Evidence for type II cells as cells of origin of K-Ras-induced distal lung adenocarcinoma

Abstract

Identifying the cells of origin of lung cancer may lead to new therapeutic strategies. Previous work has focused upon the putative bronchoalveolar stem cell at the bronchioalveolar duct junction as a cancer cell of origin when a codon 12 K-Ras mutant is induced via adenoviral Cre inhalation. In the present study, we use two "knock-in" Cre-estrogen receptor alleles to inducibly express K-RasG12D in CC10(+) epithelial cells and Sftpc(+) type II alveolar cells of the adult mouse lung. Analysis of these mice identifies type II cells, Clara cells in the terminal bronchioles, and putative bronchoalveolar stem cells as cells of origin for K-Ras-induced lung hyperplasia. However, only type II cells appear to progress to adenocarcinoma.

Fig. 1.

K-RasG12D activation in CC10⁺ cells leads to hyperplasia at the BADJ and adenocarcinoma in the alveoli. (A–C) Sections of CC10-CreER; Rosa26R-fGFP mouse lung 1 wk after tmx injection. (A and B) Green, anti-GFP, lineage label; red, anti-CC10, Clara cells. More than 80% of airway Clara cells were lineage-labeled. (C) Green, anti-GFP, lineage label; purple, anti-Sftpc, type II cells. About 90% of putative BASCs (arrow) and 10% of alveolar type II cells (arrowhead) are double-labeled, as described (7), with higher magnification (Inset). (D) H&E section of CC10-CreER; LSL-K-RasG12D mouse lung 15 wk after tmx. Tumors arise predominantly near the BADJ and in the alveoli but not in the airways.

Fig. 2.

Time course of neoplasia at the BADJ and in alveoli. Sections of CC10-CreER; LSL-K-RasG12D mouse lung at various times after four doses of tmx were stained with anti-CC10 (red) and pro-Sftpc (green) (A, B, D, E, G, H, J, and K) or H&E (C, F, I, and L). (Insets) Cells marked by arrows. (A–C) Three weeks. (A) Rare CC10⁺, Sftpc⁺ cells (arrow) at the BADJ. (B) Alveolar hyperplasia consists of Sftpc⁺ cells (arrow). (C) Hyperplasia in alveoli (arrow). (D–F) Nine weeks. (D) BADJ dual-positive cells (arrow). (E and F) Alveolar Sftpc⁺ adenomas. (G–I) Fifteen weeks. (G) BADJ dual-positive cells (arrow). (H and I) Sftpc⁺ papillary adenoma. (J–L) Twenty-one weeks. (J) Dual-positive hyperplasia at BADJ (arrow). (K and L) Sftpc⁺ papillary adenocarcinoma.

Fig. 3.

BADJ CC10⁺ cells and alveolar CC10⁺, Sftpc⁺ cells are the most abundant proliferating cells after K-RasG12D activation. (A, B, D, E, G, and H) Sections of CC10-CreER; LSL-K-RasG12D; Rosa26R-fGFP mouse lung stained with anti-BrdU (red), GFP (green), and Sftpc (purple). (A and B) Twenty-four hours after one dose of tmx. (A) Representative BADJ with one GFP⁺, Sftpc⁻, BrdU⁺ cell (arrow) surrounded by GFP⁺, Sftpc⁺ cells (arrowheads). (B) Representative GFP⁺, BrdU⁺, Sftpc⁺ cell at the BADJ (arrow). (C) Quantification of BrdU-incorporating BADJ cells at different times after K-Ras activation. (D, E, G, and H) Seventy-two hours after four doses of tmx. (D) Alveolar BrdU⁺ cells are GFP⁺, Sftpc⁺. (E) Higher magnification of boxed area in D showing BrdU⁺, GFP⁺, Sftpc⁺ cells (arrows). (F) Quantification of alveolar BrdU⁺ cells at different times after K-Ras activation. (G and H) BrdU⁺, GFP⁺, Sftpc⁻ cells in larger airway (G) and trachea (H). Error bars represent standard variation of three different sections of a mouse lung.

