Figure 9. Clonal heterogeneity in RGB-marked tumors.
(A) Schematic representation of lentiviral RGB marking of GL261 cells. Fluorescent proteins of the 3 basic colors are mixed at different but highly stable expression intensities so that all perceivable colors are generated. (B) Confocal fluorescence microscopy of RGB-marked GL261 tumors in WT and Pfp–/– Rag2–/– mice. Scale bar: 50 μm. (C) Spherical scatter plot of cells analyzed by flow cytometry. Each data point designates the chromaticity value of a cell. Note the reduced occupancy, i.e., the plot area occupied with data points in WT mice compared with Pfp–/– Rag2–/– mice and compared with the preinjection mix. (D) Quantification of occupancy after multistep dimensionality reduction validates significantly higher clonal contraction in tumors in WT mice than in Pfp–/– Rag2–/– mice, compared with the multiclonal preinjection mix. Box plots with IQR (box), mean (line), and maximum/minimum (whiskers). One-way ANOVA (P = 0.001) with Tukey’s post hoc test; *P < 0.05, ***P = 0.001; n = 4 per mouse group, n = 2 for the preinjection mix.