Jennifer L. Gilbert

Novel stem/progenitor cell population from murine tracheal submucosal gland ducts with multipotent regenerative potential.

The airway epithelium is in direct contact with the environment and therefore constantly at risk for injury. Basal cells (BCs) have been found to repair the surface epithelium (SE), but the contribution of other stem cell populations to airway epithelial repair has not been identified. We demonstrated that airway submucosal gland (SMG) duct cells, in addition to BCs, survived severe hypoxic‐ischemic injury. We developed a method to isolate duct cells from the airway. In vitro and in vivo models were used to compare the self‐renewal and differentiation potential of duct cells and BCs. We found that only duct cells were capable of regenerating SMG tubules and ducts, as well as the SE overlying the SMGs. SMG duct cells are therefore a multipotent stem cell for airway epithelial repair This is of importance to the field of lung regeneration as determining the repairing cell populations could lead to the identification of novel therapeutic targets and cell‐based therapies for patients with airway diseases. STEM CELLS 2011;29:1283–1293

Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer.

Smoking is the most important known risk factor for the development of lung cancer. Tobacco exposure results in chronic inflammation, tissue injury, and repair. A recent hypothesis argues for a stem/progenitor cell involved in airway epithelial repair that may be a tumor-initiating cell in lung cancer and which may be associated with recurrence and metastasis. We used immunostaining, quantitative real-time PCR, Western blots, and lung cancer tissue microarrays to identify subpopulations of airway epithelial stem/progenitor cells under steady-state conditions, normal repair, aberrant repair with premalignant lesions and lung cancer, and their correlation with injury and prognosis. We identified a population of keratin 14 (K14)-expressing progenitor epithelial cells that was involved in repair after injury. Dysregulated repair resulted in the persistence of K14+ cells in the airway epithelium in potentially premalignant lesions. The presence of K14+ progenitor airway epithelial cells in NSCLC predicted a poor prognosis, and this predictive value was strongest in smokers, in which it also correlated with metastasis. This suggests that reparative K14+ progenitor cells may be tumor-initiating cells in this subgroup of smokers with NSCLC.

Isolation and In Vitro Characterization of Basal and Submucosal Gland Duct Stem/Progenitor Cells from Human Proximal Airways

Basal cells and submucosal gland (SMG) duct cells have been isolated and shown to be stem/progenitor cell populations for the murine airway epithelium. However, methods for the isolation of basal and SMG duct cells from human airways have not been defined. We used an optimized two‐step enzyme digestion protocol to strip the surface epithelium from tracheal specimens separate from SMG cells, and we then sorted the basal and duct stem/progenitors using fluorescence‐activated cell sorting. We used nerve growth factor receptor, as well as a combination of CD166 and CD44, to sort basal cells and also used CD166 to isolate SMG duct cells. Sorted stem/progenitor cells were cultured to characterize their self‐renewal and differentiation ability. Both basal and SMG duct cells grew into spheres. Immunostaining of the spheres showed mostly dense spheres with little to no central lumen. The spheres expressed cytokeratins 5 and 14, with some mucus‐ and serous‐secreting cells. The sphere‐forming efficiency and the rate of growth of the spheres varied widely between patient samples and correlated with the degree of hyperplasia of the epithelium. We found that only aldehyde dehydrogenase (ALDH)hi basal and duct cells were capable of sphere formation. Global inhibition of ALDH, as well as specific inhibition of the ALDH2 isoform, inhibited self‐renewal of both basal and duct cells, thereby producing fewer and smaller spheres. In conclusion, we have developed methods to isolate basal and SMG duct cells from the surface epithelium and SMGs of human tracheas and have developed an in vitro model to characterize their self‐renewal and differentiation.

Aldehyde Dehydrogenase Activity Enriches for Proximal Airway Basal Stem Cells and Promotes Their Proliferation

Both basal and submucosal gland (SMG) duct stem cells of the airway epithelium are capable of sphere formation in the in vitro sphere assay, although the efficiency at which this occurs is very low. We sought to improve this efficiency of sphere formation by identifying subpopulations of airway basal stem cells (ABSC) and SMG duct cells based on their aldehyde dehydrogenase (ALDH) activity. ALDH(hi) ABSCs and SMG duct cells were highly enriched for the population of cells that could make spheres, while the co-culture of ALDH(hi) differentiated cells with the ALDH(hi) ABSCs increased their sphere-forming efficiency. Specific ALDH agonists and antagonists were used to show that airway specific ALDH isozymes are important for ABSC proliferation. Pathway analysis of gene expression profiling of ALDH(hi) and ALDH(lo) ABSCs revealed a significant upregulation of the arachidonic acid (AA) metabolism pathway in ALDH(hi) ABSCs. We confirmed the importance of this pathway in the metabolism of proliferating ALDH(hi) ABSCs using bioenergetics studies as well as agonists and antagonists of the AA pathway. These studies could lead to the development of novel strategies for altering ABSC proliferation in the airway epithelium.

Abstract 1042: Down-regulation of UTX contributes to lung cancer progression

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Smoking, the main risk factor leading to lung cancer, causes both genetic and epigenetic changes resulting in aberrant gene expression. UTX is a histone demethylase that has been implicated in many cancers and has been shown to regulate both cell cycle and differentiation genes. However, the role of UTX has not been elucidated in lung cancer. We hypothesized that smoking injury leads to a down regulation in UTX expression. This results in an inability of repairing airway epithelial cells to differentiate, resulting in premalignant lesions and ultimately invasive lung cancer. Examination of published lung cancer data sets revealed a down-regulation of Utx gene expression in patients with poorer prognosis, tumor grade, tumor recurrence and in smokers. To assess the involvement of UTX protein expression in lung cancer we stained a lung cancer tissue microarray and found a significant correlation between down-regulation of UTX protein expression and poorer prognosis in metastasis (p=0.0005) and smokers (p= 0.02). UTX expression was found to be significantly lower in squamous cell carcinoma compared to adenocarcinoma, linking down-regulation of UTX in a diseased caused solely by smoking. To test if smoking directly affects Utx expression we treated mouse airway epithelial cells at air-liquid interface with media infused with cigarette smoke for 9 days. After this short time point of acute smoke exposure there was a significant down regulation of Utx gene expression (p=0.03). We carried out UTX overexpression studies to assess the role of UTX in lung cancer cells lines and found up-regulation of UTX results in up-regulation of genes involved in lung development and differentiation, including Nrf2, FoxJ2 and FZD4. Our finding support the hypothesis that smoking causes a reduction in UTX that affects the proliferation and differentiation of airway epithelial cells contributing to lung cancer progression. Re-activation of UTX therefore may be a potential therapeutic target for the prevention of lung cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1042. doi:1538-7445.AM2012-1042