Human EGFR cDNA encoding the exon 19-deletion mutant (EGFR^ex19del) was generated by a polymerase chain reaction-based method (7) and subcloned into a retroviral vector, pMXs-Puro-3×HA, as described previously (7). The plasmid pBabe-puro Kras-V12 was a gift from Dr C. Counter (8). The plasmid pBabe-neo largeT cDNA was a gift from Dr R. Weinberg (9). Matrigel was obtained from BD Biosciences (Franklin Lakes, NJ, USA). Murine EGF (mEGF) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA). Erlotinib was purchased from Wako Pure Chemical Industries (Osaka, Japan). Trametinib and Captisol were purchased from ChemScene (Monmouth Junction, NJ, USA). Y-27632 was purchased from Calbiochem (Darmstadt, Germany). TrypLE Express from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

C57BL/6 mice were used at 6–9 weeks of age. Isolation, 3D culture and passage were performed similarly to methods previously described for colon epithelial cells, except that R-Spondin, Noggin and Jagged-1 were not added to culture media (10). In various analyses, mEGF and Y-27632 were added to the medium both in 3D and 2D culture, both at final concentrations of 50 nM. To passage cells in 2D culture, cells were washed with PBS, treated with Trypl Express reagent, which is a mixture of protease and collagenase, and then detached from the dish using a cell-scraper. Time-lapse imaging was performed using an Incucyte cell-analyzer (Essen Bioscience, Ann Arbor, MI, USA).

Cellular caspase activity was measured using the Caspase-Glo 3/7 Assay kit according to the manufacturer's recommendation (Promega, Madison, WI, USA). The values were normalized to cell numbers. The results shown in Fig. 2C is results from analyses of cells 2 h after the re-plating upon passage.

LE cells were transduced with retroviral vectors harboring Kras^G12V, EGFR^ex19del, or SV40 Large-T plus selection marker (Puro^r for Kras^G12V and EGFR^ex19del, and Neo^r for SV40 Large-T construct). Ecotropic viruses were packaged using PLAT-E cells (a gift from Dr T. Kitamura, Tokyo University) (11) and Fugene 6 transfection reagent. Infection of LE cells in 3D culture with retroviruses was performed as described (12).

All animal experiments were performed after approval of Miyagi Cancer Center Research Institute Animal Care and Use committee. In allograft experiments, 4×10⁵ of LE-LT/Kras^G12V cells were injected sc into the dorsal flank of nude mice. LE-LT/EGFR^ex19del cells were injected as 1:1 mixtures with Matrigel at 2×10⁶ cells per injection site.

Immunohistochemical analyses were performed using reagents from Roche Ventana systems (Basel, Switzerland). Antibodies against thyroid transcription factor 1 (TTF1) (SP141) and Ki-67 (30–9) were from Roche. Anti-cytokeratin 14 (CK14) goat antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-prosurfactant protein-C (SPC) antibody (ab40879) was obtained from Abcam (Cambridge, UK). Anti-EpCAM monoclonal antibody (2–17-F-1) was obtained from MBL (Nagoya, Japan). Frozen sections were prepared for anti-EpCAM staining only, as the antibody did not work on paraffin-embedded sections (data not shown). Flow cytometry was performed with a FACS Canto-II flow cytometer (BD Bioscences Inc.).

We used Student's t-test (2-tailed) to compare two groups. A P-value of <0.05 was considered significant. Data are presented as means with the SD (Fig. 2B and D) or SEM (all others).

Primary LE cells were isolated from adult mice and expanded in Matrigel-assisted 3D culture for 2 to 3 weeks in the presence of EGF (Fig. 1A and B). During this period, non-epithelial cells, such as fibroblasts, were likely lost or selected against, as nearly all expanded cells were positive for EpCAM, an established marker of the epithelial lineage (Fig. 1C) (13). In 3D culture, LE cells could be propagated, for at least two to three months, with passage once weekly at an approximately 1:4 dilution.

