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1.  Mesenchymal Stem Cells as Vectors for Lung Cancer Therapy 
Respiration  2013;85(6):443-451.
Despite recent advances in treatment, lung cancer accounts for one third of all cancer-related deaths, underlining the need of development of new therapies. Mesenchymal stem cells (MSCs) possess the ability to specifically home into tumours and their metastases. This property of MSCs could be exploited for the delivery of various anti-tumour agents directly into tumours. However, MSCs are not simple delivery vehicles but cells with active physiological process. This review outlines various agents which can be delivered by MSCs with substantial emphasis on TRAIL (tumour necrosis factor-related apoptosis-inducing ligand).
doi:10.1159/000351284
PMCID: PMC3929000  PMID: 23711430
Apoptosis; Delivery vectors; Homing; Lung cancer; Mesenchymal stem cells; TRAIL

4.  Stem cells as vectors for antitumour therapy 
Thorax  2010;65(4):362-369.
Recent research suggests that mesenchymal stem cells (MSCs) are able to migrate specifically to tumours and their metastases throughout the body. This has led to considerable excitement about the possibility of modifying these cells to express anticancer molecules and using them as specific targeted anticancer agents. However, there are concerns that systemically delivered MSCs may have non-desirable effects, and there are also many unanswered questions including the mechanism of tumour homing. This review investigates the different MSC-delivered anticancer agents, addresses the questions and concerns, and tries to place this potential therapy in future cancer management.
doi:10.1136/thx.2009.128025
PMCID: PMC3401681  PMID: 20388765
5.  Combination of endobronchial ultrasound-guided transbronchial needle aspiration with standard bronchoscopic techniques for the diagnosis of stage I and stage II pulmonary sarcoidosis 
Respirology (Carlton, Vic.)  2011;16(3):467-472.
Background and objective
Standard bronchoscopic techniques (transbronchial lung biopsy and endobronchial biopsy) provide a diagnosis in 70% of patients with pulmonary sarcoidosis. Previous data suggest that endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has a high sensitivity in patients with sarcoidosis. The feasibility and utility of combining EBUS-TBNA with standard bronchoscopic techniques is unknown. The aim of this study was to evaluate the feasibility, safety and efficacy of combined EBUS-TBNA and standard bronchoscopic techniques in patients with suspected sarcoidosis and enlarged mediastinal or hilar lymphadenopathy.
Methods
Forty consecutive patients with suspected pulmonary sarcoidosis and enlarged mediastinal or hilar lymph nodes (radiographical stage I and stage II) underwent EBUS-TBNA followed by transbronchial biopsies and endobronchial biopsies under conscious sedation.
Results
Thirty-nine out of 40 patients successfully underwent combined EBUS-TBNA and standard bronchoscopy. Twenty-seven patients were diagnosed with sarcoidosis, eight had tuberculosis, two had reactive lymphadenopathy, two had lymphoma and one had metastatic adenocarcinoma. In patients with sarcoidosis, the sensitivity of EBUS-TBNA for detection of noncaseating granulomas was 85%, compared with a sensitivity of 35% for standard bronchoscopic techniques (P < 0.001). The diagnostic yield of combined EBUS-TBNA and bronchoscopy was 93% (P < 0.0001).
Conclusions
Combination of EBUS-TBNA with standard bronchoscopic techniques is safe and feasible, and optimizes the diagnostic yield in patients with pulmonary sarcoidosis and enlarged intrathoracic lymphadenopathy.
doi:10.1111/j.1440-1843.2011.01933.x
PMCID: PMC3361303  PMID: 21261784
endobronchial ultrasound; mediastinal lymphadenopathy; sarcoidosis; transbronchial biopsy
6.  Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors 
eLife  2013;2:e00966.
Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to “neutral drift” of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease.
DOI: http://dx.doi.org/10.7554/eLife.00966.001
eLife digest
As air flows into our lungs, the lining of the nasal cavity, the throat and the rest of the respiratory tract prevents microbes, bacteria, dust and other small particles from entering the lungs. The lining of these airways is made up of many different types of cells, which must be continuously replaced as they become damaged. Experiments in mice have shown that cells called basal cells act as progenitor cells to keep the lining supplied with new cells. Progenitor cells are similar to stem cells: they divide to make, on average, one copy of themselves and one mature cell of another type (such as a secretory cell). This ensures that healthy supply of progenitor cells is maintained for the future. However, it is not clear whether this process takes place at the level of individual progenitor cells or as an average for a population of cells.
