HS1 KD cells show a distinct in vivo pattern of growth and localization. Rag2^−/−γ_c^−/− male mice received a transplant subcutaneously in the left flank with either GFP-expressing HS1 KD or CNTR cells (10 × 10⁶ cells/mouse; n = 4). (A) Tumor volume was evaluated by measuring perpendicular diameters by a caliper. Animals were killed when the tumor volume reached 1000 mm³ (*P ≤ .05; day 35). (B) Cells from lung, spleen, liver, kidney, axillary tumor-draining lymph node, bone marrow, and site of injection were collected. The slides were stained with anti-GFP Alexa Fluor 488 (green) and TO-PRO3 (red) for the nuclear staining (overlap yellow). Slides were analyzed by Radiance 2100 (Bio-Rad Laboratories) dual-laser confocal microscope with the use of an inverted 20× oil objective. The extent and pattern of invasion is shown by the presence of green GFP⁺ leukemic cells. (C-D) HS1^−/− mice (n = 3, repeated twice) and age-matched WT mice (n = 3, repeated twice) were given intravenous injections of a mixture of CFSE-low– and CFSE-high–labeled CD19⁺ purified splenocytes from WT and HS1^−/− mice, respectively. Histogram plots represent flow cytometric analysis of 7AAD⁻ CFSE-low (WT cells, peak T) and CFSE-high (HS1^−/− cells, peak U) splenocytes obtained from BM of representative WT (C) and HS1^−/−(D) recipient mice killed 20 hours after cell injection. Cells represented in each plot were gated on physical parameters. Numbers show percentages of cells falling in peaks T and U.