To test EGF-dependence of LE cells, we transferred cells to 2D culture, as Matrigel used for 3D culture contains substantial amounts of EGF (14). Then, we prepared single-cell suspensions from LE spheres formed in 3D culture and seeded them in tissue-culture plates in the presence of EGF and the Rock-inhibitor Y-27632. Cells adapted to 2D culture (Fig. 2A) and were passaged (1:5 dilution) several times, always replacing EGF and Y-27632 in the media (‘Control’ in Fig. 2B and C). We confirmed that LE cells passaged in 2D were still EpCAM-positive. In addition, the 2D-cultured cells as monolayer were converted into 3D-spheres again when re-transferred on Matrigel. To evaluate the requirement for either of these factors, we prepared replicates of culture when cells were passaged and then withdrew EGF or Y-27632 from the media the next day. Upon removing EGF, LE cells in 2D culture ceased proliferating and became more flattened and enlarged (Fig. 2A), exhibiting the morphology of senescent cells, and died after the next passage (Fig. 2B). Upon the passage, the LE cells deprived of EGF showed markedly higher activity of caspase, suggesting apoptosis induction, compared to control cells (Fig. 2C). We also observed that the presence of both EGF and the Rock-inhibitor was required for maximal LE cell proliferation in 2D culture (Fig. 2D), as Y-27632 withdrawal immediately decreased cell proliferation. These results overall indicate that LE cells expanded in 3D culture over a long period maintain their requirement for EGF to proliferate.

We next transformed LE cells by transduction with SV40 Large-T and the active Kras-mutant Kras^G12V, either singly or together, using retroviral vectors harboring Neo^r and Puro^r, respectively, as selection marker. Transduced cells were then drug-selected and nude mice were inoculated with drug-resistant cells. Only LE cells receiving both Large-T and Kras^G12V (LE-LT/Kras^G12V cells) formed allograft tumors, while cells harboring either Large-T or Kras^G12V alone did not (Fig. 3A and B). These results are consistent with previous observations that cellular transformation requires both oncogene activation and inactivation of tumor suppressors such as p53 and pRb (15).

Immunostaining of LE-LT/Kras^G12V tumor showed that the Ki-67 index of the tumor was very high (~85%; Fig. 3C) and that tumor cells arising from LE cells were mostly EpCAM-positive (Fig. 3D). In addition, a part of tumor cells were positive for TTF1, an established marker of pulmonary adenocarcinoma (Fig. 3E). No staining in tumors was observed for SP-C and CK14, respective markers of lung adenocarcinoma and squamous-cell carcinoma (data not shown). Together with H&E analysis (Fig. 3B), these results suggest that tumors derived from LE-LT/Kras^G12V cells are largely anaplastic (expressing neither adenocarcinoma marker nor squamous-cell carcinoma marker) and that a subpopulation of tumor cells tends to differentiate into adenocarcinoma.

In addition to Kras^G12V, we also transformed LE cells with EGFR^ex19del, the constitutively active mutant of EGFR, in combination with Large-T. Like the LE-LT/Kras^G12V cells, the LE-LT/EGFR^ex19del cells formed tumors when transplanted into nude mice (data not shown).

It is well known that tumorigenicity due to Kras^G12V activity requires activation of the classical mitogen-activated protein kinase (MAPK) cascade, in which MAPK/ERK kinase (MEK) phosphorylates and activates extracellular regulated kinase (Erk) (16). Proliferation of LE-LT/Kras^G12V cells in vitro was independent of exogenous EGF (data not shown), which activates the MEK-ERK pathway. We then tested the sensitivity of LE-LT/Kras^G12V cells in vitro to trametinib, a MEK1-inhibitor. As shown in Fig. 4A, proliferation of LE-LT/Kras^G12V cells markedly decreased following trametinib treatment, whereas comparable treatment with erlotinib, which potently inhibits EGFR kinase activity, had no effect. In contrast, both trametinib and erlotinib treatment inhibited LE-LT/EGFR^ex19del cell proliferation (Fig. 4B). These results are consistent with the idea that Kras activates MAPK signaling downstream of EGFR, making cells resistant to EGFR inhibition and that transformation by EGFR^ex19del requires its kinase activity and downstream MAPK signaling (17). Overall, these results show that transformed LE cells are addicted to oncogenic Kras- or EGFR-stimulated signaling.