Characterization of clones derived from human bronchial epithelial cells after long-term treatment with TPM from reference cigarettes versus THS 2.2

The prevailing risk factor for lung cancer in humans is cigarette smoking. Lung cancer is a complex genetic disease characterized by genetic and epigenetic aberrations that are reflected in morphological and phenotypic changes of the bronchial and alveolar epithelium. We successfully established an in vitro model to compare the effect of long-term exposure to total particulate matter (TPM) from 3R4F reference cigarettes with that from Tobacco Heating System 2.2 (THS2.2) on cellular transformation. The study demonstrated that repeated exposure of BEAS-2B cells to THS 2.2 TPM, with a 20-fold higher concentration than 3R4F TPM, induced anchorage independence. Transformed cells were not observed following prolonged treatment of BEAS-2B cells with THS 2.2 TPM at the same or even at 5-fold higher concentration than with 3R4F TPM. Viable transformed cells (clones) were recovered from a single ancestor cell, making it possible to isolate and establish uniform cell populations. In total, 27 3R4F TPM-derived clones and 31 THS 2.2 TPM-derived clones were recovered. We have established assay capabilities to characterize individual clones based on their growth properties and invasion phenotype. For example, population doubling time (PDT) was assessed using cell growth curves, expression of the EMT markers E-cadherin and vimentin was evaluated by antibody-based high-content screening, and invasion assays were run in xCelligence® CIM chambers. Metabolic activity of the clones was examined using Seahorse FX technology (Agilent, Santa Clara, CA, USA). In addition, Short Tandem Repeat (STR) profiling was performed by ATCC (Manassas, VA, USA) to verify the relatedness to the parental BEAS-2Bcells. Our data showed that all 3R4F TPM-derived clones had increased PDT, compared with 80% of THS2.2 clones. Vimentin expression was increased in >90% of 3R4F as well as in all THS 2.2 TPM-derived clones. However, high expression of vimentin and low expression of E-cadherin did not correlate with the invasive capacity of the clones as measured by the invasion assay. Twenty-five of 27 3R4F and 29 of 31 THS2.2 TPM-derived clones matched the STR profile of the parental BEAS-2B cells. Future work will entail exome sequencing as well as in vivo xenograft studies to further elucidate the tumor-initiating and metastatic potential of the 3R4F and THS 2.2 TPM-derived clones.