Teixeira et al. have now performed a study which shows that basal cells achieve this balance as a result of averaging. The study took advantage of the fact that cellular organelles called mitochondria have their own DNA, which gradually accumulates mutations over time. This makes it possible to identify groups of cells that are descended from a single progenitor cell because they will all contain the same mitochondrial mutation.
By studying lung tissue from seven individuals, Teixeira et al. were able to identify clusters of related cells and found that, as expected, the size of the clusters increased with age. And by applying a mathematical model across all the cells in the study, it was discovered that whenever one basal progenitor cell committed to a particular fate, another progenitor cell duplicated itself: however, this balancing process happened in a random manner across a large number of cells, and not at the level of individual progenitor cells. Interestingly, it was found random cell division happened among smokers too, but was accelerated. This leads to clusters of identical cells forming more quickly in smokers than in non-smokers. In addition to providing further insights into the origins of lung cancer, the statistical methods developed by Teixeira et al. could be used to analyse the behaviour of many other types of stem or progenitor cells.
DOI: http://dx.doi.org/10.7554/eLife.00966.002
doi:10.7554/eLife.00966
PMCID: PMC3804062  PMID: 24151545
Human lineage tracing; mtDNA mutations; lung basal progenitor stem cells; stochastic homeostasis; airways; Human
7.  Mesenchymal Stem Cells as Vectors for Lung Disease 
Stem cells divide asymmetrically, leading to self-renewal and the production of a daughter cell committed to differentiation. This property has engendered excitement as to the use of these cells for treatments. The majority of the work with stem cells has used the relatively accessible and well-characterized adult bone marrow stem cell compartment. Initially the focus of this research was on the potential for these stem cells to repair damaged organs by differentiating into epithelial cells to replace the injured areas. More recently it has become clear that engraftment of these stem cells as epithelial tissue is a rare event with perhaps limited clinical significance. Despite this, stem cells appear to have the ability to home to and be specifically recruited to areas of inflammation and injured tissues often characterized by excessive extracellular matrix deposition. As a consequence they are intimately involved in regions of physiological and pathological repair. Coupled with this, autologous hematopoietic stem cells, or the relatively immunoprivileged mesenchymal stem cells, can be expanded and engineered ex vivo and reintroduced without immunomodulation. The prospect of using such cells clinically as a cellular therapy holds much promise for many conditions and organ pathologies. Here we address the evidence for the incorporation of bone marrow stem cells into areas of stroma formation as a prelude to possible future treatment options for common lung diseases.
doi:10.1513/pats.200801-009AW
PMCID: PMC2643224  PMID: 18684723
stem cell; vector; gene therapy; bone marrow; chemokine
8.  Endobronchial Ultrasound–guided Transbronchial Needle Aspiration Prevents Mediastinoscopies in the Diagnosis of Isolated Mediastinal Lymphadenopathy 
Rationale: Patients with isolated mediastinal lymphadenopathy (IML) are a common presentation to physicians, and mediastinoscopy is traditionally considered the “gold standard” investigation when a pathological diagnosis is required. Endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) is established as an alternative to mediastinoscopy in patients with lung cancer.
Objective: To determine the efficacy and health care costs of EBUS-TBNA as an alternative initial investigation to mediastinoscopy in patients with isolated IML.
Methods: Prospective multicenter single-arm clinical trial of 77 consecutive patients with IML from 5 centers between April 2009 and March 2011. All patients underwent EBUS-TBNA. If EBUS-TBNA did not provide a diagnosis, then participants underwent mediastinoscopy.
