Like many organs, the airway is lined by a single layer of epithelial cells that form the barrier between the external and internal environments. The epithelium is the first line of defense against inhaled pathogens, particularly respiratory viruses that infect airway epithelial cells. The airway epithelium also encounters carcinogens present in tobacco smoke and can become transformed, initiating lung tumorigenesis.
Cells cultured on plastic do not replicate true biological conditions. Advances in culturing techniques have allowed researchers to create more relevant in vitro models to study the airway epithelium. In particular, airway epithelial cells grown in an air-liquid interface will differentiate and form a pseudo-epithelium in culture. This system can be used to study epithelial differentiation, transformation, or importantly, can be used to study viral infection as in the study described below.
Lifeline® offers three types of airway epithelial cells from different locations:
All Lifeline® airway epithelial cells are optimized for growth in BronchiaLife® medium. Lifeline® also offers Air-Liquid Interface Epithelial Differentiation Medium, optimized for air-liquid interface culture of Lifeline® bronchial/tracheal epithelial cells.
Lifeline® Cells Used to Study Lung Cancer and Viral Infection
Lung cancer is the primary cause of cancer death and smoking is a significant factor in the development of lung cancer. NNK (4-[methylnitrosamine]-1-[3-pyridyl]-1-butanone) is a carcinogen found in tobacco that is associated with lung tumorigenesis. NNK binds and activates nicotinic acetylcholine receptors (nAChRs), which are expressed in bronchial epithelial cells. Another important factor in the pathogenesis of lung cancer is insulin growth factor (IGF) signaling, which, when activated, contributes to lung tumorigenesis. In a study last year, Boo and colleagues investigated how NNK contributes to lung cancer through IGF signaling activation.
The researchers used Lifeline® human bronchial epithelial cells and Lifeline® small airway epithelial cells, as well as HB56B and BEAS-2B immortalized human bronchial cell lines, to demonstrate that NNK treatment activates IGF signaling in the airway epithelium. They show that NNK treatment of BEAS-2B cells induces upregulation of IGF2, a ligand for IGF receptors (IGF-Rs). Using premalignant HBEL/p53i cells, they found that NNK also induces IGF-1R phosphorylation. Furthermore, the group demonstrated that NNK induces IGF2 secretion by stimulated Ca2+ influx into the cell, in a nAChR-dependent manner. Finally, the researchers illustrated that in a NNK-induced mouse model of lung cancer, calcium channel blocker (CCB) treatment decreased tumor burden. Together, the results of this study suggest that NNK exposure through smoking leads to activation of IGF signaling and CCBs may be efficacious against this mechanism of lung tumorigenesis.
The common cold is triggered by infection of the airway epithelium by human coronavirus 229E (HCoV-229E). HCoV-229E gains entry into epithelial cells through its spike (S) glycoprotein, which is activated by either cell surface (TMPRSS2) or endosomal (cathepsin L) proteases. HCoV-229E was originally isolated for laboratory research in 1966, but long-term adaptation of this isolate (VR-740, or 229E/lab) to culture has changed its behavior. In a 2016 study, Shirato and colleagues set out to define which cellular proteases activate HCoV S protein, allowing HCoV to infect a cell. Using two clinical isolates (229E/clin) and the original 229E/lab strain, they found that 229E/clin had a lower replication rate than 229E/lab in parental HeLa cells, and could not persist in HeLa cells lacking TMPRSS2 expression. Using inhibitors against cathepsin L and TMPRSS2 in parental HeLa cells and HeLa cells expressing TMPRSS2, the group found that 229E/lab utilized cathepsin L, while 229E/clin utilized TMPRSS2. The researchers concluded that the lower efficiency of cathepsin L cleavage of 229E/clin might explain the decreased replication rate of that strain. To understand the molecular differences that account for the behaviors of the different strains, the researchers identified amino acid differences between the S proteins of 229E/lab and 229E/clin that may alter cathepsin L usage of the S protein of 229E/clin. Interestingly, the researchers found that the longer they passaged 229E/clin, the more that strain utilized cathepsin L and was able to replicate in parental HeLa cells, which was likely due to a point mutation in the S protein.
To determine the relevance to human airway epithelium, the researchers used Lifeline® primary human bronchial/tracheal epithelial cells and modeled the airway epithelium using an air-liquid interface. They compared early 229E/clin passages (passage 1) to later passages (passage 20) to determine their replicative capacity. They found that later passages of 229E/clin did not replicate as well as passage 1, suggesting that use of cathepsin L reduces replicative capacity in human airway epithelium. Together, the results of this study demonstrate that long-term passaging of HCoV-229E has changed the mode of S protein activation and no longer models the clinical behavior of the virus. Additionally, the researchers suggest that rather than treating HCoV infection with cathepsin inhibitors, TMPRSS2 inhibitors may be more useful.
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