Kras ^G12D mutation–carrying BM cells show increased motility in vivo. (A) Analysis of Kras-mutant cell behavior in vivo. Pdzk1ip1-CreER R26^Tom/Tom reporter mice with WT or conditional mutant (Kras^G12D) Kras alleles were administered tamoxifen to induce simultaneous Tom expression and Kras mutation in HSC. 2 or 3 wk later, their tibiae were examined by intravital microscopy using continuous frame recording for 1 h. (B) Representative snapshots of intravital imaging of reporter mice at the indicated time points. Red and green signals represent Tom⁺ cells and autofluorescent macrophages, respectively; select Tom⁺ cells are marked by gray spheres, and their tracks are indicated. Data are representative of three to four mice per group per time point. Scale bar, 10 µm. (C) Representative flower plots showing tracks of individual Tom⁺ cells (marked by different colors) relative to each cell’s starting point. (D and E) Motility of Tom⁺ cells. Shown are violin plots of track velocity and displacement velocity of individual Tom⁺Kras^G12D or WT cells at 2 or 3 wk (D) and their corresponding MSD (E). For the 2-wk experiment, individual Tom⁺ WT cells (109 cells from four mice) and Tom⁺Kras-mutant cells (40 cells from four mice) were analyzed. For the 3-wk experiment, individual Tom⁺ WT cells (206 cells from four mice) and Tom⁺Kras-mutant cells (677 cells from three mice) were analyzed. Lines on violin plots indicate median and quartiles; on MSD graphs, symbols indicate mean MSD values for all tracks at the indicated time point and ranges indicate the standard error. (F and G) CXCL12-driven migration of Tom⁺ cells in vitro. (F) Lin⁻ cells were isolated by magnetic negative selection using additional depletion of committed progenitors by antibodies to CD11c, CD115, CD16/32, and CD41. The resulting cells from reporter mice 3 wk after tamoxifen treatment were used in a transwell migration assay toward CXCL12 for 4 h. (G) Fractions of Tom⁺ or Tom⁻ Lineage⁻ cells that migrated into the bottom chamber. Symbols represent individual transwell cultures seeded in triplicates from five mice per genotype pooled from two independent experiments. (H and I) Expression of CXCR4 in Kras-mutant stem/progenitor cells. (H) Representative histograms of CXCR4 staining in Tom⁺-gated HSC or MPP from Kras^G12D or WT reporter mice 3 wk after induction. The threshold of positive staining is indicated by the dotted line. (I) Fractions of CXCR4⁺ cells in Tom⁺ HSC or MPP, and MFI of CXCR4 in MPP. Symbols represent individual mice; bars represent the mean ± SD Statistical significance was estimated by unpaired Student’s t test (D) or the Mann–Whitney test (other panels). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MFI, mean fluorescence intensities.