 hyperactivity. We optimize the concentrations of nelfinavir and mefloquine to a clinically viable range that kill cells that lack TSC2, while wild-type cells tolerate treatment. This new clinically viable drug combination causes a significant level of cell death in TSC2-deficient tumor spheroids. Furthermore, no cell recovery was apparent after drug withdrawal, revealing potent cytotoxicity. Transcriptional profiling by RNA sequencing of drug treated TSC2-deficient cells compared to wild-type cells suggested the cytotoxic mechanism of action, involving initial ER stress and an imbalance in energy homeostatic pathways. Further characterization revealed that supplementation with methyl pyruvate alleviated energy stress and reduced the cytotoxic effect, implicating energy deprivation as the trigger of cell death. This work underpins a critical vulnerability with cancer cells with aberrant signaling through the TSC2-mTORC1 pathway that lack flexibility in homeostatic pathways, which could be exploited with combined nelfinavir and mefloquine treatment.

Nuclear cytometry and chromatin organization.

The nuclear-targeting chemical probe, for the detection and quantification of DNA within cells, has been a mainstay of cytometry-from the colorimetric Feulgen stain to smart fluorescent agents with tuned functionality. The level of nuclear structure and function at which the probe aims to readout, or indeed at which a DNA-targeted drug acts, is shadowed by a wide range of detection modalities and analytical methods. These methods are invariably limited in terms of the resolution attainable versus the volume occupied by targeted chromatin structures. The scalar challenge arises from the need to understand the extent and different levels of compaction of genomic DNA and how such structures can be re-modeled, reported, or even perturbed by both probes and drugs. Nuclear cytometry can report on the complex levels of chromatin order, disorder, disassembly, and even active disruption by probes and drugs. Nuclear probes can report defining features of clinical and therapeutic interest as in NETosis and other cell death processes. New cytometric approaches continue to bridge the scalar challenges of analyzing chromatin organization. Advances in super-resolution microscopy address the resolution and depth of analysis issues in cellular systems. Typical of recent insights into chromatin organization enabled by exploiting a DNA interacting probe is ChromEM tomography (ChromEMT). ChromEMT uses the unique properties of the anthraquinone-based cytometric dye DRAQ5™ to reveal that local and global 3D chromatin structures effect differences in compaction. The focus of this review is nuclear and chromatin cytometry, with linked reference to DNA targeting probes and drugs as exemplified by the anthracenediones.

Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir-bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death.

Cancer cells lose homeostatic flexibility because of mutations and dysregulated signaling pathways involved in maintaining homeostasis. Tuberous Sclerosis Complex 1 (TSC1) and TSC2 play a fundamental role in cell homeostasis, where signal transduction through TSC1/TSC2 is often compromised in cancer, leading to aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 hyperactivation increases the basal level of endoplasmic reticulum (ER) stress via an accumulation of unfolded protein, due to heightened de novo protein translation and repression of autophagy. We exploit this intrinsic vulnerability of tumor cells lacking TSC2, by treating with nelvinavir to further enhance ER stress while inhibiting the proteasome with bortezomib to prevent effective protein removal. We show that TSC2-deficient cells are highly dependent on the proteosomal degradation pathway for survival. Combined treatment with nelfinavir and bortezomib at clinically relevant drug concentrations show synergy in selectively killing TSC2-deficient cells with limited toxicity in control cells. This drug combination inhibited tumor formation in xenograft mouse models and patient-derived cell models of TSC and caused tumor spheroid death in 3D culture. Importantly, 3D culture assays differentiated between the cytostatic effects of the mTORC1 inhibitor, rapamycin, and the cytotoxic effects of the nelfinavir/bortezomib combination. Through RNA sequencing, we determined that nelfinavir and bortezomib tip the balance of ER protein homeostasis of the already ER-stressed TSC2-deficient cells in favor of cell death. These findings have clinical relevance in stratified medicine to treat tumors that have compromised signaling through TSC and are inflexible in their capacity to restore ER homeostasis.

Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones.

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532-587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.

The unidirectional hypoxia-activated prodrug OCT1002 inhibits growth and vascular development in castrate-resistant prostate tumors.