Measurements and Main Results: EBUS-TBNA prevented 87% of mediastinoscopies (95% confidence interval [CI], 77–94%; P < 0.001) but failed to provide a diagnosis in 10 patients (13%), all of whom underwent mediastinoscopy. The sensitivity and negative predictive value of EBUS-TBNA in patients with IML were 92% (95% CI, 83–95%) and 40% (95% CI, 12–74%), respectively. One patient developed a lower respiratory tract infection after EBUS-TBNA, requiring inpatient admission. The cost of the EBUS-TBNA procedure per patient was £1,382 ($2,190). The mean cost of the EBUS-TBNA strategy was £1,892 ($2,998) per patient, whereas a strategy of mediastinoscopy alone was significantly more costly at £3,228 ($5,115) per patient (P < 0.001). The EBUS-TBNA strategy is less costly than mediastinoscopy if the cost per EBUS-TBNA procedure is less than £2,718 ($4,307) per patient.
Conclusions: EBUS-TBNA is a safe, highly sensitive, and cost-saving initial investigation in patients with IML.
Clinical trial registered with ClinicalTrials.gov (NCT00932854).
doi:10.1164/rccm.201203-0393OC
PMCID: PMC3423452  PMID: 22652031
endobronchial ultrasound; mediastinal lymphadenopathy; sarcoidosis; tuberculosis; lymphoma
9.  Myd88 deficiency influences murine tracheal epithelial metaplasia and submucosal gland abundance 
The Journal of pathology  2011;224(2):190-202.
Tracheal epithelial remodelling, excess mucus production, and submucosal gland hyperplasia are features of numerous lung diseases, yet their origins remain poorly understood. Previous studies have suggested that NF-κB signalling may regulate airway epithelial homeostasis. The purpose of this study was to determine whether deletion of the NF-κB signalling pathway protein myeloid differentiation factor 88 (Myd88) influenced tracheal epithelial cell phenotype. We compared wild-type and Myd88-deficient or pharmacologically inhibited adult mouse tracheas and determined that in vivo Myd88 deletion resulted in increased submucosal gland number, secretory cell metaplasia, and excess mucus cell abundance. We also found that Myd88 was required for normal resolution after acute tracheal epithelial injury. Microarray analysis revealed that uninjured Myd88-deficient tracheas contained 103 transcripts that were differentially expressed relative to wild-type and all injured whole tracheal samples. These clustered into several ontologies and networks that are known to functionally influence epithelial cell phenotype. Comparing these transcripts to those expressed in airway progenitor cells revealed only five common genes, suggesting that Myd88 influences tracheal epithelial homeostasis through an extrinsic mechanism. Overall, this study represents the first identification of Myd88 as a regulator of adult tracheal epithelial cell phenotype.
doi:10.1002/path.2876
PMCID: PMC3434371  PMID: 21557220
Myd88; mucus; trachea; secretion; injury; homeostasis
10.  β-Catenin determines upper airway progenitor cell fate and preinvasive squamous lung cancer progression by modulating epithelial–mesenchymal transition 
The Journal of pathology  2012;226(4):575-587.
Human lung cancers, including squamous cell carcinoma (SCC) are a leading cause of death and, whilst evidence suggests that basal stem cells drive SCC initiation and progression, the mechanisms regulating these processes remain unknown. In this study we show that β-catenin signalling regulates basal progenitor cell fate and subsequent SCC progression. In a cohort of preinvasive SCCs we established that elevated basal cell β-catenin signalling is positively associated with increased disease severity, epithelial proliferation and reduced intercellular adhesiveness. We demonstrate that transgene-mediated β-catenin inhibition within keratin 14-expressing basal cells delayed normal airway repair while basal cell-specific β-catenin activation increased cell proliferation, directed differentiation and promoted elements of early epithelial-mesenchymal transition (EMT), including increased Snail transcription and reduced E-cadherin expression. These observations are recapitulated in normal human bronchial epithelial cells in vitro following both pharmacological β-catenin activation and E-cadherin inhibition, and mirrored our findings in preinvasive SCCs. Overall, the data show that airway basal cell β-catenin determines cell fate and its mis-expression is associated with the development of human lung cancer.
doi:10.1002/path.3962
PMCID: PMC3434372  PMID: 22081448
β-catenin; E-cadherin; Snail; airway; stem cell; pre-invasive; lung cancer; squamous
11.  Stem Cell Implants for Cancer Therapy: TRAIL-Expressing Mesenchymal Stem Cells Target Cancer Cells In Situ 
Journal of Breast Cancer  2012;15(3):273-282.
