Fig. 2.
Mychigh and Myclow cells exhibit clonal mutual interdependence. Unless otherwise indicated, analysis of mixed WM+T/WM−T tumors was carried out after 3 d of treatment with tamoxifen (100 μg/mouse, twice daily) or oil (vehicle) for the respective controls. Abbreviations in graphs: C, mixed tumors plus vehicle; T, mixed tumors plus tamoxifen. Error bars represent SD. (A) Representative picture of the gross morphology of mixed (in this case, 30%/70%) WM+T/WM−T tumors and representative fluorescent image of mixed tumor tissue. (B) Caliperimetric measurement of tumor growth of mixed WM+T/WM−T tumors and oil controls. (C) Necrotic burden of mixed WM+T/WM−T tumors. (D) Quantification of the cellularity of mixed WM+T/WM−T tumors. (E) Quantification of hypoxia in mixed WM+T/WM−T tumors. (F) Average area of blood vessel in mixed WM+T/WM−T tumors. (G) Quantification of cell death, by presence of cleaved caspase 3, in mixed WM+T/WM−T tumors. Clones were distinguished via GFP staining. (H) Quantification of proliferation, by IDU incorporation, in mixed WM+T/WM−T tumors. Clones were distinguished via GFP staining. (I) Immunofluorescent staining for GFP (WM+T) and p19ARF of intermingled areas in mixed WM+T/WM−T tumors and controls (in D–I: n = 8 mixed WM+T/WM−T tumors treated with tamoxifen and 5 vehicle controls). (J) Caliperimetric measurement of tumor growth of individual WM+T, WM−T, mixed WM+T/WM−T mixed tumors, and controls on long-term tamoxifen treatment. (K) Representative images of immunofluorescence staining for Myc, dtTomato, and GFP on frozen tissue sections from long-term–treated WM+T and mixed WM+T/WM−T tumors showing loss of Myc in the WM+T tumors, but no such phenomenon in the mixed WM+T/WM−T tumors. (L) Digital droplet PCR on genomic DNA comparing the recombination status of the R26CMER allele in 20% WM+T and 100% WM+T tumor after long-term tamoxifen treatment (n = 1 20% WM+T and 1 100% WM+T tumor). For box-and-whisker plots, the error bars represent min to max values, the box represents the interquartile range, and the horizontal line represents the median. P values are based on Student’s t test: *P < 0.05, **P < 0.01, and ***P < 0.001. In I, the Myclow clone is outlined by a dotted line. Areas that are neither GFP positive nor marked up by dotted lines are nontumor tissues such as stroma and necrotic areas.