BACKGROUND: OCT1002 is a unidirectional hypoxia-activated prodrug (uHAP) OCT1002 that can target hypoxic tumor cells. Hypoxia is a common feature in prostate tumors and is known to drive disease progression and metastasis. It is, therefore, a rational therapeutic strategy to directly target hypoxic tumor cells in an attempt to improve treatment for this disease. Here we tested OCT1002 alone and in combination with standard-of-care agents in hypoxic models of castrate-resistant prostate cancer (CRPC). METHODS: The effect of OCT1002 on tumor growth and vasculature was measured using murine PC3 xenograft and dorsal skin fold (DSF) window chamber models. The effects of abiraterone, docetaxel, and cabazitaxel, both singly and in combination with OCT1002, were also compared. RESULTS: The hypoxia-targeting ability of OCT1002 effectively controls PC3 tumor growth. The effect was evident for at least 42 days after exposure to a single dose (30 mg/kg) and was comparable to, or better than, drugs currently used in the clinic. In DSF experiments OCT1002 caused vascular collapse in the PC3 tumors and inhibited the revascularization seen in controls. In this model OCT1002 also enhanced the anti-tumor effects of abiraterone, cabazitaxel, and docetaxel; an effect which was accompanied by a more prolonged reduction in tumor vasculature density. CONCLUSIONS: These studies provide the first evidence that OCT1002 can be an effective agent in treating hypoxic, castrate-resistant prostate tumors, either singly or in combination with established chemotherapeutics for prostate cancer.

Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells.

Uncontrolled cell growth in Tuberous Sclerosis Complex occurs due to inappropriate activation of mechanistic (mammalian) target of rapamycin complex 1 (mTORC1). The current therapy, rapamycin, produced promising clinical trial results, but patient tumours regrow if treatment is discontinued, revealing rapamycin has cytostatic properties rather than a cytotoxic effect. Taking advantage of the enhanced levels of endoplasmic reticulum (ER) stress present in TSC2-null cells, we investigated drug combinations producing a cytotoxic response. We found a nelfinavir and salinomycin combination specifically killed TSC2-deficient, mTORC1 hyperactive cells. Cytotoxicity was rescued by reducing protein synthesis, either through mTORC1 inhibition or cycloheximide treatment. This indicates that the drug combination targets the cells by tipping the protein homeostasis balance of the already metabolically stressed TSC2-deficient cells in favour of cell death. Furthermore, this drug combination also inhibited tumour formation in TSC2-deficient cell models and caused tumour spheroid death in 3D culture. Importantly, the 3D assay could differentiate the cytostatic agent, rapamycin, from the cytotoxic nelfinavir/salinomycin combination. Sporadic cancer cell lines with hyperactive mTORC1 signalling were also susceptible to this nelfinavir/salinomycin drug combination. This work indicates that the protein homeostasis pathway is an attractive therapeutic target in both Tuberous Sclerosis Complex and mTORC1-driven sporadic cancers.

Targeting Hypoxic Prostate Tumors Using the Novel Hypoxia-Activated Prodrug OCT1002 Inhibits Expression of Genes Associated with Malignant Progression.

Purpose: To understand the role of hypoxia in prostate tumor progression and to evaluate the ability of the novel unidirectional hypoxia-activated prodrug OCT1002 to enhance the antitumor effect of bicalutamide.Experimental Design: The effect of OCT1002 on prostate cancer cells (LNCaP, 22Rv1, and PC3) was measured in normoxia and hypoxia in vitroIn vivo, tumor growth and lung metastases were measured in mice treated with bicalutamide, OCT1002, or a combination. Dorsal skin fold chambers were used to image tumor vasculature in vivo Longitudinal gene expression changes in tumors were analyzed using PCR.Results: Reduction of OCT1002 to its active form (OCT1001) decreased prostate cancer cell viability. In LNCaP-luc spheroids, OCT1002 caused increased apoptosis and decreased clonogenicity. In vivo, treatment with OCT1002 alone, or with bicalutamide, showed significantly greater tumor growth control and reduced lung metastases compared with controls. Reestablishment of the tumor microvasculature following bicalutamide-induced vascular collapse is inhibited by OCT1002. Significantly, the upregulation of RUNX2 and its targets caused by bicalutamide alone was blocked by OCT1002.Conclusions: OCT1002 selectively targets hypoxic tumor cells and enhances the antitumor efficacy of bicalutamide. Furthermore, bicalutamide caused changes in gene expression, which indicated progression to a more malignant genotype; OCT1002 blocked these effects, emphasizing that more attention should be attached to understanding genetic changes that may occur during treatment. Early targeting of hypoxic cells with OCT1002 can provide a means of inhibiting prostate tumor growth and malignant progression. This is of importance for the design and refinement of existing androgen-deprivation regimens in the clinic. Clin Cancer Res; 23(7); 1797-808. ©2016 AACR.

Tracking the Cyclin B1-GFP Sensor to Profile the Pattern of Mitosis Versus Mitotic Bypass.

This chapter provides a method for quantitative single cell analysis to track the transition of single cells from G2, indicated by high cyclin B1 levels, to G1 polyploidy phase (G1(p)), indicated by low cyclin B1 levels, in a 4n population. The cell tracking methodology described provides a fluorescence fingerprint suitable for deriving G2/M or G2/G1p transitions. Notably, during late G2 the absolute cyclin B1-eGFP reporter levels obtained were high and the switch-off point identifiable, with destruction rates of a similar order across all cell cycle routing avenues. The three principle parameters extracted were defined as (1) G2-to-G1(p) transition duration (tGFP(off)); (2) rate of sensor destruction (kGFP(off)), and (3) peak sensor expression (GFP(peak)).

