Lung Adenocarcinoma Mouse Models Based on Orthotopic Transplantation of Syngeneic Tumor-Initiating Cells Expressing EpCAM, SCA-1, and Ly6d

Abstract

Somatic mutations in EGFR and KRAS as well as chromosome rearrangements affecting ALK, ROS1, and RET have been identified in human lung adenocarcinoma (LUAD). We here developed organoid-based orthotopic and syngeneic mouse models for studies of the pathogenesis and treatment of LUAD. We isolated EpCAM-positive epithelial cells from mouse lungs and cultured them as organoids to maintain epithelial stem cell properties. These cells were transformed by KRAS(G12V) or EML4-ALK and then transplanted via the trachea into the lungs of the syngeneic mice, where they formed tumors that expressed the lung lineage marker TTF-1 and which closely recapitulated the pathology of human LUAD. Treatment with crizotinib suppressed the growth of tumors formed by the EML4-ALK-expressing lung epithelial cells in a subcutaneous transplantation model. Organoid culture of normal lung epithelial cells resulted in enrichment of EpCAM⁺SCA-1(Ly6a)⁺ cells as well as in that of cells expressing another member of the Ly6 protein family, Ly6d, which was found to be required for the growth of the LUAD-initiating cells expressing KRAS(G12V) or EML4-ALK. We also found that a high expression level of LY6D was associated with poor prognosis in human LUAD. Our results thus suggest that LY6D is a potential lung cancer stem cell marker.

Keywords: EpCAM; Ly6d; SCA-1; lung cancer; orthotopic transplantation; syngeneic mouse model.

Figure 1

Three-dimensional culture of mouse lung epithelial cells. (A) Representative immunofluorescence staining of epithelial cells in a mouse lung section with antibodies to EpCAM. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 100 µm. (B) Gating strategy for sorting of EpCAM⁺CD31⁻CD45⁻ lung epithelial cells by FACS. SSC, side scatter; FSC, forward scatter; PI, propidium iodide; APC, allophycocyanin; PE, phycoerythrin. (C) Schematic representation of the 3D culture system. (D) Representative phase-contrast images of primary EpCAM⁺CD31⁻CD45⁻ lung epithelial cells expanded in 3D culture. (E) Representative phase-contrast microscopy (upper row), hematoxylin-eosin staining (middle row), and immunocytochemical staining of TTF-1, SP-C, AQP5, or CC-10 (lower row) for colonies of lung epithelial cells. Scale bars, 100 µm. Three types of colony morphology—cystic, spherical, and branched—were apparent. (F) Representative fluorescent multiplex immunostaining of SP-C and CC-10 in a primary (p0) lung epithelial cell colony. Nuclei were stained with DAPI. Scale bar, 50 µm. (G) Quantification of colony types for lung epithelial cells before (p0) and after consecutive serial passages (p1, p2). Data are means of triplicates from a representative experiment.

Figure 2

Expansion of oncogene-transformed mouse lung epithelial cells supported by Cdkn2a knockout. (A) Schematic representation of oncogene transduction in lung epithelial cells. Cells dissociated from colonies in 3D culture were infected with retroviruses harboring GFP and either KRAS^G12V or EML4-ALK cDNAs, after which GFP-positive cells were isolated by FACS and subjected to organoid culture. (B) Colony formation efficiency for lung epithelial cells from wild-type (WT) or Cdkn2a^−/− mice after retroviral transduction of KRAS^G12V or EML4-ALK. Data are means + SD (n = 3 independent experiments). Representative phase-contrast images of culture plates are also shown. (C) Representative phase-contrast and fluorescence microscopy as well as hematoxylin-eosin (HE) and TTF-1 immunocytochemical staining of colonies formed by KC cells or AC cells. Scale bars, 100 µm.

