Pre‐clinical non‐small cell lung cancer (NSCLC) models are poorly representative of the considerable inter‐ and intra‐tumor heterogeneity of the disease in patients. Primary cell‐based in vitro models of NSCLC are therefore desirable for novel therapy development and personalized cancer medicine. Methods have been described to generate rapidly proliferating epithelial cell cultures from multiple human epithelia using 3T3‐J2 feeder cell culture in the presence of Y‐27632, a RHO‐associated protein kinase (ROCK) inhibitor, in what are known as “conditional reprograming conditions” (CRC) or 3T3 + Y. In some cancer studies, variations of this methodology have allowed primary tumor cell expansion across a number of cancer types but other studies have demonstrated the preferential expansion of normal epithelial cells from tumors in such conditions. Here, we report our experience regarding the derivation of primary NSCLC cell cultures from 12 lung adenocarcinoma patients enrolled in the Tracking Cancer Evolution through Therapy (TRACERx) clinical study and discuss these in the context of improving the success rate for in vitro cultivation of cells from NSCLC tumors.

Background Iron is required for cell growth, and various cancers have been shown to proliferate more readily when iron replete. We have shown this previously in lung cancer and further demonstrated that this was reduced by either iron chelation or knockdown of IREB2, an iron regulatory gene. 1 Differences in iron content of bronchoalveolar lavage (BAL) fluid have been reported in smokers compared to non-smokers, 2 so we hypothesised that iron dysregulation might be an active mechanism of cancer progression in smokers. Methods Two lung cancer cell lines were cultured with either ferrous (Fe2+) or ferric (Fe3+) forms of iron, or with cigarette smoke extract (CSE). Proliferation, apoptosis, necrosis and migration were assessed by BRDU assay, FACS and scratch wound assay respectively. Iron regulation was assessed by means of gene expression and Western blot for IREB2 (protein product IRP2), ferritin and transferrin receptor. Resected lung cancers (n = 78) were stained for iron regulatory protein 2 (IRP2) and staining related to clinical features such as tumour size and survival. Results Cancer cells proliferated more in the presence of ferrous iron or 5% CSE (p Cancers staining positive for IRP2 tended to be larger (p = 0.045) and survival poorer (p = 0.079). Conclusions Proliferation of cancer cells driven by iron dysregulation may be a clinically relevant mechanism in lung cancer, particularly in smokers.

BackgroundAberrations of EGFR signalling drive cancer development. In squamous cell lung cancer (SqCLC), EGFR is overexpressed. LRIG1 is a negative regulator of EGFR and patient pre-invasive SqCLC samples show LRIG1 loss, suggesting involvement in early disease pathogenesis. In skin and gut homeostasis, LRIG1 regulates stem cells. In the upper airway, basal cells act as stem cells and are the putative origin of SqCLC. We hypothesise LRIG1 has a key role in airway homeostasis and its loss promotes pre-invasive SqCLC development.Methods Lrig1 EGFP-ires-CreERT2 mice were used to delineate airway LRIG1 expression. Flow sorted LRIG1-positive and -negative murine basal cells were used in 2D and 3D colony-forming, spheroid and proliferation assays. A murine SqCLC model was set up through application of N-Nitrosotris-(2-chloroethyl)urea (NTCU). Pre-invasive lesions and tumour development were compared between wild-type (WT), heterozygous and LRIG1-knockout (KO) animals. Human basal cells obtained from bronchoscopy were sorted according to LRIG1 expression and used directly in colony-forming assays or maintained in primary culture to assess the effect of shRNA knockdown of LRIG1. LRIG1-knockdown cells were assessed in colony-forming and proliferation assays, and differentiation and invasion were assessed using organotypic models.ResultsLRIG1 is expressed by 40% of airway basal cells. LRIG1-expressing murine basal cells exhibit increased colony-forming capacity (p = 0.0286), spheroid formation (p = 0.0043) and proliferation (p = 0.0043) compared with LRIG1-negative cells. Similarly, LRIG1-expressing human airway basal cells isolated from endobronchial brush biopsy samples exhibit increased colony-forming capacity (p = 0.0469). Topical application of NTCU to mice recapitulates the development of human pre-invasive and SCLC lesions after 23 weeks. Results show lesions in LRIG1-KO mice to be larger than those of WT animals. Knock down of LRIG1 in cultured human airway basal cells alters cell phenotype, leading to an increased colony-forming efficiency and greater proliferation at cell confluence.ConclusionsLRIG1 has an important role in stem cell homeostasis of the human and murine airway epithelium. Loss of LRIG1 promotes pre-cancerous lesion development in a murine SqCLC mouse model and behaviour of human epithelial cells in culture, indicating a potential target for chemoprevention of SqCLC in humans.

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