Fig. 4.

K-RasG12D activation in Sftpc-expressing cells leads to lung adenocarcinoma in alveoli. Arrows in A and B denote putative BASCs. (A) Section of Sftpc-CreER; Rosa26R-fGFP mouse lung 2 wk after one dose of tmx stained with anti-GFP (green) and CC10 (purple). GFP labels the majority of type II cells and putative BASCs. (B) BADJ area in A. (C) H&E section of Sftpc-CreER; LSL-K-RasG12D; Rosa26R-fGFP mouse lung 14 wk after one dose of tmx. The majority of tumors arise in the peripheral lung and near the BADJ but not in the airways.

Fig. 5.

Time course of lung adenocarcinoma progression in Sftpc-CreER; LSL-K-RasG12D; Rosa26R-fGFP mice after one dose of tmx. Insets are higher magnification images of cells denoted by arrows. (A–E) Two weeks. (A) Section of Sftpc-CreER; Rosa26R-fGFP mouse lung stained with anti-Sftpc (green) and CC10 (red). At the BADJ, putative BASCs stain Sftpc⁺ and CC10⁺. (Inset) Putative BASC at higher magnification. (B–E) Sections of Sftpc-CreER; LSL-K-RasG12D; Rosa26R-fGFP mouse lungs. (B) BADJ CC10⁺, Sftpc⁺ cells (arrow) do not expand. (C) Representative small tumor is Sftpc⁺, CC10⁻. (D) Normal BADJ (arrow) and hyperplasia in the peripheral lung. (E) Representative small tumor. (F–J) Four weeks. (F) In Sftpc-CreER; Rosa26R-fGFP mouse lung, alveolar CC10⁺ type II cell. (G–J) Sections of Sftpc-CreER; LSL-K-RasG12D; Rosa26R-fGFP mouse lung. (G) Normal BADJ. (H) Sftpc⁺, CC10⁻ alveolar tumor cells. (I) Normal BADJ (arrows) and alveolar tumors. (J) Peripheral lung tumor. (K–O) Eight weeks. (K) In Sftpc-CreER; Rosa26R-fGFP mouse lung, alveolar CC10⁺, Sftpc⁺ cells. (L–O) Sections of Sftpc-CreER; LSL-K-RasG12D; Rosa26R-fGFP mouse lungs. (L) Normal BADJ staining. CC10⁺, Sftpc⁺ cells (arrow) do not expand. (M) Tumor with few very low CC10⁺ cells (arrow). (N) Normal BADJ (arrows) and alveolar tumors. (O) Representative tumor. (P–T) Fourteen weeks. (P) In Sftpc-CreER; Rosa26R-fGFP mouse lung, alveolar CC10⁺, Sftpc⁺ cells. (Q–T) Sections of Sftpc-CreER; LSL-K-RasG12D; Rosa26R-fGFP mouse lungs. (Q) Normal BADJ staining. (R) Sftpc⁺, CC10⁻ alveolar tumor cells. (S) Normal BADJ (arrows) and widespread alveolar tumors. (T) Large adenocarcinoma in the peripheral lung.

Fig. 6.

Model for response of various respiratory epithelial cells to K-RasG12D induction. In the steady-state normal lung (Upper), there are type II cells that express Sftpc (red) and those that are dual-positive for CC10 and Sftpc (purple). When K-RasG12D is expressed in CC10-CreER mice (Left) there is hyperplasia in the BADJ that involves not only putative BASCs but also Clara cells. We propose that adenocarcinomas arise in the alveoli exclusively from dual-positive type II cells but that these become only Sftpc⁺. When K-RasG12D is expressed in Sftpc-CreER mice (Right), the BADJ is normal or contains rare small hyperplastic areas, and tumors arise only in the alveoli. We propose that they can arise from either dual-positive or Sftpc⁺ cells.