Healthy hematopoietic cell clusters and increased vascular leakiness in leukemic mice. (A) Representative frames from time-lapse IVM of calvarium BM in healthy (left) and AML-burdened (right) Flk1-GFP mice. Green: ECs. Red: mTomato⁺ healthy hematopoietic cells. AML cells are not shown. Arrows point at circulating healthy cells (left) or cell clusters (right). (B) Quantification of circulating healthy single cells and clusters in healthy and AML-burdened mice in 3–hour-long timelapse images. Data pooled from 5 healthy and 4 leukemic mice from 3 independent experiments. Eight fields of view were imaged simultaneously per mouse. (C) Percentage of doublets and cell clusters in peripheral blood (PB) of healthy and AML-burdened mice. Data obtained from 15 healthy and 25 leukemic mice from 3 independent experiments. (D) Vascular leakiness was assessed by time-lapse IVM of randomly selected regions within the calvarium of Flk1-GFP mice following administration of TRITC-dextran. Representative frames selected from 3 healthy and 4 leukemic (<10% AML blood infiltration) mice are shown. AML cells are not shown. (E) Quantification of the fold change in TRITC-dextran intensity in the BM parenchyma of healthy and early infiltrated (<10% AML blood infiltration) leukemic mice. n = 3 control and 4 leukemic mice, 3 areas measured/mice (eg, white boxes in [D]). (F) Quantification of average vessel diameter in healthy vs early infiltrated mice, with vessels measured in 3 fields of view/mice. n = 5 mice/condition. (G) Gene expression heatmap for MMP-8, MMP-9, and MMP-19 for AML cells and GMPs. n = 9 control and n = 9 leukemic mice. (H) Stroma and hematopoietic cell types expressing MMPs in healthy murine BM based on published single-cell RNAseq datasets. Circle size and color represent the proportion of expressing cells within the population, and average expression, respectively. All scale bars represent 80 µm. All data are mean ± SEM. **P < .01; ***P < .001; NS, not significant. P values determined by t tests.