Figure 4. Twist1 accelerates conditional Kras^G12D-induced lung tumorigenesis.

(A) Crosses (CT×LSL) to produce CCSP-rtTA/Twist1-tetO₇-luc/LSL-Kras^G12D (CT-LSL) mice. CT-LSL mice are infected with intranasal Cre to activate Kras^G12D. Addition of Dox enables Twist1 and luc transcription. In contrast to CRT OFF mice, CT-LSL OFF mice have Kras^G12D still active and only Twist1 expression is inactivated. (B) Kaplan-Meier tumor free survival by serial microCT of F1 littermates with CT, LSL and CT-LSL genotypes. The double oncogene animals (CT-LSL, n = 18) developed multiple tumors at a median tumor latency that was significantly shorter than the single LSL (n = 14) animals, 5 versus 6 weeks (CT-LSL versus LSL, by log-rank analysis p = 0.0121). CT animals (n = 17) and littermate controls not infected with AdCMVCre (n = 5) never developed lung tumors. (C) Lung tumor burden is increased at 9 weeks post-AdCMVCre in CT-LSL versus LSL mice qualitatively by representative microCT. Blue arrowheads denote lung tumors. H – heart; and S – spine. (D) H&E stained sections of lung tumors from a CT-LSL mouse. Black bars equal 200 and 50 µm. (E) Immunohistochemical (IHC) phenotyping of CT-LSL lung tumors indicate a type II pneumocyte cell of origin using CCSP and proSp-C markers. (F) Ki-67 IHC of LSL versus CT-LSL lung tumors (n = 2). Low - <5%; Med – 5–25%; and High - >25%. (G) pErk1/2 and p19^ARF IHC staining in serial sections demonstrate overlap. Note the nucleolar staining of p19^ARF, specific nuclei are denoted by blue arrowheads.