Purpose
Tumor-specific delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis-inducing peptide, at effective doses remains challenging. Herein we demonstrate the utility of a scaffold-based delivery system for sustained therapeutic cell release that capitalizes on the tumor-homing properties of mesenchymal stem cells (MSCs) and their ability to express genetically-introduced therapeutic genes.
Methods
Implants were formed from porous, biocompatible silk scaffolds seeded with full length TRAIL-expressing MSCs (FLT-MSCs). under a doxycycline inducible promoter. In vitro studies with FLT-MSCs demonstrated TRAIL expression and antitumor effects on breast cancer cells. Next, FLT-MSCs were administered to mice using three administration routes (mammary fat pad co-injections, tail vein injections, and subcutaneous implantation on scaffolds).
Results
In vitro cell-specific bioluminescent imaging measured tumor cell specific growth in the presence of stromal cells and demonstrated FLT-MSC inhibition of breast cancer growth. FLT-MSC implants successfully decreased bone and lung metastasis, whereas liver metastasis decreased only with tail vein and co-injection administration routes. Average tumor burden was decreased when doxycycline was used to induce TRAIL expression for co-injection and scaffold groups, as compared to controls with no induced TRAIL expression.
Conclusion
This implant-based therapeutic delivery system is an effective and completely novel method of anticancer therapy and holds great potential for clinical applications.
doi:10.4048/jbc.2012.15.3.273
PMCID: PMC3468780  PMID: 23091539
Breast neoplasms; Mesenchymal stem cells; Tissue engineering; Tissue therapy; TNF-related apoptosis-inducing ligand
12.  Mediastinal staging of NSCLC with endoscopic and endobronchial ultrasound 
Mediastinal staging of non-small-cell lung cancer (NSCLC) is of paramount importance. it distinguishes operable from inoperable disease, guides prognosis and allows accurate comparison of outcomes in clinical trials. Noninvasive imaging modalities for mediastinal staging include CT, PET and integrated PET-CT. Mediastinoscopy is considered the current gold standard; however, each of these techniques has limitations in sensitivity or specificity. These inadequacies mean that 10% of operations performed with curative intent in patients with NSCLC are futile, owing to inaccurate locoregional lymph-node staging. endoscopic and endobronchial ultrasound-guided mediastinal lymph-node aspiration are important and promising innovative techniques with reported sensitivities and specificities higher than standard investigations. The role of these techniques in mediastinal lymph-node staging is evolving rapidly and early data suggest that they may diminish the need for invasive surgical staging of the mediastinum. Furthermore, these are outpatient procedures that do not require general anesthesia and may be combined safely in the same sitting, for optimal accuracy of mediastinal staging. we propose a new algorithm for the diagnosis and staging of NSCLC, based on the current evidence, which incorporates endoscopic and endobronchial ultrasound as a first investigation after CT in patients with intrathoracic disease.
doi:10.1038/nrclinonc.2009.39
PMCID: PMC3401685  PMID: 19390554
13.  Suitability of EBUS-TBNA Specimens for Subtyping and Genotyping of NSCLC: A Multi-Centre Study of 774 Patients 
Rationale
The current management of advanced non-small cell lung cancer (NSCLC) requires differentiation between squamous and non-squamous sub-types as well as epidermal growth factor receptor (EGFR) mutation status. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used for the diagnosis and staging of lung cancer. However, it is unclear whether cytology specimens obtained with EBUS-TBNA are suitable for the sub-classification and genotyping of NSCLC.
Objectives
To determine whether cytology specimens obtained from EBUS-TBNA in routine practice are suitable for phenotyping and genotyping of NSCLC.
Methods
Cytological diagnoses from EBUS-TBNA were recorded from 774 patients with known or suspected lung cancer across 5 centres in the United Kingdom between 2009 and 2011.