A 3D ex vivo mandible slice system for longitudinal culturing of transplanted dental pulp progenitor cells.

Harnessing mesenchymal stem cells for tissue repair underpins regenerative medicine. However, how the 3D tissue matrix maintains such cells in a quiescent state whilst at the same time primed to respond to tissue damage remains relatively unknown. Developing more physiologically relevant 3D models would allow us to better understand the matrix drivers and influence on cell-lineage differentiation in situ. In this study, we have developed an ex vivo organotypic rat mandible slice model; a technically defined platform for the culture and characterization of dental pulp progenitor cells expressing GFP driven by the ß-actin promoter (cGFP DPPCs). Using confocal microscopy we have characterized how the native environment influences the progenitor cells transplanted into the dental pulp. Injected cGFP-DPPCs were highly viable and furthermore differentially proliferated in unique regions of the mandible slice; in the dentine region, cGFP-DPPCs showed a columnar morphology indicative of expansion and lineage differentiation. Hence, we demonstrated the systematic capacity for establishing a dental pulp cell-micro-community, phenotypically modified in the tooth (the "biology"); and at the same time addressed technical challenges enabling the mandible slice to be accessible on platforms for high-content imaging (the biology in a "multiplex" format).

A multi-stage random forest classifier for phase contrast cell segmentation.

We present a machine learning based approach to automatically detect and segment cells in phase contrast images. The proposed method consists of a multi-stage classification scheme based on random forest (RF) classifier. Both low level and mid level image features are used to determine meaningful cell regions. Pixel-wise RF classification is first carried out to categorize pixels into 4 classes (dark cell, bright cell, halo artifact, and background) and generate a probability map for cell regions. K-means clustering is then applied on the probability map to group similar pixels into candidate cell regions. Finally, cell validation is performed by another RF to verify the candidate cell regions. The proposed method has been tested on U2-OS human osteosarcoma phase contrast images. The experimental results show better performance of the proposed method with precision 92.96% and recall 96.63% compared to a state-of-the-art segmentation technique.

Endoplasmic reticulum stress and cell death in mTORC1-overactive cells is induced by nelfinavir and enhanced by chloroquine.

Inappropriate activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is common in cancer and has many cellular consequences including elevated endoplasmic reticulum (ER) stress. Cells employ autophagy as a critical compensatory survival mechanism during ER stress. This study utilised drug-induced ER stress through nelfinavir in order to examine ER stress tolerance in cell lines with hyper-active mTORC1 signalling. Our initial findings in wild type cells showed nelfinavir inhibited mTORC1 signalling and upregulated autophagy, as determined by decreased rpS6 and S6K1 phosphorylation, and SQTSM1 protein expression, respectively. Contrastingly, cells with hyper-active mTORC1 displayed basally elevated levels of ER stress which was greatly exaggerated following nelfinavir treatment, seen through increased CHOP mRNA and XBP1 splicing. To further enhance the effects of nelfinavir, we introduced chloroquine as an autophagy inhibitor. Combination of nelfinavir and chloroquine significantly increased ER stress and caused selective cell death in multiple cell line models with hyper-active mTORC1, whilst control cells with normalised mTORC1 signalling tolerated treatment. By comparing chloroquine to other autophagy inhibitors, we uncovered that selective toxicity invoked by chloroquine was independent of autophagy inhibition yet entrapment of chloroquine to acidified lysosomal/endosomal compartments was necessary for cytotoxicity. Our research demonstrates that combination of nelfinavir and chloroquine has therapeutic potential for treatment of mTORC1-driven tumours.

ProtocolNavigator: emulation-based software for the design, documentation and reproduction biological experiments.

MOTIVATION: Experimental reproducibility is fundamental to the progress of science. Irreproducible research decreases the efficiency of basic biological research and drug discovery and impedes experimental data reuse. A major contributing factor to irreproducibility is difficulty in interpreting complex experimental methodologies and designs from written text and in assessing variations among different experiments. Current bioinformatics initiatives either are focused on computational research reproducibility (i.e. data analysis) or laboratory information management systems. Here, we present a software tool, ProtocolNavigator, which addresses the largely overlooked challenges of interpretation and assessment. It provides a biologist-friendly open-source emulation-based tool for designing, documenting and reproducing biological experiments. AVAILABILITY AND IMPLEMENTATION: ProtocolNavigator was implemented in Python 2.7, using the wx module to build the graphical user interface. It is a platform-independent software and freely available from http://protocolnavigator.org/index.html under the GPL v2 license.