Fig. 4: Early leukemia progression by Image-seq.
From: Image-seq: spatially resolved single-cell sequencing guided by in situ and in vivo imaging
a, In vivo imaging timepoints. IVM, intravital microscopy. b, Cell counts per bone marrow cavity on day 0 (n = 143 cavities, n = 6 independent mice), day 1 (n = 103 cavities, n = 6 independent mice) and day 3 (n = 132 cavities, n = 7 independent mice) after transplantation. Error bars represent the standard deviation and are centered at the arithmetic mean. The 99.9% confidence interval for proliferating (P) cells is shown in red, that for intermediate (IM) cells in blue and that for non-proliferating (NP) cells in green. c, Number of cells per cavity on day 3 plotted for P, IM, and NP regions. The dashed line represents the median and the dotted line, the quartiles in each distribution. Statistical significance was determined using Welch’s ANOVA test and the adjusted P values were determined using Dunnett’s multiple comparisons test. NP, 10 biological replicates, n = 3 independent mice; IM, 32 biological replicates, n = 3 independent mice; P, 107 biological replicates, n = 3 independent mice. d, Tiled, maximum intensity projection image of leukemic bone marrow on day 3 after transplantation. Red square and inset, P cells; blue square and inset, IM cells; green square and inset, NP cell (marked by white arrow). Scale bars: main image, 500 μm; insets, 100 μm. Bone, bone SHG. Imaging of the entire calvarium bone marrow at day 3 was performed in 17 independent mice, including 122 biological replicates with P, 35 biological replicates with IM, and 79 biological replicates with NP cells. e, Images of proliferating (top, red border; scale bars, 100 μm) and non-proliferating (bottom, green border; scale bars, 50 μm) AML cells imaged longitudinally in the same animal and bone cavity on day 1 and 3 after transplantation. Bone SHG signal is shown in grey, AML-GFP signal is shown in green. The same depth along the z dimension is shown (Zrel = 0) on both days, along with a maximum intensity projection for P cells. For the NP cell a single Z slice (Zrel = 28 µm) shows that the AML cell has moved deeper into the bone marrow on day 3. A total of 107 biological replicates with P, 10 biological replicates with NP and 32 biological replicates with IM cells (n = 3 independent mice, respectively) were identified by longitudinal imaging (definition of P, NP, IM based on the fold-change difference between day 1 and day 3). f, Distribution of the cell cycle in the three imaging phenotypes (P value obtained using a two-sided Fisher’s exact test). g, Bar-plot representing the fraction of P, NP, and IM cells in each AML cluster. AP1, AML-AP1; GMP, AML-GMP; Mono, AML-mono (P value obtained using a two-sided Fisher’s exact test). h, Leiden clustering of 84 AML cells isolated by Image-seq after regression of cell cycle genes using Seurat, visualized as UMAP embedding. i, Heatmap of scaled normalized expression for the 10 most strongly upregulated genes (based on logFoldChange) in each AML cluster.
