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. 2021 Apr 6;10:e66788. doi: 10.7554/eLife.66788

Figure 2. Nkx2-1 deletion in established BRAF-mutant LUAD induces mucinous adenocarcinoma without altering tumor growth.

(A–C) All mice are BrafFSF-V600E/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2. Viral FlpO-recombinase expression simultaneously activated BRAFV600E and CreERT2 and deleted Trp53 by excision of Frt sites. Three weeks post-tumor initiation, mice were injected with corn oil (as vehicle control) or with tamoxifen to recombine LoxP-sites within Nkx2-1 alleles by CreERT2 recombinase. Lungs were harvested 3 weeks thereafter. (A) Experimental scheme. (B) H and E, Alcian Blue, NKX2-1 and HNF4A staining of lung neoplasia arising 6 weeks after initiation with Ad5CMV-FlpO adenovirus (2 × 107 pfu/mouse). Scale bar: 100 µm. (C) Quantitation of tumor burden at 3 weeks (n = 4 mice), and at 6 weeks after initiation, following corn oil treatment (n = 10 mice) or tamoxifen injections (n = 8 mice). Tamoxifen administration consisted of six intraperitoneal doses over 9 days. Graphs represent mean ± S.D. p Values are not statistically significant. (D) Mice are KrasFSF-G12D/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2 and were administered with corn oil (n = 6 mice) or tamoxifen (n = 6 mice) injections as described in (A). Shown is the quantitation of tumor burden at 3 weeks and at 6 weeks post-initiation. Graphs represent mean ± S.D. **p=0.0014 by Student’s t-test. (E) Survival of BrafFSF-V600E/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2 mice that were treated with tamoxifen or vehicle starting at 3 weeks following tumor initiation. Tamoxifen administration consisted of six intraperitoneal doses over 9 days, followed by tamoxifen-containing chow for 1 month.

Figure 2.

Figure 2—figure supplement 1. Nkx2-1 deletion in established BRAF-mutant LUAD induces mucinous adenocarcinoma without altering tumor growth.

Figure 2—figure supplement 1.

(A) Immunostaining for indicated mucin/gastric markers in NKX2-1-positive and -negative LUADs. Lung tumor sections were obtained from corn oil or tamoxifen treated BrafFSF-V600E/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2 mice 6 weeks post-initiation with Ad5CMV-FlpO adenovirus (2 × 107 pfu/mouse). Scale bar: 100 µm. (B, C) Quantitation of MCM2-positive cells in lung tumors at 6 weeks after initiation, and following corn oil/tamoxifen injections at 3 weeks. Lung neoplasia was initiated with Ad5CMV-FlpO adenovirus (2 × 107 pfu/mouse) in BrafFSF-V600E/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2 and KrasFSF-G12D/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2 mice. Graphs represent mean ± S.D. Multiple tumors in BrafFSF-V600E/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2 oil (n = 7 mice); tamoxifen administered (n = 5 mice); KrasFSF-G12D/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2 oil (n = 4 mice) and tamoxifen administered (n = 5 mice) were analyzed. (D) Representative images of high-grade tumors obtained from a BrafFSF-V600E/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2 mouse that had received tamoxifen injections. Scale bar: 100 µm. (E) Grades for individual tumors based on criteria described for Figure 1D. Histopathologic assessment was performed for mice enrolled in survival study (Figure 2E).
Figure 2—figure supplement 2. Percentage of incomplete recombinant tumors in BrafFSF-V600E/+;Trp53frt/frt;Nkx2-1f/f;Rosa26FSF-CreERT2/FSF-CreERT2 mice that were treated with Tamoxifen from Figure 2E.

Figure 2—figure supplement 2.

Graph represents mean ± S.D.