Measurements and Main Results
The proportion of patients with a final diagnosis by EBUS-TBNA in whom subtype was classified was 77% (95% CI 73% - 80%). The rate of NSCLC not otherwise specified (NSCLC-NOS) was significantly reduced in patients who underwent immunohistochemistry (adjusted OR 0.50 95% CI 0.28 – 0.82, P=0.016). EGFR mutation analysis was possible in 107 (90%) of the 119 patients in whom mutation analysis was requested. The sensitivity, negative predictive value and diagnostic accuracy of EBUS-TBNA in patients with NSCLC was 88% (95% CI 86% - 91%), 72% (95% CI 66% - 77%) and 91% (95% CI 89% - 93%) respectively.
Conclusions
This large multi-centre pragmatic study demonstrates that cytology samples obtained from EBUS-TBNA in routine practice are suitable for sub-typing of NSCLC and EGFR mutation analysis and that use of immunohistochemistry reduces the rate of NSCLC-NOS.
doi:10.1164/rccm.201202-0294OC
PMCID: PMC3378660  PMID: 22505743
Endobronchial ultrasound; non-small cell lung cancer; adenocarcinoma; EGFR mutation; squamous cell carcinoma; NSCLC-NOS
14.  The learning curve for EBUS-TBNA 
Thorax  2010;66(4):352-353.
doi:10.1136/thx.2010.146407
PMCID: PMC3361006  PMID: 21097815
15.  Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration for the Diagnosis of Intrathoracic Lymphadenopathy in Patients with Extrathoracic Malignancy 
Introduction
Mediastinal lymphadenopathy in patients with an extrathoracic malignancy is a common clinical scenario. Invasive sampling of intrathoracic lymph nodes may be performed by mediastinoscopy or endoscopic ultrasound-guided fine needle aspiration. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is an alternative to mediastinoscopy and endoscopic ultrasound in patients with lung cancer and sarcoidosis. The utility of EBUS-TBNA in patients with extrathoracic malignancy was evaluated.
Methods
Consecutive patients who were suspected to have intrathoracic lymph node metastases from an extrathoracic malignancy underwent EBUS-TBNA. When EBUS-TBNA did not provide a specific diagnosis, patients underwent mediastinoscopy or clinical follow-up of at least 6 months duration.
Results
One hundred sixty-one patients meeting the inclusion criteria underwent EBUS-TBNA in five UK centers over a 3-year period. EBUS-TBNA diagnosed mediastinal or hilar metastases in 71 (44%) patients, new lung cancer in 20 (12%) patients, and sarcoidosis in 14 (9%) patients. The sensitivity, negative predictive value for malignancy, and overall accuracy for EBUS-TBNA were 87%, 73% and 88%, respectively. One hundred ten (68%) patients in the study had a final diagnosis of malignant intrathoracic lymphadenopathy.
Conclusion
Because of the high prevalence of alternative diagnoses, pathological evaluation is important in patients with extrathoracic malignancy and suspected mediastinal or hilar lymph node metastases. EBUS-TBNA is a safe and sensitive technique and may be considered a first-line investigation in these patients.
doi:10.1097/JTO.0b013e318223c3fe
PMCID: PMC3361007  PMID: 21792077
Endobronchial ultrasound; Mediastinal lymphadenopathy; Breast cancer; Lung cancer
16.  Utility of endobronchial ultrasound-guided transbronchial needle aspiration in patients with tuberculous intrathoracic lymphadenopathy: a multicentre study 
Thorax  2011;66(10):889-893.
Background
Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has emerged as an important tool for the diagnosis and staging of lung cancer but its role in the diagnosis of tuberculous intrathoracic lymphadenopathy has not been established. The aim of this study was to describe the diagnostic utility of EBUS-TBNA in patients with intrathoracic lymphadenopathy due to tuberculosis (TB).
Methods
156 consecutive patients with isolated intrathoracic TB lymphadenitis were studied across four centres over a 2-year period. Only patients with a confirmed diagnosis or unequivocal clinical and radiological response to antituberculous treatment during follow-up for a minimum of 6 months were included. All patients underwent routine clinical assessment and a CT scan prior to EBUS-TBNA. Demographic data, HIV status, pathological findings and microbiological results were recorded.