Figure 3

Establishment of clinically relevant syngeneic mouse models of LUAD. (A) Representative hematoxylin-eosin staining of the lungs of C57BL/6 mice at 1, 2, and 3 weeks after intratracheal bleomycin administration. Scale bars, 100 µm. (B) Representative hematoxylin-eosin staining of the lungs of PBS- or bleomycin-pretreated mice at 28 days after intratracheal transfer of KC or AC cells. Scale bars, 1 cm. (C) Lung tumor incidence in PBS- or bleomycin-pretreated mice at 28 days after KC or AC cell transfer. Data are means + SD (n = 5 recipient mice). The p values were calculated with the two-tailed Student’s t test. (D) Kaplan-Meier survival curves for KC or AC lung tumor–bearing mice (n = 5 for each model) from the time of cell transfer. (E) Representative hematoxylin-eosin staining as well as immunohistochemical staining of GFP or TTF-1 in lung tumors isolated 28 days after KC or AC cell transfer. Scale bars, 100 μm. (F) Viability of KC or AC cells cultured in the presence of the indicated concentrations of crizotinib for 72 h. Data are means ± SD of triplicates from a representative experiment. (G) Nude mice bearing subcutaneous tumors formed by injected AC cells were treated with vehicle or crizotinib for 14 days, and tumor volume was determined at the indicated times after the onset of treatment. CR indicates complete tumor regression. Data are means ± SD (n = 5 mice), and the p value was calculated with the two-tailed Student’s t test.

Figure 4

Increased expression of SCA-1 and Ly6d in lung epithelial cells with culture and in oncogene-expressing tumorigenic lung epithelial cells. (A) Representative immunofluorescence staining of SP-C and CC-10 (nuclei were stained with DAPI) as well as immunochemical staining of TTF-1 in p2 colonies of mouse lung epithelial cells. Scale bars, 100 µm. (B) Flow cytometric analysis of the proportion of EpCAM⁺SCA-1⁺ cells among primary lung epithelial cells (p0) or cells of subsequent serial passages (p1, p2). Data are means + SD of triplicates from a representative experiment, and the p values were calculated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test. (C) Representative flow cytometric analysis of EpCAM and SCA-1 expression on KC or AC cells. (D) Representative immunohistochemical staining for EpCAM, SCA-1, or TTF-1 in serial sections of lung tumors from KC and AC mouse models. Scale bars, 100 µm. (E) Heatmap showing the changes in mRNA abundance for Ly6/uPAR family genes in mouse lung epithelial cells during serial passage in 3D culture. Data are shown for three independent experiments. (F) Flow cytometric analysis of the proportion of EpCAM⁺Ly6d⁺ cells among primary lung epithelial cells (p0) or cells of subsequent serial passages (p1, p2). Data are means + SD of triplicates from a representative experiment, and the p values were calculated by one-way ANOVA followed by Tukey’s multiple-comparison test. (G,H) Representative flow cytometric analysis of EpCAM and Ly6d expression (G) and of SCA-1 and Ly6d expression (H) on KC or AC cells. (I) Representative immunofluorescence staining of KC or AC lung tumor sections for Ly6d and SCA-1. Nuclei were stained with DAPI. Scale bars, 100 µm.

Figure 5

Ly6d is required for colony formation by oncogene-transformed lung epithelial cells in 3D culture. (A) Ly6d^high and Ly6d^low cells isolated by FACS from KC or AC cells were seeded in 3D culture for determination of colony formation efficiency. (B) Colony formation in 3D culture by KC or AC cells transfected with expression vectors for control (shCtrl) or Ly6d (shLy6d #2 or #5) shRNAs. All quantitative data are means + SD (n = 5), and the p values were calculated with the two-tailed Student’s t test (A) or by one-way ANOVA followed by Tukey’s multiple-comparison test (B).

Figure 6

Expression of Ly6d is associated with poor prognosis in LUAD patients. (A) Representative immunohistochemical staining of Ly6d and Ki67 in a LUAD tumor sample. Scale bars, 50 µm. (B) Quantitation of the percentage of Ki67⁺ tumor cells per field in Ly6d^low (n = 60) and Ly6d^high (n = 60) LUAD tumors. Ly6d^low and Ly6d^high subpopulations of LUAD patients were classified based on the Ly6d expression levels with the mean values used for the cutoff points. Data are presented as box-and-whisker plots, with the boxes indicating the quartile values and the whiskers indicating minimum and maximum. The p value was calculated by Mann-Whitney test. (C) Kaplan-Meier analysis of overall survival according to LY6D gene expression in LUAD patients (n = 719) generated using Kaplan-Meier Plotter, with auto select cut-off selected. The hazard ratio (HR) with its 95% confidence interval as well as the log-rank p value are indicated.