Purpose: To develop an in vitro model system to study the multi-step pathogenesis of lung cancer. Introduction: We established a series of cdk4/hTERT-immortalized human bronchial epithelial cell lines (HBECs) which can be genetically manipulated, are able to differentiate into mature airway cells in organotypic cultures, but do not form colonies in soft agar or tumors in nude mice. Previously, we observed that combinations of p53 knockdown with mutant EGFR or with physiological levels of oncogenic KRASV12 progressed HBECs part but not all of the way towards malignancy. These cells form colonies in soft agar, but fail to form tumors in nude mice. In the present study we evaluated various combinations of oncogenic manipulation, higher levels of expression of oncogenic KRASV12, and the biologic behavior of clones selected to grow to large size in soft agar. Methods: HBEC3 immortalized with cdk4 and hTERT was engineered with various vectors systems to have stable: p53 knockdown (with shRNA); PTEN knockdown; express oncogenic KRASV12 at physiologic or 10 fold higher; express mutant EGFR (deletion and missense mutants). These were made alone and in combination. These and selected colonies were then tested for soft agar colony formation and subcutaneous and orthotopic tumor formation in nude mice. Results: Without oncogenic manipulation soft agar colony forming efficiency (CFE) was <∼0.1%. With various oncogenic manipulations including higher expression of KRASV12 alone soft agar colony CFEs were ∼3%, and the combination of higher KRASV12 and p53 knockdown increased CFE to 8%. None of these were tumorigenic. However, the combination of higher KRASV12 and p53 knockdown but not high KRASV12 alone led to development of a subset of very large soft agar colonies (∼0.1-0.2%). These large colonies were isolated and found to have CFE of 10∼15% with large colony numbers increased 10 fold. 5 of the large colonies were expanded and tested for tumorgenicity and 2 were capable of forming subcutaneous and orthotopic (bronchus) tumors. Of interest one was an adenocarcinoma and the other was a squamous cell carcinoma. In addition, the adenocarcinoma expressed high levels of KRASV12 and the squamous carcinoma expressed normal levels of KRASV12. Conclusion: Multiple oncogenic changes found in lung cancer (telomerase expression, p16 bypass, p53 ablation, mutant EGFR, oncogenic KRAS) introduced into HBECs are still not capable of full oncogenic transformation. However, a subset of cells selected for the ability to form large colonies in soft agar are capable of tumor formation and tend to differentiate into two non-small cell lung cancer phenotypes (adenocarcinoma and squamous cell cancer). The expression, genetic, and epigenetic changes as well as the stem cell like characteristics of these subsets of cells are being investigated to delineate the key final steps in lung cancer pathogenesis.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]