Results
EBUS-TBNA was diagnostic of TB in 146 patients (94%; 95% CI 88% to 97%). Pathological findings were consistent with TB in 134 patients (86%). Microbiological investigations yielded a positive culture of TB in 74 patients (47%) with a median time to positive culture of 16 days (range 3–84) and identified eight drug-resistant cases (5%). Ten patients (6%) did not have a specific diagnosis following EBUS; four underwent mediastinoscopy which confirmed the diagnosis of TB while six responded to empirical antituberculous therapy. There was one complication requiring an inpatient admission.
Conclusions
EBUS-TBNA is a safe and effective first-line investigation in patients with tuberculous intrathoracic lymphadenopathy.
doi:10.1136/thoraxjnl-2011-200063
PMCID: PMC3361304  PMID: 21813622
17.  Pulmonary mass in a 19-year-old male 
Thorax  2011;67(5):468-470.
doi:10.1136/thoraxjnl-2011-200732
PMCID: PMC3361305  PMID: 22106020
18.  Correction: Rac1 Deletion Causes Thymic Atrophy 
PLoS ONE  2011;6(6):10.1371/annotation/5c665e96-0303-438f-afb0-c257c3481692.
doi:10.1371/annotation/5c665e96-0303-438f-afb0-c257c3481692
PMCID: PMC3107337
19.  Magnetic resonance imaging of mesenchymal stem cells homing to pulmonary metastases using biocompatible magnetic nanoparticles 
Cancer research  2009;69(23):8862-8867.
The ability of mesenchymal stem cells (MSCs) to specifically home to tumors has suggested their potential use as a delivery vehicle for cancer therapeutics. MSC integration into tumors has been demonstrated in animal models using histopathological techniques after animal sacrifice. Tracking the delivery and engraftment of MSCs into human tumors will need in vivo imaging techniques. We hypothesized that labeling MSCs with iron oxide nanoparticles would enable in vivo tracking with magnetic resonance imaging (MRI).
Human MSCs were labeled in vitro with superparamagnetic iron oxide nanoparticles, with no effect on differentiation potential, proliferation, survival or migration of the cells. In initial experiments we demonstrated that as few as 1000 MSCs carrying iron oxide nanoparticles can be detected by MRI one month after their coinjection with breast cancer cells which formed sub-cutaneous tumors. Subsequently we show that intravenously injected iron-labeled MSCs could be tracked in vivo to multiple lung metastases using MRI, observations that were confirmed histologically.
This is the first study to utilize MRI to track MSCs to lung metastases in vivo. This technique has the potential to demonstrate MSC integration into human tumors, allowing early phase clinical studies examining MSC homing in patients with metastatic tumors.
doi:10.1158/0008-5472.CAN-09-1912
PMCID: PMC2833408  PMID: 19920196
Cancer; Magnetic; Metastases; MRI; Nanoparticle
20.  Bone Marrow Stem Cells Expressing Keratinocyte Growth Factor via an Inducible Lentivirus Protects against Bleomycin-Induced Pulmonary Fibrosis 
PLoS ONE  2009;4(11):e8013.
Many common diseases of the gas exchange surface of the lung have no specific treatment but cause serious morbidity and mortality. Idiopathic Pulmonary Fibrosis (IPF) is characterized by alveolar epithelial cell injury, interstitial inflammation, fibroblast proliferation and collagen accumulation within the lung parenchyma. Keratinocyte Growth Factor (KGF, also known as FGF-7) is a critical mediator of pulmonary epithelial repair through stimulation of epithelial cell proliferation. During repair, the lung not only uses resident cells after injury but also recruits circulating bone marrow-derived cells (BMDC). Several groups have used Mesenchymal Stromal Cells (MSCs) as therapeutic vectors, but little is known about the potential of Hematopoietic Stem cells (HSCs). Using an inducible lentiviral vector (Tet-On) expressing KGF, we were able to efficiently transduce both MSCs and HSCs, and demonstrated that KGF expression is induced in a regulated manner both in vitro and in vivo. We used the in vivo bleomycin-induced lung fibrosis model to assess the potential therapeutic effect of MSCs and HSCs. While both populations reduced the collagen accumulation associated with bleomycin-induced lung fibrosis, only transplantation of transduced HSCs greatly attenuated the histological damage. Using double immunohistochemistry, we show that the reduced lung damage likely occurs through endogenous type II pneumocyte proliferation induced by KGF. Taken together, our data indicates that bone marrow transplantation of lentivirus-transduced HSCs can attenuate lung damage, and shows for the first time the potential of using an inducible Tet-On system for cell based gene therapy in the lung.
doi:10.1371/journal.pone.0008013
PMCID: PMC2779453  PMID: 19956603
21.  Mesenchymal Stem Cell delivery of TRAIL can eliminate Metastatic Cancer 
Cancer research  2009;69(10):4134-4142.
Cancer is a leading cause of mortality throughout the world and new treatments are urgently needed. Recent studies suggest that bone marrow-derived mesenchymal stem cells (MSCs) home to and incorporate within tumor tissue. We hypothesised that MSCs engineered to produce and deliver TNF-related apoptosis-inducing ligand (TRAIL), a transmembrane protein which causes selective apoptosis of tumor cells, would home to and kill cancer cells in a lung metastatic cancer model.
Human MSCs were transduced with TRAIL and the IRES-eGFP reporter gene, under the control of a tetracycline promoter using a lentiviral vector. Transduced and activated MSCs caused lung (A549), breast (MDAMB231), squamous (H357), and cervical (Hela) cancer cell apoptosis and death in co-culture experiments. Subcutaneous xenograft experiments confirmed directly delivered TRAIL-expressing MSCs were able to significantly reduce tumor growth (0.12 cm3 (0.04-0.21) vs 0.66 cm3 (0.21-1.11) (p<0.001)). We then found using a pulmonary metatastasis model, systemically delivered MSCs localised to lung metastases and the controlled local delivery of TRAIL completely cleared the metastatic disease in 38% of mice compared to 0% of controls (p<0.05).
This is the first study to demonstrate a significant reduction in metastatic tumor burden with frequent eradication of metastases using inducible TRAIL-expressing MSCs. This has a wide potential therapeutic role, which includes the treatment of both primary tumors and their metastases, possibly as an adjuvant therapy in clearing micrometastatic disease following primary tumor resection.
doi:10.1158/0008-5472.CAN-08-4698
PMCID: PMC2699841  PMID: 19435900
Mesenchymal Stem Cell; Lung Cancer; Breast Cancer; TRAIL; Apoptosis
22.  PI3-kinase dependent activation of apoptotic machinery occurs on commitment of epidermal keratinocytes to terminal differentiation 
Cell research  2009;19(3):328-339.
We have investigated the earliest events in commitment of human epidermal keratinocytes to terminal differentiation. Phosphorylated Akt and caspase activation were detected in cells exiting the basal layer of the epidermis. Activation of Akt by retroviral transduction of primary cultures of human keratinocytes resulted in an increase in abortive clones founded by transit amplifying cells, while inhibition of the upstream kinase, PI3 kinase, inhibited suspension-induced terminal differentiation. Caspase inhibition also blocked differentiation, the primary mediator being caspase 8. Caspase activation was initiated by 2 hours in suspension, preceding the onset of expression of the terminal differentiation marker involucrin by several hours. Incubation of suspended cells with fibronectin or inhibition of PI3 kinase prevented caspase induction. At 2 hours in suspension, keratinocytes that had committed to terminal differentiation had increased side scatter, were 7AAD positive and annexin V negative; they exhibited loss of mitochondrial membrane potential and increased cardiolipin oxidation, but with no increase in reactive oxygen species. These properties indicate that the onset of terminal differentiation, while regulated by PI3 kinase and caspases, is not a classical apoptotic process.
doi:10.1038/cr.2008.281
PMCID: PMC2650684  PMID: 18766172
epidermis; integrins; extracellular matrix
23.  Switch from αvβ5 to αvβ6 integrin expression protects squamous cell carcinomas from anoikis 
The Journal of Cell Biology  2004;166(3):419-431.
Stratified squamous epithelia express the αvβ5 integrin, but in squamous cell carcinomas (SCCs) there is down-regulation of αvβ5 and up-regulation of αvβ6. To investigate the significance of this finding, we transduced an αv-negative human SCC line with retroviral vectors encoding αv integrins. αvβ5-expressing cells underwent suspension-induced apoptosis (anoikis), whereas αv-negative cells and cells expressing αvβ6 did not. Resistance to anoikis correlated with PKB/Akt activation in suspension, but not with changes in PTEN or p110α PI3 kinase levels. Anoikis was induced in parental and αvβ6-expressing cells by inhibiting PI3 kinase. Conversely, activation of Akt or inhibition of caspases in αvβ5-expressing cells suppressed anoikis. Caspase inhibition resulted in increased phosphoAkt, placing caspase activation upstream of decreased Akt activation. Anoikis required the cytoplasmic domain of β5 and was independent of the death receptor pathway. These results suggest that down-regulation of αvβ5 through up-regulation of αvβ6 may protect SCCs from anoikis by activating an Akt survival signal.
doi:10.1083/jcb.200312074
PMCID: PMC2172256  PMID: 15289499
keratinocyte; apoptosis; differentiation; Akt; extracellular matrix
24.  Genomic evidence of pre-invasive clonal expansion, dispersal and progression in bronchial dysplasia 
The Journal of Pathology  2011;224(2):153-159.
The term ‘field cancerization’ is used to describe an epithelial surface that has a propensity to develop cancerous lesions, and in the case of the aerodigestive tract this is often as a result of chronic exposure to carcinogens in cigarette smoke 1, 2. The clinical endpoint is the development of multiple tumours, either simultaneously or sequentially in the same epithelial surface. The mechanisms underlying this process remain unclear; one possible explanation is that the epithelium is colonized by a clonal population of cells that are at increased risk of progression to cancer. We now address this possibility in a short case series, using individual genomic events as molecular biomarkers of clonality. In squamous lung cancer the most common genomic aberration is 3q amplification. We use a digital PCR technique to assess the clonal relationships between multiple biopsies in a longitudinal bronchoscopic study, using amplicon boundaries as markers of clonality. We demonstrate that clonality can readily be defined by these analyses and confirm that field cancerization occurs at a pre-invasive stage and that pre-invasive lesions and subsequent cancers are clonally related. We show that while the amplicon boundaries can be shared between different biopsies, the degree of 3q amplification and the internal structure of the 3q amplicon varies from lesion to lesion. Finally, in this small cohort, the degree of 3q amplification corresponds to clinical progression. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
doi:10.1002/path.2887
PMCID: PMC3378694  PMID: 21506132
clonality; bronchial dysplasia; molecular copy-number counting
25.  Myd88 deficiency influences murine tracheal epithelial metaplasia and submucosal gland abundance 
The Journal of Pathology  2011;224(2):190-202.
Tracheal epithelial remodelling, excess mucus production, and submucosal gland hyperplasia are features of numerous lung diseases, yet their origins remain poorly understood. Previous studies have suggested that NF-κB signalling may regulate airway epithelial homeostasis. The purpose of this study was to determine whether deletion of the NF-κB signalling pathway protein myeloid differentiation factor 88 (Myd88) influenced tracheal epithelial cell phenotype. We compared wild-type and Myd88-deficient or pharmacologically inhibited adult mouse tracheas and determined that in vivo Myd88 deletion resulted in increased submucosal gland number, secretory cell metaplasia, and excess mucus cell abundance. We also found that Myd88 was required for normal resolution after acute tracheal epithelial injury. Microarray analysis revealed that uninjured Myd88-deficient tracheas contained 103 transcripts that were differentially expressed relative to wild-type and all injured whole tracheal samples. These clustered into several ontologies and networks that are known to functionally influence epithelial cell phenotype. Comparing these transcripts to those expressed in airway progenitor cells revealed only five common genes, suggesting that Myd88 influences tracheal epithelial homeostasis through an extrinsic mechanism. Overall, this study represents the first identification of Myd88 as a regulator of adult tracheal epithelial cell phenotype. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
doi:10.1002/path.2876
PMCID: PMC3434371  PMID: 21557220
Myd88; mucus; trachea; secretion; injury; homeostasis

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